US20110312622A1 - Microfluidic device with low-volume hybridization chambers for electrochemiluminescent detection of target sequences - Google Patents

Abstract

A microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device having an array of hybridization chambers, each containing electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes, wherein, the hybridization chambers each have a volume less than 900000 cubic microns.

Description

FIELD OF THE INVENTION
• The present invention relates to diagnostic devices that use microsystems technologies (MST). In particular, the invention relates to microfluidic and biochemical processing and analysis for molecular diagnostics.
CO-PENDING APPLICATIONS
• The following applications have been filed by the Applicant which relate to the present application:

• GBS001US	GBS002US	GBS003US	GBS005US	GBS006US
  GSR001US	GSR002US	GAS001US	GAS002US	GAS003US
  GAS004US	GAS006US	GAS007US	GAS008US	GAS009US
  GAS010US	GAS012US	GAS013US	GAS014US	GAS015US
  GAS016US	GAS017US	GAS018US	GAS019US	GAS020US
  GAS021US	GAS022US	GAS023US	GAS024US	GAS025US
  GAS026US	GAS027US	GAS028US	GAS030US	GAS031US
  GAS032US	GAS033US	GAS034US	GAS035US	GAS036US
  GAS037US	GAS038US	GAS039US	GAS040US	GAS041US
  GAS042US	GAS043US	GAS044US	GAS045US	GAS046US
  GAS047US	GAS048US	GAS049US	GAS050US	GAS054US
  GAS055US	GAS056US	GAS057US	GAS058US	GAS059US
  GAS060US	GAS061US	GAS062US	GAS063US	GAS065US
  GAS066US	GAS067US	GAS068US	GAS069US	GAS070US
  GAS080US	GAS081US	GAS082US	GAS083US	GAS084US
  GAS085US	GAS086US	GAS087US	GAS088US	GAS089US
  GAS090US	GAS091US	GAS092US	GAS093US	GAS094US
  GAS095US	GAS096US	GAS097US	GAS098US	GAS099US
  GAS100US	GAS101US	GAS102US	GAS103US	GAS105US
  GAS106US	GAS108US	GAS109US	GAS110US	GAS111US
  GAS112US	GAS113US	GAS114US	GAS115US	GAS117US
  GAS118US	GAS119US	GAS120US	GAS121US	GAS122US
  GAS123US	GAS124US	GAS125US	GAS126US	GAS127US
  GAS128US	GAS129US	GAS130US	GAS131US	GAS132US
  GAS133US	GAS134US	GAS135US	GAS136US	GAS137US
  GAS138US	GAS139US	GAS140US	GAS141US	GAS142US
  GAS143US	GAS144US	GAS146US	GAS147US	GRR001US
  GRR002US	GRR003US	GRR004US	GRR005US	GRR006US
  GRR007US	GRR008US	GRR009US	GRR010US	GVA001US
  GVA002US	GVA004US	GVA005US	GVA006US	GVA007US
  GVA008US	GVA009US	GVA010US	GVA011US	GVA012US
  GVA013US	GVA014US	GVA015US	GVA016US	GVA017US
  GVA018US	GVA019US	GVA020US	GVA021US	GVA022US
  GHU001US	GHU002US	GHU003US	GHU004US	GHU006US
  GHU007US	GHU008US	GWM001US	GWM002US	GDI001US
  GDI002US	GDI003US	GDI004US	GDI005US	GDI006US
  GDI007US	GDI009US	GDI010US	GDI011US	GDI013US
  GDI014US	GDI015US	GDI016US	GDI017US	GDI019US
  GDI023US	GDI028US	GDI030US	GDI039US	GDI040US
  GDI041US	GPC001US	GPC002US	GPC003US	GPC004US
  GPC005US	GPC006US	GPC007US	GPC008US	GPC009US
  GPC010US	GPC011US	GPC012US	GPC014US	GPC017US
  GPC018US	GPC019US	GPC023US	GPC027US	GPC028US
  GPC029US	GPC030US	GPC031US	GPC033US	GPC034US
  GPC035US	GPC036US	GPC037US	GPC038US	GPC039US
  GPC040US	GPC041US	GPC042US	GPC043US	GLY001US
  GLY002US	GLY003US	GLY004US	GLY005US	GLY006US
  GIN001US	GIN002US	GIN003US	GIN004US	GIN005US
  GIN006US	GIN007US	GIN008US	GMI001US	GMI002US
  GMI005US	GMI008US	GLE001US	GLE002US	GLE003US
  GLE004US	GLE005US	GLE006US	GLE007US	GLE008US
  GLE009US	GLE010US	GLE011US	GLE012US	GLE013US
  GLE014US	GLA001US	GGA001US	GGA003US	GRE001US
  GRE002US	GRE003US	GRE004US	GRE005US	GRE006US
  GRE007US	GCF001US	GCF002US	GCF003US	GCF004US
  GCF005US	GCF006US	GCF007US	GCF008US	GCF009US
  GCF010US	GCF011US	GCF012US	GCF013US	GCF014US
  GCF015US	GCF016US	GCF020US	GCF021US	GCF022US
  GCF023US	GCF024US	GCF025US	GCF027US	GCF028US
  GCF029US	GCF030US	GCF031US	GCF032US	GCF033US
  GCF034US	GCF035US	GCF036US	GCF037US	GSA001US
  GSA002US	GSE001US	GSE002US	GSE003US	GSE004US
  GDA001US	GDA002US	GDA003US	GDA004US	GDA005US
  GDA006US	GDA007US	GPK001US	GMO001US	GMV001US
  GMV002US	GMV003US	GMV004US	GRD001US	GRD002US
  GRD003US	GRD004US	GPD001US	GPD003US	GPD004US
  GPD005US	GPD006US	GPD007US	GPD008US	GPD009US
  GPD010US	GPD011US	GPD012US	GPD013US	GPD014US
  GPD015US	GPD016US	GPD017US	GAL001US	GPA001US
  GPA003US	GPA004US	GPA005US	GSS001US	GSL001US
  GCA001US	GCA002US	GCA003US

• The disclosures of these co-pending applications are incorporated herein by reference. The above applications have been identified by their filing docket number, which will be substituted with the corresponding application number, once assigned.
BACKGROUND OF THE INVENTION
• Molecular diagnostics has emerged as a field that offers the promise of early disease detection, potentially before symptoms have manifested. Molecular diagnostic testing is used to detect:
• • Inherited disorders
  • Acquired disorders
  • Infectious diseases
  • Genetic predisposition to health-related conditions.
• With high accuracy and fast turnaround times, molecular diagnostic tests have the potential to reduce the occurrence of ineffective health care services, enhance patient outcomes, improve disease management and individualize patient care. Many of the techniques in molecular diagnostics are based on the detection and identification of specific nucleic acids, both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), extracted and amplified from a biological specimen (such as blood or saliva). The complementary nature of the nucleic acid bases allows short sequences of synthesized DNA (oligonucleotides) to bond (hybridize) to specific nucleic acid sequences for use in nucleic acid tests. If hybridization occurs, then the complementary sequence is present in the sample. This makes it possible, for example, to predict the disease a person will contract in the future, determine the identity and virulence of an infectious pathogen, or determine the response a person will have to a drug.
Nucleic Acid Based Molecular Diagnostic Test
• A nucleic acid based test has four distinct steps:
• 1. Sample preparation
• 2. Nucleic acid extraction
• 3. Nucleic acid amplification (optional)
• 4. Detection
• Many sample types are used for genetic analysis, such as blood, urine, sputum and tissue samples. The diagnostic test determines the type of sample required as not all samples are representative of the disease process. These samples have a variety of constituents, but usually only one of these is of interest. For example, in blood, high concentrations of erythrocytes can inhibit the detection of a pathogenic organism. Therefore a purification and/or concentration step at the beginning of the nucleic acid test is often required.
• Blood is one of the more commonly sought sample types. It has three major constituents: leukocytes (white blood cells), erythrocytes (red blood cells) and thrombocytes (platelets). The thrombocytes facilitate clotting and remain active in vitro. To inhibit coagulation, the specimen is mixed with an agent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration. Erythrocytes are usually removed from the sample in order to concentrate the target cells. In humans, erythrocytes account for approximately 99% of the cellular material but do not carry DNA as they have no nucleus. Furthermore, erythrocytes contain components such as haemoglobin that can interfere with the downstream nucleic acid amplification process (described below). Removal of erythrocytes can be achieved by differentially lysing the erythrocytes in a lysis solution, leaving remaining cellular material intact which can then be separated from the sample using centrifugation. This provides a concentration of the target cells from which the nucleic acids are extracted.
• The exact protocol used to extract nucleic acids depends on the sample and the diagnostic assay to be performed. For example, the protocol for extracting viral RNA will vary considerably from the protocol to extract genomic DNA. However, extracting nucleic acids from target cells usually involves a cell lysis step followed by nucleic acid purification. The cell lysis step disrupts the cell and nuclear membranes, releasing the genetic material. This is often accomplished using a lysis detergent, such as sodium dodecyl sulfate, which also denatures the large amount of proteins present in the cells.
• The nucleic acids are then purified with an alcohol precipitation step, usually ice-cold ethanol or isopropanol, or via a solid phase purification step, typically on a silica matrix in a column, resin or on paramagnetic beads in the presence of high concentrations of a chaotropic salt, prior to washing and then elution in a low ionic strength buffer. An optional step prior to nucleic acid precipitation is the addition of a protease which digests the proteins in order to further purify the sample.
• Other lysis methods include mechanical lysis via ultrasonic vibration and thermal lysis where the sample is heated to 94° C. to disrupt cell membranes.
• The target DNA or RNA may be present in the extracted material in very small amounts, particularly if the target is of pathogenic origin. Nucleic acid amplification provides the ability to selectively amplify (that is, replicate) specific targets present in low concentrations to detectable levels.
• The most commonly used nucleic acid amplification technique is the polymerase chain reaction (PCR). PCR is well known in this field and comprehensive description of this type of reaction is provided in E. van Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Springer, 2008.
• PCR is a powerful technique that amplifies a target DNA sequence against a background of complex DNA. If RNA is to be amplified (by PCR), it must be first transcribed into cDNA (complementary DNA) using an enzyme called reverse transcriptase. Afterwards, the resulting cDNA is amplified by PCR.
• PCR is an exponential process that proceeds as long as the conditions for sustaining the reaction are acceptable. The components of the reaction are:
• 1. pair of primers—short single strands of DNA with around 10-30 nucleotides complementary to the regions flanking the target sequence
• 2. DNA polymerase—a thermostable enzyme that synthesizes DNA
• 3. deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotides that are incorporated into the newly synthesized DNA strand
• 4. buffer—to provide the optimal chemical environment for DNA synthesis
• PCR typically involves placing these reactants in a small tube (˜10-50 microlitres) containing the extracted nucleic acids. The tube is placed in a thermal cycler; an instrument that subjects the reaction to a series of different temperatures for varying amounts of time. The standard protocol for each thermal cycle involves a denaturation phase, an annealing phase, and an extension phase. The extension phase is sometimes referred to as the primer extension phase. In addition to such three-step protocols, two-step thermal protocols can be employed, in which the annealing and extension phases are combined. The denaturation phase typically involves raising the temperature of the reaction to 90-95° C. to denature the DNA strands; in the annealing phase, the temperature is lowered to ˜50-60° C. for the primers to anneal; and then in the extension phase the temperature is raised to the optimal DNA polymerase activity temperature of 60-72° C. for primer extension. This process is repeated cyclically around 20-40 times, the end result being the creation of millions of copies of the target sequence between the primers.
• There are a number of variants to the standard PCR protocol such as multiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCR and reverse-transcriptase PCR, amongst others, which have been developed for molecular diagnostics.
• Multiplex PCR uses multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several experiments. Optimization of multiplex PCR is more difficult though and requires selecting primers with similar annealing temperatures, and amplicons with similar lengths and base composition to ensure the amplification efficiency of each amplicon is equivalent.
• Linker-primed PCR, also known as ligation adaptor PCR, is a method used to enable nucleic acid amplification of essentially all DNA sequences in a complex DNA mixture without the need for target-specific primers. The method firstly involves digesting the target DNA population with a suitable restriction endonuclease (enzyme). Double-stranded oligonucleotide linkers (also called adaptors) with a suitable overhanging end are then ligated to the ends of target DNA fragments using a ligase enzyme. Nucleic acid amplification is subsequently performed using oligonucleotide primers which are specific for the linker sequences. In this way, all fragments of the DNA source which are flanked by linker oligonucleotides can be amplified.
• Direct PCR describes a system whereby PCR is performed directly on a sample without any, or with minimal, nucleic acid extraction. It has long been accepted that PCR reactions are inhibited by the presence of many components of unpurified biological samples, such as the haem component in blood. Traditionally, PCR has required extensive purification of the target nucleic acid prior to preparation of the reaction mixture. With appropriate changes to the chemistry and sample concentration, however, it is possible to perform PCR with minimal DNA purification, or direct PCR. Adjustments to the PCR chemistry for direct PCR include increased buffer strength, the use of polymerases which have high activity and processivity, and additives which chelate with potential polymerase inhibitors.
• Tandem PCR utilises two distinct rounds of nucleic acid amplification to increase the probability that the correct amplicon is amplified. One form of tandem PCR is nested PCR in which two pairs of PCR primers are used to amplify a single locus in separate rounds of nucleic acid amplification. The first pair of primers hybridize to the nucleic acid sequence at regions external to the target nucleic acid sequence. The second pair of primers (nested primers) used in the second round of amplification bind within the first PCR product and produce a second PCR product containing the target nucleic acid, that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake during the first round of nucleic acid amplification, the probability is very low that it would also be amplified a second time by a second pair of primers and thus ensures specificity.
• Real-time PCR, or quantitative PCR, is used to measure the quantity of a PCR product in real time. By using a fluorophore-containing probe or fluorescent dyes along with a set of standards in the reaction, it is possible to quantitate the starting amount of nucleic acid in the sample. This is particularly useful in molecular diagnostics where treatment options may differ depending on the pathogen load in the sample.
• Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA from RNA. Reverse transcriptase is an enzyme that reverse transcribes RNA into complementary DNA (cDNA), which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. It is also used to amplify RNA viruses such as human immunodeficiency virus or hepatitis C virus.
• Isothermal amplification is another form of nucleic acid amplification which does not rely on the thermal denaturation of the target DNA during the amplification reaction and hence does not require sophisticated machinery. Isothermal nucleic acid amplification methods can therefore be carried out in primitive sites or operated easily outside of a laboratory environment. A number of isothermal nucleic acid amplification methods have been described, including Strand Displacement Amplification, Transcription Mediated Amplification, Nucleic Acid Sequence Based Amplification, Recombinase Polymerase Amplification, Rolling Circle Amplification, Ramification Amplification, Helicase-Dependent Isothermal DNA Amplification and Loop-Mediated Isothermal Amplification.
• Isothermal nucleic acid amplification methods do not rely on the continuing heat denaturation of the template DNA to produce single stranded molecules to serve as templates for further amplification, but instead rely on alternative methods such as enzymatic nicking of DNA molecules by specific restriction endonucleases, or the use of an enzyme to separate the DNA strands, at a constant temperature.
• Strand Displacement Amplification (SDA) relies on the ability of certain restriction enzymes to nick the unmodified strand of hemi-modified DNA and the ability of a 5′-3′ exonuclease-deficient polymerase to extend and displace the downstream strand. Exponential nucleic acid amplification is then achieved by coupling sense and antisense reactions in which strand displacement from the sense reaction serves as a template for the antisense reaction. The use of nickase enzymes which do not cut DNA in the traditional manner but produce a nick on one of the DNA strands, such as N. Alw1, N. BstNB1 and Mly1, are useful in this reaction. SDA has been improved by the use of a combination of a heat-stable restriction enzyme (Aval) and heat-stable Exo-polymerase (Bst polymerase). This combination has been shown to increase amplification efficiency of the reaction from 10⁸ fold amplification to 10¹⁰ fold amplification so that it is possible using this technique to amplify unique single copy molecules.
• Transcription Mediated Amplification (TMA) and Nucleic Acid Sequence Based Amplification (NASBA) use an RNA polymerase to copy RNA sequences but not corresponding genomic DNA. The technology uses two primers and two or three enzymes, RNA polymerase, reverse transcriptase and optionally RNase H (if the reverse transcriptase does not have RNase activity). One primer contains a promoter sequence for RNA polymerase. In the first step of nucleic acid amplification, this primer hybridizes to the target ribosomal RNA (rRNA) at a defined site. Reverse transcriptase creates a DNA copy of the target rRNA by extension from the 3′ end of the promoter primer. The RNA in the resulting RNA:DNA duplex is degraded by the RNase activity of the reverse transcriptase if present or the additional RNase H. Next, a second primer binds to the DNA copy. A new strand of DNA is synthesized from the end of this primer by reverse transcriptase, creating a double-stranded DNA molecule. RNA polymerase recognizes the promoter sequence in the DNA template and initiates transcription. Each of the newly synthesized RNA amplicons re-enters the process and serves as a template for a new round of replication.
• In Recombinase Polymerase Amplification (RPA), the isothermal amplification of specific DNA fragments is achieved by the binding of opposing oligonucleotide primers to template DNA and their extension by a DNA polymerase. Heat is not required to denature the double-stranded DNA (dsDNA) template. Instead, RPA employs recombinase-primer complexes to scan dsDNA and facilitate strand exchange at cognate sites. The resulting structures are stabilised by single-stranded DNA binding proteins interacting with the displaced template strand, thus preventing the ejection of the primer by branch migration. Recombinase disassembly leaves the 3′ end of the oligonucleotide accessible to a strand displacing DNA polymerase, such as the large fragment of Bacillus subtilis Pol I (Bsu), and primer extension ensues. Exponential nucleic acid amplification is accomplished by the cyclic repetition of this process.
• Helicase-dependent amplification (HDA) mimics the in vivo system in that it uses a DNA helicase enzyme to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. In the first step of the HDA reaction, the helicase enzyme traverses along the target DNA, disrupting the hydrogen bonds linking the two strands which are then bound by single-stranded binding proteins. Exposure of the single-stranded target region by the helicase allows primers to anneal. The DNA polymerase then extends the 3′ ends of each primer using free deoxyribonucleoside triphosphates (dNTPs) to produce two DNA replicates. The two replicated dsDNA strands independently enter the next cycle of HDA, resulting in exponential nucleic acid amplification of the target sequence.
• Other DNA-based isothermal techniques include Rolling Circle Amplification (RCA) in which a DNA polymerase extends a primer continuously around a circular DNA template, generating a long DNA product that consists of many repeated copies of the circle. By the end of the reaction, the polymerase generates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA. This allows for spatial resolution of target and rapid nucleic acid amplification of the signal. Up to 10¹² copies of template can be generated in 1 hour. Ramification amplification is a variation of RCA and utilizes a closed circular probe (C-probe) or padlock probe and a DNA polymerase with a high processivity to exponentially amplify the C-probe under isothermal conditions.
• Loop-mediated isothermal amplification (LAMP), offers high selectivity and employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10⁹ copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand.
• After completion of the nucleic acid amplification, the amplified product must be analysed to determine whether the anticipated amplicon (the amplified quantity of target nucleic acids) was generated. The methods of analyzing the product range from simply determining the size of the amplicon through gel electrophoresis, to identifying the nucleotide composition of the amplicon using DNA hybridization.
• Gel electrophoresis is one of the simplest ways to check whether the nucleic acid amplification process generated the anticipated amplicon. Gel electrophoresis uses an electric field applied to a gel matrix to separate DNA fragments. The negatively charged DNA fragments will move through the matrix at different rates, determined largely by their size. After the electrophoresis is complete, the fragments in the gel can be stained to make them visible. Ethidium bromide is a commonly used stain which fluoresces under UV light.
• The size of the fragments is determined by comparison with a DNA size marker (a DNA ladder), which contains DNA fragments of known sizes, run on the gel alongside the amplicon. Because the oligonucleotide primers bind to specific sites flanking the target DNA, the size of the amplified product can be anticipated and detected as a band of known size on the gel. To be certain of the identity of the amplicon, or if several amplicons have been generated, DNA probe hybridization to the amplicon is commonly employed.
• DNA hybridization refers to the formation of double-stranded DNA by complementary base pairing. DNA hybridization for positive identification of a specific amplification product requires the use of a DNA probe around 20 nucleotides in length. If the probe has a sequence that is complementary to the amplicon (target) DNA sequence, hybridization will occur under favourable conditions of temperature, pH and ionic concentration. If hybridization occurs, then the gene or DNA sequence of interest was present in the original sample.
• Optical detection is the most common method to detect hybridization. Either the amplicons or the probes are labelled to emit light through fluorescence or electrochemiluminescence. These processes differ in the means of producing excited states of the light-producing moieties, but both enable covalent labelling of nucleotide strands. In electrochemiluminescence (ECL), light is produced by luminophore molecules or complexes upon stimulation with an electric current. In fluorescence, it is illumination with excitation light which leads to emission.
• Fluorescence is detected using an illumination source which provides excitation light at a wavelength absorbed by the fluorescent molecule, and a detection unit. The detection unit comprises a photosensor (such as a photomultiplier tube or charge-coupled device (CCD) array) to detect the emitted signal, and a mechanism (such as a wavelength-selective filter) to prevent the excitation light from being included in the photosensor output. The fluorescent molecules emit Stokes-shifted light in response to the excitation light, and this emitted light is collected by the detection unit. Stokes shift is the frequency difference or wavelength difference between emitted light and absorbed excitation light.
• ECL emission is detected using a photosensor which is sensitive to the emission wavelength of the ECL species being employed. For example, transition metal-ligand complexes emit light at visible wavelengths, so conventional photodiodes and CCDs are employed as photosensors. An advantage of ECL is that, if ambient light is excluded, the ECL emission can be the only light present in the detection system, which improves sensitivity.
• Microarrays allow for hundreds of thousands of DNA hybridization experiments to be performed simultaneously. Microarrays are powerful tools for molecular diagnostics with the potential to screen for thousands of genetic diseases or detect the presence of numerous infectious pathogens in a single test. A microarray consists of many different DNA probes immobilized as spots on a substrate. The target DNA (amplicon) is first labelled with a fluorescent or luminescent molecule (either during or after nucleic acid amplification) and then applied to the array of probes. The microarray is incubated in a temperature controlled, humid environment for a number of hours or days while hybridization between the probe and amplicon takes place. Following incubation, the microarray must be washed in a series of buffers to remove unbound strands. Once washed, the microarray surface is dried using a stream of air (often nitrogen). The stringency of the hybridization and washes is critical. Insufficient stringency can result in a high degree of nonspecific binding. Excessive stringency can lead to a failure of appropriate binding, which results in diminished sensitivity. Hybridization is recognized by detecting light emission from the labelled amplicons which have formed a hybrid with complementary probes.
• Fluorescence from microarrays is detected using a microarray scanner which is generally a computer controlled inverted scanning fluorescence confocal microscope which typically uses a laser for excitation of the fluorescent dye and a photosensor (such as a photomultiplier tube or CCD) to detect the emitted signal. The fluorescent molecules emit Stokes-shifted light (described above) which is collected by the detection unit.
• The emitted fluorescence must be collected, separated from the unabsorbed excitation wavelength, and transported to the detector. In microarray scanners, a confocal arrangement is commonly used to eliminate out-of-focus information by means of a confocal pinhole situated at an image plane. This allows only the in-focus portion of the light to be detected. Light from above and below the plane of focus of the object is prevented from entering the detector, thereby increasing the signal to noise ratio. The detected fluorescent photons are converted into electrical energy by the detector which is subsequently converted to a digital signal. This digital signal translates to a number representing the intensity of fluorescence from a given pixel. Each feature of the array is made up of one or more such pixels. The final result of a scan is an image of the array surface. The exact sequence and position of every probe on the microarray is known, and so the hybridized target sequences can be identified and analysed simultaneously.
• More information regarding fluorescent probes can be found at: http://www.premierbiosoft.com/tech_notes/FRET_probe.html and http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.html
Point-of-Care Molecular Diagnostics
• Despite the advantages that molecular diagnostic tests offer, the growth of this type of testing in the clinical laboratory has been slower than expected and remains a minor part of the practice of laboratory medicine. This is primarily due to the complexity and costs associated with nucleic acid testing compared with tests based on methods not involving nucleic acids. The widespread adaptation of molecular diagnostics testing to the clinical setting is intimately tied to the development of instrumentation that significantly reduces the cost, provides a rapid and automated assay from start (specimen processing) to finish (generating a result) and operates without major intervention by personnel.
• A point-of-care technology serving the physician's office, the hospital bedside or even consumer-based, at home, would offer many advantages including:
• • rapid availability of results enabling immediate facilitation of treatment and improved quality of care.
  • ability to obtain laboratory values from testing very small samples.
  • reduced clinical workload.
  • reduced laboratory workload and improved office efficiency by reducing administrative work.
  • improved cost per patient through reduced length of stay of hospitalization, conclusion of outpatient consultation at the first visit, and reduced handling, storing and shipping of specimens.
  • facilitation of clinical management decisions such as infection control and antibiotic use.
Lab-on-a-Chip (LOC) Based Molecular Diagnostics
• Molecular diagnostic systems based on microfluidic technologies provide the means to automate and speed up molecular diagnostic assays. The quicker detection times are primarily due to the extremely low volumes involved, automation, and the low-overhead inbuilt cascading of the diagnostic process steps within a microfluidic device. Volumes in the nanoliter and microliter scale also reduce reagent consumption and cost. Lab-on-a-chip (LOC) devices are a common form of microfluidic device. LOC devices have MST structures within a MST layer for fluid processing integrated onto a single supporting substrate (usually silicon). Fabrication using the VLSI (very large scale integrated) lithographic techniques of the semiconductor industry keeps the unit cost of each LOC device very low. However, controlling fluid flow through the LOC device, adding reagents, controlling reaction conditions and so on necessitate bulky external plumbing and electronics. Connecting a LOC device to these external devices effectively restricts the use of LOC devices for molecular diagnostics to the laboratory setting. The cost of the external equipment and complexity of its operation precludes LOC-based molecular diagnostics as a practical option for point-of-care settings.
• In view of the above, there is a need for a molecular diagnostic system based on a LOC device for use at point-of-care.
SUMMARY OF THE INVENTION
• Various aspects of the present invention are now described in the following numbered paragraphs.
• GBS001.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate;
• a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer.
• GBS001.2 Preferably, the cap has a channel connecting at least some of the fluidic connections, the channel being configured to draw fluid flow between the fluidic connections by capillary action.
• GBS001.3 Preferably, the cap has a reservoir in fluid communication with the channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GBS001.4 Preferably, the reservoir is in fluid communication with the channel via the MST layer and at least two of the fluidic connections.
• GBS001.5 Preferably, the channel and the reservoir are formed in a unitary layer of material.
• GBS001.6 Preferably, the channel is formed in one surface of the layer such that an outer surface of the MST layer encloses the channel.
• GBS001.7 Preferably, the cap has an exterior surface opposite said one surface, the exterior surface having an inlet for receiving fluid and feeding the fluid to the channel.
• GBS001.8 Preferably, the microfluidic device also has a dialysis section wherein the fluid contains constituents of different sizes and the dialysis section is configured to separate constituents smaller than a size threshold from constituents larger than the size threshold.
• GBS001.9 Preferably, the dialysis section includes at least one of the fluid connections which is configured as an array of holes to filter out cells larger than the threshold.
• GBS001.10 Preferably, the fluid is blood and the cap has a waste reservoir for collecting erythrocytes removed from the blood by the dialysis section.
• GBS001.11 Preferably, the reagent is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GBS001.12 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid.
• GBS001.13 Preferably, the cap has a lysis reagent reservoir for containing a lysis reagent to lyse cells in the blood and release nucleic acid sequences within.
• GBS001.14 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the blood prior to amplifying the nucleic acid sequences.
• GBS001.15 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GBS001.16 Preferably, the microfluidic device also has a hybridization section with hybridization chambers containing probe nucleic acid sequences for annealing to target nucleic acid sequences in the fluid.
• GBS001.17 Preferably, the probe nucleic acid sequences are contained in fluorescence resonance energy transfer (FRET) probes.
• GBS001.18 Preferably, each of the hybridization chambers has a photosensor for detecting fluorescence from the FRET probes in response to annealing with target nucleic acid sequences following fluorescence excitation.
• GBS001.19 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the fluid.
• GBS001.20 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the heaters.
• The surface-micromachined layers provide the smaller features at high density. The cap provides the larger features that are required. The surface-micromachined layers and the cap, together, provide the requisite plurality of structures.
• GBS002.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer overlying the supporting substrate, the MST layer defining MST channels for fluid flow within the MST layer;
• a cap overlying the MST layer, the cap defining cap channels for fluid flow within the cap and reservoirs for holding a quantity of fluid; wherein,
• the MST channels and the cap channels are in fluid communication.
• GBS002.2 Preferably, the reservoirs are in fluid communication with the MST channels.
• GBS002.3 Preferably, at least one of the reservoirs is configured to retain liquid within the reservoir by pinning a meniscus such that during use, the liquid is retained in the reservoir until fluid flow in the MST channels contacts and removes the meniscus.
• GBS002.4 Preferably, the cap has a lower seal for enclosing the cap channels, the lower seal having a plurality of openings providing fluid communication between the cap channels and the MST channels.
• GBS002.5 Preferably, at least some of the openings are configured to pin a meniscus until removed by contact with fluid flow.
• GBS002.6 Preferably, the microfluidic device also has CMOS circuitry between the supporting substrate and the MST layer, the CMOS circuitry having feedback sensors for sensing characteristics of the fluid flow through the MST channels.
• GBS002.7 Preferably, the MST layer has heater elements for heating the fluid flow.
• GBS002.8 Preferably, the cap has an upper seal which forms an outer layer, the upper seal having vent holes sized to allow air inflow as the reservoirs empty of liquid but retain liquid within the reservoir.
• GBS002.9 Preferably, the microfluidic device also has an actuator valve having an inlet and an outlet configured to draw liquid flow along a flow direction from the inlet to the outlet by capillary action; and,
• an actuator valve also having a movable member intermediate the inlet and the outlet, the movable member configured for movement between a quiescent position and an actuated position displaced from the quiescent position; and,
• an aperture configured to arrest the liquid flow by pinning a meniscus at the aperture; wherein during use,
• displacement of the movable member to the actuated position generates a pressure pulse to break the meniscus and force liquid through the aperture.
• GBS002.10 Preferably, the movable member at least partially defines the aperture.
• GBS002.11 Preferably, the actuator valve has an actuator for moving the movable member, the actuator having a resistive element for causing differential thermal expansion to move the movable member.
• GBS002.12 Preferably, the actuator valve reciprocates the movable member between the quiescent and displaced positions until the outlet immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GBS002.13 Preferably, the CMOS circuitry operatively controls the actuator valve.
• GBS002.14 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying target nucleic acid sequences in a biological sample.
• GBS002.15 Preferably, the microfluidic device also has a dialysis section for removing some cells from the biological sample on the basis of cell size.
• GBS002.16 Preferably, the microfluidic device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GBS002.17 Preferably, the CMOS circuitry has an array of photodiodes for detecting the probe-target hybrids.
• GBS002.18 Preferably, the reservoirs store an assay of reagents selected from:
• anticoagulant;
• lysis reagent;
• dNTPs, buffer and primers;
• polymerase; and,
• water.
• GBS002.19 Preferably, the actuator valve is positioned at a downstream end of the PCR section such that the CMOS circuitry opens the actuator valve to allow flow into the array of probes upon predetermined thermal cycling by the heater elements.
• GBS002.20 Preferably, the CMOS circuitry has memory for storing a list of the reagents loaded in the reservoirs and the probe types and location within the array of probes.
• The surface-micromachined layers provide the smaller features at high density. The cap provides the larger features that are required. The surface-micromachined layers and the cap, together, provide the requisite plurality of features.
• GBS003.1 This aspect of the invention provides a microfluidic device for molecular diagnostic analysis of a biological sample containing cells, the microfluidic device comprising:
• a supporting substrate;
• a plurality of microsystems technology (MST) channels each with a cross-sectional area between 1 square micron and 400 square microns for biochemical processing of constituents within the biological sample; and,
• a plurality of cell transport channels, each with a cross-sectional area greater than 400 square microns for receiving the biological sample and transporting cells within the biological sample to predetermined sites in the MST channels.
• GBS003.2 Preferably, the microfluidic device also has a MST layer on the supporting substrate and a cap overlying the MST layer wherein the MST channels are formed in the MST layer and the cell transport channels are formed in the cap, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer.
• GBS003.3 Preferably, the cell transport channel is a cap channel formed in a surface of the cap facing the MST layer, the cap channels being configured for fluidic propulsion of the sample by capillary action.
• GBS003.4 Preferably, the cap has a reservoir in fluid communication with the cap channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GBS003.5 Preferably, the cap channels and the reservoir are formed in a unitary layer of material.
• GBS003.6 Preferably, the cap has an interface layer between the MST layer and the cap channels, the interface layer being configured to provide fluid connections between the cap channels and the MST layer.
• GBS003.7 Preferably, the cap has an exterior surface opposite the MST layer, the exterior surface having an inlet for receiving the sample and drawing the sample to the cap channels by capillary action.
• GBS003.8 Preferably, the cap channels and the MST channels connect via fluid connections, at least one of the fluid connections having a valve for arresting flow of the sample until activation of the valve allows the sample flow to resume.
• GBS003.9 Preferably, the microfluidic device also has a dialysis section with apertures configured to separate cells larger than a threshold from constituents smaller than the threshold.
• GBS003.10 Preferably, the biological sample is blood and the apertures are configured to separate leukocytes from erythrocytes.
• GBS003.11 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GBS003.12 Preferably, the microfluidic device also has a lysis section in fluid communication with a lysis reagent reservoir containing a lysis reagent, the lysis section being configured to lyse cells and release genetic material within.
• GBS003.13 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid.
• GBS003.14 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplifying the nucleic acid sequences.
• GBS003.15 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GBS003.16 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GBS003.17 Preferably, the microfluidic device also has a hybridization section with hybridization chambers containing probe nucleic acid sequences for annealing to target nucleic acid sequences in the fluid.
• GBS003.18 Preferably, the probe nucleic acid sequences are contained in fluorescence resonance energy transfer (FRET) probes.
• GBS003.19 Preferably, each of the hybridization chambers has a photodiode for detecting fluorescence from the FRET probes in response to annealing with the target nucleic acid sequences.
• GBS003.20 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the sample.
• The large channels provide the capability for cell (larger specimens) transport. The small channels are more suitable for processes like mixing and heating where a need to accommodate large specimens doesn't exits, and the small channels would allow consumption of small reagent quantities. This LOC device incorporates both channel sizes to provide for both groups of advantages outlined above.
• GBS005.1 This aspect of the invention provides a microfluidic device for diagnostic analysis of a sample fluid, the microfluidic device comprising:
• a plurality of lab-on-a-chip (LOC) devices each having a supporting substrate and a microsystems technology (MST) layer for processing the sample fluid; and,
• a cap interconnecting the plurality of LOC devices, the cap having a cap channel for establishing fluid communication between the MST layers on at least two of the LOC devices.
• GBS005.2 Preferably, the MST layer in each of the LOC devices defines at least one microchannel for processing the sample fluid and the cap is configured to establish a fluid connection between one of the microchannels on one of the LOC devices, and one of the microchannels on at least one other of the LOC devices.
• GBS005.3 Preferably, the cap is configured to draw fluid flow between the LOC devices by capillary action.
• GBS005.4 Preferably, the cap has a reservoir in fluid communication with the cap channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GBS005.5 Preferably, the plurality of LOC devices is a first LOC device and a second LOC device, the first LOC device having a dialysis section for receiving the sample and separating constituents larger than a threshold size from constituents smaller than the threshold.
• GBS005.6 Preferably, the dialysis section has apertures corresponding to the threshold size and the constituents larger than the threshold include leukocytes and the constituents smaller than the threshold include pathogens.
• GBS005.7 Preferably, the second LOC device has a hybridization section with probe nucleic acid sequences for hybridization of target nucleic acid sequences from the sample to form probe-target hybrids, the hybridization section being configured for detecting the probe-target hybrids.
• GBS005.8 Preferably, the cap has a laminar structure with the cap channel formed in one layer and the reservoir formed in an adjacent layer.
• GBS005.9 Preferably, the laminar structure includes an interface layer for establishing fluid communication between the cap channel and the LOC devices.
• GBS005.10 Preferably, the MST layer on the second LOC has an active valve for arresting flow of the sample from the MST channel to the cap channel until activation of the active valve allows the sample flow to resume.
• GBS005.11 Preferably, the sample is blood and the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GBS005.12 Preferably, the microfluidic device also has a lysis section in fluid communication with a lysis reagent reservoir containing a lysis reagent, the lysis section being configured to lyse cells and release genetic material within.
• GBS005.13 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid.
• GBS005.14 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplifying the nucleic acid sequences.
• GBS005.15 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GBS005.16 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GBS005.17 Preferably, the microfluidic device also has a hybridization section with hybridization chambers containing probe nucleic acid sequences for annealing to target nucleic acid sequences in the fluid.
• GBS005.18 Preferably, the probe nucleic acid sequences are contained in electrochemiluminescence resonance energy transfer (ERET) probes.
• GBS005.19 Preferably, each of the hybridization chambers has a photodiode for detecting luminescence from the ERET probes in response to annealing with target nucleic acid sequences.
• GBS005.20 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the sample.
• Multiple surface-micromachined chips are integrated fluidically to provide more extensive and improved functionality. The microfluidic multichip assemblies provide for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, eg, a bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GBS006.1 This aspect of the invention provides a test module comprising:
• a casing for hand held portability;
• a receptacle for receiving a sample fluid; and,
• a microfluidic device in fluid communication with the inlet, the microfluidic device having a plurality of lab-on-a-chip (LOC) devices and a cap in engagement with each of the plurality of LOC devices; wherein,
• the cap is configured to establish fluid communication between at least two of the LOC devices.
• GBS006.2 Preferably, each of the LOC devices have a supporting substrate and a microsystems technologies (MST) layer on the supporting substrate and a cap overlying the MST layer, the MST layer incorporating MST channels and a plurality of fluidic connections for fluid communication with the cap, and the cap having cap channels for fluid communication with the fluidic connections of each LOC device.
• GBS006.3 Preferably, the sample fluid is a biological sample and the MST channels each have a cross-sectional area between 1 square micron and 400 square microns for biochemical processing of constituents within the biological sample and the cap channels each have a cross-sectional area greater than 400 square microns for receiving the biological sample and transporting cells within the biological sample to predetermined sites in the MST channels.
• GBS006.4 Preferably, the outer casing has a lancet for finger pricking a patient to obtain a blood sample for insertion in the sample inlet.
• GBS006.5 Preferably, the lancet is movable between a retracted and extended position, and the outer casing has a biasing mechanism to bias the lancet into the extended position and a user actuated catch for retaining the lancet in the retracted position until user actuation.
• GBS006.6 Preferably, the cap is configured to draw fluid flow through the cap channels by capillary action.
• GBS006.7 Preferably, the cap has a reservoir in fluid communication with the cap channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GBS006.8 Preferably, the plurality of LOC devices is a first LOC device and a second LOC device, the first LOC device having a dialysis section for receiving the sample and separating constituents larger than a threshold size from constituents smaller than the threshold.
• GBS006.9 Preferably, the dialysis section has apertures corresponding to the threshold size and the constituents larger than the threshold include leukocytes and the constituents smaller than the threshold include pathogens.
• GBS006.10 Preferably, the second LOC device has a hybridization section with probe nucleic acid sequences for hybridization of target nucleic acid sequences from the sample to form probe-target hybrids, the hybridization section being configured for detecting the probe-target hybrids.
• GBS006.11 Preferably, the cap has a laminar structure with the cap channel formed in one layer and the reservoir formed in an adjacent layer.
• GBS006.12 Preferably, the laminar structure includes an interface layer for establishing fluid communication between the cap channel and the LOC devices.
• GBS006.13 Preferably, the MST layer on the second LOC has an active valve for arresting flow of the sample from the MST channel to the cap channel until activation of the active valve allows the sample flow to resume.
• GBS006.14 Preferably, the sample is blood and the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GBS006.15 Preferably, the microfluidic device also has a lysis section in fluid communication with a lysis reagent reservoir containing a lysis reagent, the lysis section being configured to lyse cells and release genetic material within.
• GBS006.16 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid.
• GBS006.17 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplifying the nucleic acid sequences.
• GBS006.18 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GBS006.19 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GBS006.20 Preferably, the microfluidic device also has a hybridization section with hybridization chambers containing probe nucleic acid sequences for annealing to target nucleic acid sequences in the fluid.
• Multiple surface-micromachined chips are integrated fluidically to provide more extensive and improved functionality. The microfluidic multichip assemblies provide for higher modularity. Surface-micromachined chips in the assembly are each much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, eg, a bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass substrate.
• GSR001.1 This aspect of the invention provides a test module comprising:
• a receptacle for receiving an unprocessed biological sample through an opening;
• a cover movable between open and closed positions, the cover exposing the opening when in the open position and closing the opening when in the closed position; and,
• a microfluidic device for processing the biological sample, the microfluidic device having a sample inlet; wherein,
• the receptacle is configured for fluid communication with the sample inlet such that the biological sample flows to the sample inlet by capillary action.
• GSR001.2 Preferably, the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
• GSR001.3 Preferably, the test module also has an outer casing for hand held portability.
• GSR001.4 Preferably, the microfluidic device has a lab-on-a-chip (LOC) device with a cap, the LOC device having a supporting substrate and a microsystems technologies (MST) layer on the supporting substrate, the MST layer incorporating MST channels and a plurality of fluidic connections for fluid communication with the cap, and the cap having the sample inlet and cap channels for fluid communication with the fluidic connections.
• GSR001.5 Preferably, the MST channels each have a cross-sectional area between 1 square micron and 400 square microns for biochemical processing of constituents within the biological sample and the cap channels each have a cross-sectional area greater than 400 square microns for receiving the biological sample and transporting cells within the biological sample to predetermined sites in the MST channels.
• GSR001.6 Preferably, the outer casing has a lancet for pricking a patient to obtain a blood sample for insertion in the sample inlet.
• GSR001.7 Preferably, the lancet is movable between a retracted and extended position, and the outer casing has a biasing mechanism to bias the lancet into the extended position and a user actuated catch for retaining the lancet in the retracted position until user actuation.
• GSR001.8 Preferably, the cap is configured to draw fluid flow through the cap channels by capillary action.
• GSR001.9 Preferably, the cap has a reservoir in fluid communication with the cap channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GSR001.10 Preferably, the LOC device has a dialysis section for receiving a sample fluid with constituents of different sizes and separating the sample fluid into two fluid flows on the basis of constituent size.
• GSR001.11 Preferably, the constituents within the sample include cells and one of the two fluid flows has only cells smaller than a predetermined threshold size.
• GSR001.12 Preferably, the LOC device has a hybridization section with nucleic acid probe sequences, the hybridization section being configured for detecting hybridization of target nucleic acid sequences in the cells of the sample fluid and the nucleic acid probe sequences.
• GSR001.13 Preferably, the cap has a laminar structure with the cap channels formed in an outer layer and the reservoir formed in the opposing outer layer.
• GSR001.14 Preferably, the cap channels are formed in an outer surface of the cap, the outer surface being in contact with the MST layer of the LOC device to enclose the cap channel.
• GSR001.15 Preferably, the cap has a waste reservoir for collecting one of the two fluid flows.
• GSR001.16 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GSR001.17 Preferably, the LOC device has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample fluid.
• GSR001.18 Preferably, the reservoir is a lysis reagent reservoir for containing a lysis reagent to lyse cells in the fluid and release any nucleic acid sequences within.
• GSR001.19 Preferably, the cap has a PCR reagent reservoir containing dNTPs, buffer and primers for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GSR001.20 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• The sample receptacle permits the introduction of the sample into the microfluidic test module, funneling the sample into the microscopic sample inlet of the microfluidic device incorporated in the test module.
• GSR002.1 This aspect of the invention provides a test module comprising:
• an outer casing for hand held portability; and,
• a microfluidic device for processing a biological sample mounted in the outer casing, the microfluidic device having a lab-on-a-chip (LOC) device with a cap, the LOC device having a supporting substrate and a microsystems technologies (MST) layer on the supporting substrate, the MST layer incorporating MST channels and a plurality of fluidic connections for fluid communication with the cap, and the cap having a sample inlet and cap channels for fluid communication with the fluidic connections.
• GSR002.2 Preferably, the outer casing has a receptacle for receiving an unprocessed biological sample through an opening, and a cover movable between open and closed positions, the cover exposing the opening when in the open position and closing the opening when in the closed position, the receptacle being configured for fluid communication with the sample inlet such that the biological sample flows to the sample inlet by capillary action.
• GSR002.3 Preferably, the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
• GSR002.4 Preferably, the MST layer incorporates heaters for heating fluid in the MST channels.
• GSR002.5 Preferably, the MST channels each have a cross-sectional area between 1 square micron and 400 square microns for biochemical processing of constituents within the biological sample and the cap channels each have a cross-sectional area greater than 400 square microns for receiving the biological sample and transporting cells within the biological sample to predetermined sites in the MST channels.
• GSR002.6 Preferably, the outer casing has a lancet for pricking a patient to obtain a blood sample to insertion in the sample inlet.
• GSR002.7 Preferably, the lancet is movable between a retracted and extended position, and the outer casing has a biasing mechanism to bias the lancet into the extended position and a user actuated catch for retaining the lancet in the retracted position until user actuation.
• GSR002.8 Preferably, the cap is configured to draw fluid flow through the cap channels by capillary action.
• GSR002.9 Preferably, the cap has a reservoir in fluid communication with the cap channel, the reservoir being configured to retain a reagent by surface tension in a meniscus of the reagent.
• GSR002.10 Preferably, the LOC device has a dialysis section for receiving a sample fluid with constituents of different sizes and separating the sample fluid into two fluid flows on the basis of constituent size.
• GSR002.11 Preferably, the constituents within the sample include cells and one of the two fluid flows has only cells smaller than a predetermined threshold size.
• GSR002.12 Preferably, the LOC device has a hybridization section with probe nucleic acid sequences, the hybridization section being configured for detecting hybridization of target nucleic acid sequences in the cells of the sample fluid and the probe nucleic acid sequences.
• GSR002.13 Preferably, the cap has a laminar structure with the cap channels formed in an outer layer and the reservoir formed in the opposing outer layer.
• GSR002.14 Preferably, the cap channels are formed in an outer surface of the cap, the outer surface being in contact with the MST layer of the LOC device to enclose the cap channel.
• GSR002.15 Preferably, the cap has a waste reservoir for collecting one of the two fluid flows.
• GSR002.16 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GSR002.17 Preferably, the LOC device has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample fluid.
• GSR002.18 Preferably, the reservoir is a lysis reagent reservoir for containing a lysis reagent to lyse cells in the fluid and release any nucleic acid sequences within.
• GSR002.19 Preferably, the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GSR002.20 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• The surface-micromachined layers provide the smaller features at high density. The cap provides the larger features that are required. The surface-micromachined layers and the cap, together, provide the requisite plurality of features. The sample receptacle permits the introduction of the sample into the microfluidic test module, funneling the sample into the microscopic sample inlet of the microfluidic device incorporated in the test module.
• GAS001.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer for processing a fluid sample; and,
• a photosensor adjacent the MST layer for detecting photons emitted from the MST layer.
• GAS001.2 Preferably, the fluid sample has target molecules and the MST layer has an array of probes for reaction with the target molecules to form probe-target complexes that emit photons when excited.
• GAS001.3 Preferably, the target molecules are target nucleic acid sequences and the probes are configured to form probe-target hybrids such that the photosensor detects the probe-target hybrids within the array of probes.
• GAS001.4 Preferably, the microfluidic device also has CMOS circuitry between the MST layer and the supporting substrate and the probes are fluorescence resonance energy transfer (FRET) probes wherein the photosensor is an array of photodiodes incorporated in the CMOS circuitry.
• GAS001.5 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes, and an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS001.6 Preferably, the photodiodes in the array of photodiodes are positioned in registration with each of the hybridization chambers respectively.
• GAS001.7 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and memory for storing identity data identifying each FRET probe type, the CMOS circuitry being configured to convert output from the photodiodes into a signal indicative of the FRET probes that have formed probe-target hybrids, and provide the signal to the bond-pads for transmission to the external device.
• GAS001.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS001.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS001.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS001.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS001.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS001.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS001.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS001.15 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GAS001.16 Preferably, the hybridization section has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS001.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS001.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS001.19 Preferably, the photodiodes are less than 249 microns from the FRET probes.
• GAS001.20 Preferably, the microfluidic device also has a sample inlet for receiving the fluid sample, and a plurality of reagent reservoirs for different reagents required to process the fluid sample wherein the fluid sample is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the microfluidic device.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GAS002.1 This aspect of the invention provides a microfluidic device for detecting hybridization of probes with target nucleic acid sequences, the microfluidic device comprising:
• a sample inlet for receiving a fluid sample with target nucleic acid sequences;
• an array of probes for hybridization with the target nucleic acid sequences; and,
• a flow-path from the sample inlet to the array; wherein,
• the flow-path is configured to draw the fluid sample from the sample inlet to the probes by capillary action.
• GAS002.2 Preferably, the microfluidic device also has:
• a supporting substrate;
• a microsystems technologies (MST) layer for processing a fluid sample, the MST layer incorporating the flow-path; and,
• a photosensor adjacent the MST layer for detecting photons emitted from the MST layer; wherein,
• the probes are configured to hybridize the target nucleic acid sequences to form probe-target hybrids that emit photons.
• GAS002.3 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS002.4 Preferably, the microfluidic device also has CMOS circuitry between the MST layer and the supporting substrate and the probes are fluorescence resonance energy transfer (FRET) probes wherein the photosensor is an array of photodiodes incorporated in the CMOS circuitry.
• GAS 002.5 Preferably, the microfluidic device also has an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS002.6 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization section by capillary action.
• GAS002.7 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and memory for storing identity data identifying each FRET probe type, the CMOS circuitry being configured to convert output from the photodiodes into a signal indicative of the FRET probes that have formed probe-target hybrids, and provide the signal to the bond-pads for transmission to the external device.
• GAS002.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS002.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS002.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS002.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS002.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS002.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS002.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS002.15 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GAS002.16 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS002.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS002.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS002.19 Preferably, the photodiodes are less than 249 microns from the FRET probes.
• GAS002.20 Preferably, the microfluidic device also has a sample inlet for receiving the fluid sample, and a plurality of reagent reservoirs for different reagents required to process the fluid sample wherein the fluid sample is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the microfluidic device.
• The sample inlet permits the introduction of the sample into the microfluidic test module, delivering small sample quantities with high volumetric efficiency to the required sections of the microfluidic device. The probe hybridization section provides for analysis of the targets via hybridization.
• GAS003.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer with a hybridization section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids; and,
• a photosensor for detecting the probe-target hybrids.
• GAS003.2 Preferably, the microfluidic device also has CMOS circuitry between the MST layer and the supporting substrate wherein the photosensor is an array of photodiodes incorporated in the CMOS circuitry.
• GAS003.3 Preferably, the microfluidic device also has an array of hybridization chambers containing the probes, wherein the probes are fluorescence resonance energy transfer (FRET) probes and the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
• GAS003.4 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the FRET probes.
• GAS003.5 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GAS003.6 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS003.7 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS003.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS003.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS003.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS003.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS003.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS003.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS003.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS003.15 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GAS003.16 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS003.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS003.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS003.19 Preferably, the photodiodes are less than 249 microns from the FRET probes.
• GAS003.20 Preferably, the microfluidic device also has a plurality of reagent reservoirs for different reagents required to process the fluid wherein the fluid is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the microfluidic device.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GAS004.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer overlying the supporting substrate for processing a fluid containing target nucleic acid sequences, the MST layer having an array of hybridization chambers, each containing probes for hybridization with the target nucleic acid sequences;
• a temperature sensor for sensing the temperature at the array of hybridization chambers; and,
• a hybridization heater for heating the array of hybridization chambers; wherein during use,
• output from the temperature sensor is used for feedback control of the hybridization heater.
• GAS004.2 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating a photosensor.
• GAS004.3 Preferably, the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
• GAS004.4 Preferably, the CMOS circuitry has a digital memory for storing data relating to processing of the fluid, the data including the probe details and location of each of the probes in the array.
• GAS004.5 Preferably, the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
• GAS004.6 Preferably, each of the hybridization chambers has a heater controlled by the CMOS circuitry for maintaining the probes and target nucleic acid sequences at a hybridization temperature.
• GAS004.7 Preferably, the photodiodes are less than 249 microns from the hybridization chambers.
• GAS004.8 Preferably, the hybridization chamber has a volume less than 900,000 cubic microns.
• GAS004.9 Preferably, the hybridization chamber has a volume less than 200,000 cubic microns.
• GAS004.10 Preferably, the hybridization chamber has a volume less than 40,000 cubic microns.
• GAS004.11 Preferably, the hybridization chamber has a volume less than 9000 cubic microns.
• GAS004.12 Preferably, the CMOS derives a single result from the photodiodes corresponding to the hybridization chambers that contain identical probes.
• GAS004.13 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS004.14 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
• GAS004.15 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photosensor in response to an excitation light when the FRET probe has hybridized to one of the target nucleic acid probes, the CMOS circuitry being configured to enable the photosensor after a predetermined delay following the excitation light being extinguished.
• GAS004.16 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
• GAS004.17 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS004.18 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the array of hybridization chambers by capillary action.
• GAS004.19 Preferably, the LOC device has a cap in which the reagent reservoirs are defined.
• GAS004.20 Preferably, each of the reagent reservoirs have a surface tension valve, each of the surface tension valve having a meniscus anchor for pinning a meniscus to retain reagents therein.
• The probe hybridization section provides for analysis of the targets via hybridization. The temperature feedback control assures control of the temperature in the hybridization chambers for optimal hybridization temperature and the subsequent optimal detection temperature.
• GAS006.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer overlying the supporting substrate for processing a fluid containing target nucleic acid sequences, the MST layer having an array of hybridization chambers, each containing probes for hybridization with the target nucleic acid sequences, the probes being configured to emit a fluorescence signal in response to an excitation light upon hybridization; wherein,
• the hybridization chamber has a wall section that is optically transparent to the fluorescence signal.
• GAS006.2 Preferably, the microfluidic device also has a photosensor wherein the wall section is between the probes and the photosensor.
• GAS006.3 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating the photosensor.
• GAS006.4 Preferably, the microfluidic device also has an array of the hybridization chambers wherein the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
• GAS006.5 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array.
• GAS006.6 Preferably, the CMOS circuitry has a temperature sensor for sensing the temperature at the array of hybridization chambers wherein output from the temperature sensor is used for feedback control of the hybridization heater.
• GAS006.7 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry for maintaining the probes and target nucleic acid sequences at a hybridization temperature.
• GAS006.8 Preferably, the photosensor positioned less than 249 microns from the hybridization chambers.
• GAS006.9 Preferably, the wall section is opaque to the excitation light.
• GAS006.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS006.11 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
• GAS006.12 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal in response to an excitation light when the FRET probe has hybridized to one of the target nucleic acid sequences, and the CMOS circuitry is configured to enable the photosensor after a predetermined delay following the excitation light being extinguished.
• GAS006.13 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
• GAS006.14 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS006.15 Preferably, the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
• GAS006.16 Preferably, the reagent reservoirs each have a volume less than 20,000,000 cubic microns.
• GAS006.17 Preferably, the microfluidic device also has a cap in which the reagent reservoirs are defined.
• GAS006.18 Preferably, each of the reagent reservoirs have a surface tension valve, each of the surface tension valve having a meniscus anchor for pinning a meniscus to retaining reagents therein.
• GAS006.19 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the array of hybridization chambers by capillary action.
• The probe hybridization section provides for analysis of the targets via hybridization. The optically transparent hybridization chambers provide for the transmission of the fluorescence signals used for the detection of hybridization of the targets to the probes.
• GAS007.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer overlying the supporting substrate for processing a fluid containing target nucleic acid sequences, the MST layer having a plurality of probe spots, each of the probe spots containing probes for hybridization with the target nucleic acid sequences; wherein,
• the mass of the probes in each of the probe spots is less than 270 picograms.
• GAS007.2 Preferably, the mass of the probes in each of the probe spots is less than 60 picograms.
• GAS007.3 Preferably, the mass of the probes in each of the probe spots is less than 12 picograms.
• GAS007.4 Preferably, the mass of the probes in each of the probe spots is less than 2.7 picograms.
• GAS007.5 Preferably, the microfluidic device also has an array of photodiodes wherein the MST layer has an array of hybridization chambers, each of the hybridization chambers containing one of the probe spots respectively and, the array of photodiodes is positioned such that at least one of the photodiodes corresponds to each of the hybridization chambers respectively.
• GAS007.6 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating the array of photodiodes.
• GAS007.7 Preferably, each of the hybridization chambers has a volume less than 200,000 cubic microns.
• GAS007.8 Preferably, each of the hybridization chambers has a volume less than 40,000 cubic microns.
• GAS007.9 Preferably, each of the hybridization chambers has a volume less than 9000 cubic microns.
• GAS007.10 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array.
• GAS007.11 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry for maintaining the probes and target nucleic acid sequences at a hybridization temperature.
• GAS007.12 Preferably, the CMOS circuitry has a temperature sensor for sensing the temperature at the array of hybridization chambers wherein output from the temperature sensor is used for feedback control of the hybridization heater.
• GAS007.13 Preferably, the photosensor is positioned less than 249 microns from the hybridization chambers.
• GAS007.14 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS007.15 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
• GAS007.16 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal in response to an excitation light when the FRET probe has hybridized to one of the target nucleic acid sequences, the CMOS circuitry being configured to enable the photosensor after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GAS007.17 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
• GAS007.18 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS007.19 Preferably, the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
• GAS007.20 Preferably, the reagent reservoirs each have a volume less than 20,000,000 cubic microns.
• The low probe volume provides for low probe cost, in turn, permitting the inexpensive assay system.
• GAS008.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer with a hybridization section that has an array of hybridization chambers, each containing a different probe type for hybridization with target nucleic acid sequences; wherein,
• each of the hybridization chambers has a volume less than 900,000 cubic microns.
• GAS008.2 Preferably, each of the hybridization chambers has a volume less than 200,000 cubic microns.
• GAS008.3 Preferably, each of the hybridization chambers has a volume less than 40,000 cubic microns.
• GAS008.4 Preferably, each of the hybridization chambers has a volume less than 9000 cubic microns.
• GAS008.5 Preferably, the microfluidic device also has CMOS circuitry between the MST layer and the supporting substrate; wherein,
• • the CMOS circuitry has an array of photodiodes for detecting hybridization of probes within the array of hybridization chambers.
• GAS008.6 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS008.7 Preferably, the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
• GAS008.8 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS008.9 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization section by capillary action.
• GAS008.10 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS008.11 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS008.12 Preferably, the CMOS circuitry is configured to enable the sensors after a predetermined delay following the excitation light being extinguished.
• GAS008.13 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS008.14 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS008.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS008.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS008.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS008.18 Preferably, the hybridization chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
• GAS008.19 Preferably, the hybridization section has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS008.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• The low-volume hybridization chambers, in part, provide for the low probe volumes, which in turn provide for low probe cost and the inexpensive assay system.
• GAS009.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material containing target nucleic acid sequences; and,
• a microsystems technologies (MST) layer with a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences, and an array of probes for hybridization with the target nucleic acid sequences.
• GAS009.2 Preferably, the microfluidic device also has a supporting substrate and CMOS circuitry; wherein,
• the probes are configured to form probe-target hybrids when hybridized with the target nucleic acid sequences, the probe-target hybrids being configured to emit photons in response to an excitation source, and the CMOS circuitry is between the MST layer and the supporting substrate and has an array of photodiodes for detecting the probe-target hybrids.
• GAS009.3 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS009.4 Preferably, the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
• GAS009.5 Preferably, the MST layer has a plurality of MST channels configured to draw a fluid containing the sample through the PCR section and into the hybridization chambers by capillary action.
• GAS009.6 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS009.7 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS009.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS009.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS009.10 Preferably, the PCR section has at least one elongate PCR chamber; and,
• at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR chamber; wherein,
• the elongate heater element extends parallel with the longitudinal extent of the PCR chamber.
• GAS009.11 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chamber is a section of the microchannel.
• GAS009.12 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GAS009.13 Preferably, the PCR section has a plurality of the elongate PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GAS009.14 Preferably, each of the channel section along each of the wide meanders has a plurality of the elongate heaters.
• GAS009.15 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GAS009.16 Preferably, each of the plurality of elongate heaters is independently operable.
• GAS009.17 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heaters.
• GAS009.18 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, polymerase, dNTPs and buffer to amplify the nucleic acid sequences.
• GAS009.19 Preferably, the active valve has a meniscus anchor for anchoring a meniscus such that the liquid is retained in the PCR section, and a heater for boiling the liquid at the meniscus anchor to unpin the meniscus such that capillary driven flow out of the PCR section resumes.
• GAS009.20 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• The PCR section, via the amplification of the target, provides the requisite sensitivity for target detection. The probe hybridization section provides for analysis of the targets via hybridization. The integrated PCR and probe hybridization sections substantially reduce the possibility of the introduction of contaminants into the assay, simplify the analysis stages, and provide for a small, light, and inexpensive single-device analytical solution.
• GAS010.1 This aspect of the invention provides a microfluidic device comprising:
• a sample flow-path for a biological sample containing target nucleic acid sequences;
• an array of hybridization chambers each containing probes for hybridization with the target nucleic acid sequences, each of the hybridization chambers having a chamber inlet for fluid communication between the sample flow-path and the probes; wherein,
• the chamber inlet is configured as a diffusion barrier to prevent diffusion of hybridized probes between the hybridization chambers to an extent that causes erroneous hybridization detection results.
• GAS010.2 Preferably, the chamber inlet defines a tortuous flow-path.
• GAS010.3 Preferably, the tortuous flow-path has a serpentine configuration.
• GAS010.4 Preferably, the microfluidic device also has a supporting substrate, a microsystems technologies (MST) layer incorporating the sample flow-path and the array of hybridization chambers, and CMOS circuitry between the MST layer and the supporting substrate; wherein,
• the CMOS circuitry has an array of photodiodes for detecting hybridization of the probes.
• GAS010.5 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS010.6 Preferably, the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
• GAS010.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS010.8 Preferably, the sample flow-path is configured to draw the sample fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS010.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS010.10 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS010.11 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS010.12 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS010.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS010.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS010.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS010.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS010.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS010.18 Preferably, the hybridization chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
• GAS010.19 Preferably, the sample fluid flow-path has an end-point liquid sensor, such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the end-point liquid sensor indicating that the sample fluid has reached the liquid end point sensor.
• GAS010.20 Preferably, the photodiodes and the probes are spaced apart by less than 249 microns.
• The probe hybridization section provides for analysis of the targets via hybridization. The diffusion barrier virtually eliminates a backflow of the probes, both before and after hybridization, preventing the loss of signal and providing high assay sensitivity.
• GAS012.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• CMOS circuitry on the substrate, the CMOS circuitry having a photosensor for generating an output signal in response to the fluorescence signal; wherein,
• the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photosensor.
• GAS012.2 Preferably, the photosensor is a photodiode and the probe is spaced less than 249 microns from the photodiode.
• GAS012.3 Preferably, the microfluidic device also has an array of the probes and a corresponding array of the photodiodes.
• GAS012.4 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS012.5 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS012.6 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS012.7 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS012.8 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS012.9 Preferably, the quencher has no native emission in response to the excitation light.
• GAS012.10 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
• GAS012.11 Preferably, the microfluidic device also has an array of hybridization chambers for containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to the excitation light.
• GAS012.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS012.13 Preferably, the probe array has more than 1000 probes.
• GAS012.14 Preferably, the time delay is between 100 picoseconds and 10 milliseconds.
• GAS012.15 Preferably, the microfluidic device also has a trigger photodiode that is responsive to the excitation light and configured to provide an indication when the excitation light has deactivated.
• GAS012.16 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS012.17 Preferably, the hybridization chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
• GAS012.18 Preferably, the sample fluid flow-path has an end-point liquid sensor, such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the end-point liquid sensor indicating that the sample fluid has reached the end-point liquid sensor.
• GAS012.19 Preferably, the PCR section has an active valve for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GAS012.20 Preferably, the active valve has a meniscus anchor for anchoring a meniscus such that the liquid is retained in the PCR section, and a heater for boiling the liquid at the meniscus anchor to unpin the meniscus such that capillary driven flow out of the PCR section resumes.
• The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS013.1 This aspect of the invention provides a microfluidic device comprising:
• a probe for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid having a reporting fluorophore for emitting a fluorescence signal in response to an excitation light;
• a detection photodiode for exposure to the excitation light and the fluorescence signal; and,
• a trigger photodiode for exposure to the excitation light, the trigger photodiode being configured to activate the detection photodiode after the excitation light has been extinguished.
• GAS013.2 Preferably, the microfluidic device also has a hybridization chamber containing the probe for hybridization with the target nucleic acid sequence, the probe being anchored to an internal surface of the hybridization chamber and the detection photodiode and the trigger photodiode are adjacent the internal surface.
• GAS013.3 Preferably, the hybridization chamber has an optical window opposite the internal surface on which the probe is anchored such that the probe is exposed to the excitation light.
• GAS013.4 Preferably, the microfluidic device also has an array of the hybridization chambers containing different probes for hybridization with respective target nucleic acid sequences and a corresponding array of the detection photodiodes and the trigger photodiodes.
• GAS013.5 Preferably, the detection photodiodes are larger than the trigger photodiodes.
• GAS013.6 Preferably, the microfluidic device also has:
• a supporting substrate;
• a microsystems technologies (MST) layer incorporating the array of hybridization chambers; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry incorporating the array of detection photodiodes and the trigger photodiodes; wherein,
• the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS013.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences.
• GAS013.8 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS013.9 Preferably, the microfluidic device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to generate a signal indicative of the FRET probes that hybridized with the target sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS013.10 Preferably, the CMOS circuitry has memory storing identity data for the different FRET probe types.
• GAS013.11 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS013.12 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS013.13 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS013.14 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS013.15 Preferably, the quencher has no native emission in response to the excitation light.
• GAS013.16 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS013.17 Preferably, the hybridization chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
• GAS013.18 Preferably, the microfluidic device also has a fluid flow-path from the PCR section, through the hybridization chambers, to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS013.19 Preferably, the fluid flow-path is configured to draw fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS013.20 Preferably, the PCR section has an active valve for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• Sensor-triggering of the photosensor provides accurate sensor timing, increasing the signal-to-noise ratio and sensitivity. The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS014.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a hybridization chamber containing a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• CMOS circuitry between the supporting substrate and the hybridization chamber, the CMOS circuitry having a photodiode for generating an output signal in response to the fluorescence signal, and a trigger photodiode for generating an output in response to the excitation light; wherein,
• the CMOS circuitry is configured to activate the photodiode when the trigger photodiode indicates the excitation light has deactivated.
• GAS014.2 Preferably, the CMOS circuitry provides a time delay between deactivation of the excitation light and activation of the photodiode.
• GAS014.3 Preferably, the microfluidic device also has an array of the hybridization chambers, each containing probes and a corresponding array of the photodiodes.
• GAS014.4 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS014.5 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS014.6 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS014.7 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS014.8 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS014.9 Preferably, the quencher has no native emission in response to the excitation light.
• GAS014.10 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
• GAS014.11 Preferably, the hybridization chambers each have an optical window to expose the FRET probes to the excitation light.
• GAS014.12 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS014.13 Preferably, the probe array has more than 1000 probes.
• GAS014.14 Preferably, the time delay is between 100 picoseconds and 10 milliseconds.
• GAS014.15 Preferably, the microfluidic device also has:
• at least one calibration source configured to generate a calibration emission;
• a calibration photodiode for sensing the calibration emission; wherein,
• the CMOS circuitry has a differential circuit for subtracting the calibration photodiode output from the detection photodiode output.
• GAS014.16 Preferably, the microfluidic device also has:
• a plurality of the calibration sources distributed throughout the array of hybridization chambers; wherein,
• the calibration sources are calibration probes without a fluorophore.
• GAS014.17 Preferably, the microfluidic device also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS014.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS014.19 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS014.20 Preferably, the quencher has no native emission in response to the excitation light.
• Sensor-triggering of each photosensor with its dedicated high-proximity trigger photosensor provides optimal sensor timing, improving the signal-to-noise ratio and sensitivity. The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS015.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an array of probes, each of the probes having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• CMOS circuitry on the substrate, the CMOS circuitry having an array of photodiodes for sensing deactivation of the excitation light and generating an output signal in response to the fluorescence signals from the probe-target hybrids within the array of probes.
• GAS015.2 Preferably, the microfluidic device also has an array of hybridization chambers, each of the photodiodes corresponding to one of the hybridization chambers respectively.
• GAS015.3 Preferably, the hybridization chambers each have a volume less than 900,000 cubic microns.
• GAS015.4 Preferably, the CMOS circuitry provides a time delay between sensing deactivation of the excitation light and sensing the fluorescence signal.
• GAS015.5 Preferably, the photodiodes are spaced less than 249 microns from the probes in the corresponding hybridization chamber.
• GAS015.6 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS015.7 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS015.8 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS015.9 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS015.10 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS015.11 Preferably, the quencher has no native emission in response to the excitation light.
• GAS015.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
• GAS015.13 Preferably, the hybridization chambers each have an optical window to expose the FRET probes to the excitation light.
• GAS015.14 Preferably, the CMOS circuitry has memory for identity data for the FRET probe.
• GAS015.15 Preferably, the probe array has more than 1000 probes.
• GAS015.16 Preferably, the time delay is between 100 picoseconds and 10 milliseconds.
• GAS015.17 Preferably, the microfluidic device also has:
• a plurality of calibration sources distributed throughout the array of hybridization chambers wherein the CMOS is configured to use the calibration sources to calibrate the output from the photodiodes.
• GAS015.18 Preferably, the calibration sources are calibration probes without a fluorophore.
• GAS015.19 Preferably, the microfluidic device also has a plurality of calibration chambers containing the calibration sources, the calibration chambers being distributed throughout the array of hybridization chambers, wherein during use, output from any one of the photodiodes is compared to output from the calibration photodiode most proximate to that photodiode.
• GAS015.20 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• Sensor-triggering of each photosensor with its dedicated high-proximity trigger photosensor provides optimal sensor timing, improving the signal-to-noise ratio and sensitivity. The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light. Using the hybridization detection photosensor for triggering the photodetection phase provides for a large portion of the advantages of sensor-triggered photodetection while permitting the use of a larger hybridization detection photosensor, improving the sensitivity.
• GAS016.1 THIS ASPECT OF THE INVENTION PROVIDES a microfluidic device comprising:
• a supporting substrate;
• a probe having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• CMOS circuitry on the substrate, the CMOS circuitry having a photosensor for generating an output signal in response to the fluorescence signal; wherein,
• the CMOS circuitry is configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
• GAS016.2 Preferably, the CMOS circuitry is configured for controlling the excitation light such that deactivation of the excitation light triggers the CMOS circuitry to initiate the time delay preceding activation of the photosensor.
• GAS016.3 Preferably, the CMOS circuitry has a trigger photodiode for generating an output in response to the excitation light such that the CMOS circuitry initiates the time delay when the trigger photodiode indicates the excitation light has deactivated.
• GAS016.4 Preferably, the photosensor is a photodiode and the probe is spaced less than 249 microns from the photodiode.
• GAS016.5 Preferably, the microfluidic device also has an array of the probes and a corresponding array of the photodiodes.
• GAS016.6 Preferably, the microfluidic device also has an excitation sensor photodiode configured to indicate when the excitation light deactivates.
• GAS016.7 Preferably, the microfluidic device also has an array of hybridization chambers, each of the photodiodes corresponding to one of the hybridization chambers respectively and the excitation light photodiode also corresponding to one of the hybridization chambers, wherein the hybridization chamber corresponding to the excitation light photodiode does not contain a fluorescent reporter molecule.
• GAS016.8 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS016.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS016.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS016.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS016.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS016.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS016.14 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
• GAS016.15 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS016.16 Preferably, the probe array has more than 1000 probes.
• GAS016.17 Preferably, the time delay is between 100 picoseconds and 10 milliseconds.
• GAS016.18 Preferably, the microfluidic device also has a cap overlaying the PCR section, the cap defining a plurality of reagent reservoirs containing reagents for addition to the target nucleic acid sequences.
• GAS016.19 Preferably, the reagent in the reagent reservoirs are selected from:
• anticoagulant;
• lysis reagent;
• restriction enzymes;
• buffer;
• dNTPs and primers; and,
• polymerase.
• The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light. Using a delay for triggering the photodetection phase reduces the area of trigger circuitry, permitting the use of a larger hybridization detection photosensor, improving the sensitivity.
• GAS017.1 This aspect of the invention provides a microfluidic device for detecting a target nucleic acid sequence, the microfluidic device comprising:
• a probe for hybridization with the target nucleic acid sequence to form a probe-target hybrid, the probe having a fluorophore for generating fluorescence emissions in response to an excitation light;
• a photodiode for detecting the fluorescence emissions;
• a shunt transistor between the photodiode and a voltage source; and,
• CMOS circuitry for controlling the shunt transistor to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS017.2 Preferably, the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS017.3 Preferably, the microfluidic device also has a transmission transistor between the photodiode anode and an output node, wherein the transmission transistor is configured to deactivate when the excitation light is activated and to activate after the excitation light deactivates.
• GAS017.4 Preferably, the microfluidic device also has a reset transistor for removing carriers leaking through the transmission transistor during excitation by the excitation light, the reset transistor configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS017.5 Preferably, the microfluidic device also has a supporting substrate, the CMOS circuitry being positioned between the supporting substrate and the probe for controlling the shunt transistor, the transmission transistor and the reset transistor wherein the photodiode is incorporated in the CMOS circuitry and the probe is a fluorescence resonance energy transfer (FRET) probe.
• GAS017.6 Preferably, the microfluidic device also has an array of the FRET probes, and an array of the photodiodes corresponding to each of the FRET probes respectively, each of the FRET probes being positioned in a respective hybridization chamber and configured for hybridization with different target nucleic acid sequences, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS017.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the FRET probes.
• GAS017.8 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of microchannels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS017.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS017.10 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS017.11 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS017.12 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS017.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS017.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS017.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS017.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS017.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS017.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS017.19 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS017.20 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• Photosensors with controllable shunts obviate the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS018.1 This aspect of the invention provides a microfluidic device for detecting a target nucleic acid sequence, the microfluidic device comprising:
• a probe for hybridization with the target nucleic acid sequence to form a probe-target hybrid, the probe having a fluorophore for generating fluorescence emissions in response to an excitation light;
• a photodiode for detecting the fluorescence emissions; and,
• CMOS circuitry activating the photodiode; wherein,
• the fluorophore has a fluorescence lifetime longer than 100 nanoseconds.
• GAS018.2 Preferably, the microfluidic device of claim 1 further comprising a shunt transistor between the photodiode and a voltage source to remove carriers generated by absorption of photons of the excitation light in the photodiode wherein the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS018.3 Preferably, the microfluidic device also has a transmission transistor between the photodiode anode and an output node, wherein the transmission transistor is configured to deactivate when the excitation light is activated and to activate after the excitation light deactivates.
• GAS018.4 Preferably, the microfluidic device also has a reset transistor for removing carriers leaking through the transmission transistor during excitation by the excitation light, the reset transistor configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS018.5 Preferably, the microfluidic device also has a supporting substrate, the CMOS circuitry being positioned between the supporting substrate and the probe for controlling the shunt transistor, the transmission transistor and the reset transistor wherein the photodiode is incorporated in the CMOS circuitry and the probe is a fluorescence resonance energy transfer (FRET) probe.
• GAS018.6 Preferably, the microfluidic device also has an array of the FRET probes, and a corresponding array of the photodiodes, each of the FRET probes being positioned in a respective hybridization chamber and configured for hybridization with different target nucleic acid sequences, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS018.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the FRET probes.
• GAS018.8 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS018.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photosensors into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS018.10 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS018.11 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS018.12 Preferably, the FRET probes each have a quencher for quenching fluorescence emissions from the fluorophore.
• GAS018.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS018.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS018.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS018.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS018.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS018.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS018.19 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS018.20 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• Photosensors with controllable shunts obviate the need for any wavelength dependent filter components, making the design inexpensive, small, and light. Low shunt resistance increases the signal-to-noise ratio and sensitivity.
• GAS019.1 This aspect of the invention provides a microfluidic device comprising:
• a probe for hybridization with a target nucleic acid sequence;
• a photodiode for detecting fluorescence emissions generated by hybridization of the probe in response to an excitation light; and,
• a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode; wherein,
• a peripheral edge of the photodiode and a peripheral edge of the shunt transistor contiguously abut each other.
• GAS019.2 Preferably, the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS019.3 Preferably, the microfluidic device also has a transmission transistor between the photodiode anode and an output node, wherein the transmission transistor is configured to deactivate when the excitation light is activated and to activate after the excitation light deactivates.
• GAS019.4 Preferably, the microfluidic device also has a reset transistor for removing carriers leaking through the transmission transistor during excitation by the excitation light, the reset transistor configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS019.5 Preferably, the microfluidic device also has CMOS circuitry between the probe and a supporting substrate, the CMOS circuitry for controlling the shunt transistor, the transmission transistor and the reset transistor wherein the photosensors are incorporated in the CMOS circuitry and the probe is a fluorescence resonance energy transfer (FRET) probe.
• GAS019.6 Preferably, the microfluidic device also has an array of the FRET probes, each of the FRET probes being positioned in a respective hybridization chamber and configured for hybridization with different target nucleic acid sequences, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS019.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS019.8 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS019.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photosensors into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS019.10 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS019.11 Preferably, the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
• GAS019.12 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS019.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS019.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS019.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS019.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS019.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS019.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS019.19 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS019.20 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• Photosensors with controllable shunts obviate the need for any wavelength dependent filter components, making the design inexpensive, small, and light. Shunts peripheral to the photodetectors have low resistance, increasing the signal-to-noise ratio and sensitivity.
• GAS020.1 This aspect of the invention provides a microfluidic device comprising:
• a probe for hybridization with a target nucleic acid sequence to form a probe-target hybrid;
• a photodiode having an active area for detecting fluorescence emissions generated by the probe-target hybrid in response to an excitation light; and,
• a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode; wherein,
• the shunt transistor is encompassed by the active area.
• GAS020.2 Preferably, the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS020.3 Preferably, the microfluidic device also has a transmission transistor between the photodiode anode and an output node, wherein the transmission transistor is configured to turn on when the excitation light is activated and to turn off after the excitation light deactivates.
• GAS020.4 Preferably, the microfluidic device also has a reset transistor for removing carriers leaking through the transmission transistor during excitation by the excitation light, the reset transistor configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
• GAS020.5 Preferably, the microfluidic device also has CMOS circuitry between the probe and a supporting substrate, the CMOS circuitry for controlling the shunt transistor, the transmission transistor and the reset transistor wherein the photosensors are incorporated in the CMOS circuitry and the probe is a fluorescence resonance energy transfer (FRET) probe.
• GAS020.6 Preferably, the microfluidic device also has an array of the FRET probes, each of the FRET probes being positioned in a respective hybridization chamber and configured for hybridization with different target nucleic acid sequences, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS020.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS020.8 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS020.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photosensors into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS020.10 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS020.11 Preferably, the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
• GAS020.12 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS020.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS020.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS020.15 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS020.16 Preferably, the quencher has no native emission in response to the excitation light.
• GAS020.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS020.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS020.19 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS020.20 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• Photosensors with controllable shunts obviate the need for any wavelength dependent filter components, making the design inexpensive, small, and light. Shunts internal to the photodetectors have low resistance, increasing the signal-to-noise ratio and sensitivity.
• GAS021.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a hybridization chamber containing probes having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form probe-target hybrids, the probe-target hybrids being configured to generate a fluorescence signal in response to an excitation light; and,
• a photodiode with an active area and an optical axis extending normal to the active area and through the hybridization chamber; wherein,
• the active area is less than 249 microns from the probe-target hybrids.
• GAS021.2 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS021.3 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS021.4 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• GAS021.5 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the photodiode being a component of the CMOS circuitry; wherein,
• the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
• GAS021.6 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS021.7 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS021.8 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS021.9 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS021.10 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS021.11 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS021.12 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS021.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS021.14 Preferably, the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
• GAS021.15 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS021.16 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS021.17 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS021.18 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS021.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS021.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The nonimaging optics provide for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The nonimaging optics increase the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GAS022.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a hybridization chamber containing probes having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form probe-target hybrids and generate a fluorescence signal in response to an excitation light; and,
• a photodiode with an active area and an optical axis extending normal to the active area and through the hybridization chamber; wherein,
• the hybridization chamber has a floor surface positioned parallel to the active area of the photodiode, the floor surface having a centroid and the active area being encompassed within a cone having the centroid at its vertex, and a vertex angle less than 173°.
• GAS022.2 Preferably, the active area is less than 249 microns from the floor surface.
• GAS022.3 Preferably, the photodiode has a field of view such that the fluorescence signal from the probe-target hybrids is incident on the active area.
• GAS022.4 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the photodiode being a component of the CMOS circuitry; wherein,
• the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
• GAS022.5 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS022.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS022.7 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS022.8 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS022.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS022.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS022.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS022.12 Preferably, the CMOS circuitry has memory for identity data for the FRET probes.
• GAS022.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS022.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS022.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS022.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS022.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS022.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS022.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS022.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The large emission light angle of collection provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The large emission light angle of collection increases the readout sensitivity and obviates the need for optical components in the optical collection train.
• GAS023.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequence;
• a probe with a nucleic acid sequence for hybridization with the target nucleic acid sequence to form a probe-target hybrid and generate a fluorescence signal in response to an excitation light; and,
• a photosensor for sensing the fluorescence signal; wherein during use,
• addition of the biological sample to the inlet prevents fluid communication between a fluid subsequently added to the inlet and the probe.
• GAS023.2 Preferably, the microfluidic device also has CMOS circuitry formed on the supporting substrate wherein the photosensor is a photodiode incorporated as a component of the CMOS circuitry.
• GAS023.3 Preferably, the CMOS circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS023.4 Preferably, the probe is less than 249 microns from the photodiode.
• GAS023.5 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS023.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS023.7 Preferably, the microfluidic device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes.
• GAS023.8 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS023.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS023.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the sample through the PCR section and into the hybridization chambers by capillary action.
• GAS023.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS023.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS023.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS023.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS023.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS023.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS023.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate
• a terbium chelate; or,
• a europium chelate.
• GAS023.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS023.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS023.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light microfluidic device accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads.
• GAS024.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an array of probes for hybridization with a target nucleic acid sequence to form a probe-target hybrid and generate a fluorescence signal in response to an excitation light; and,
• a photosensor positioned adjacent the array of probes; wherein,
• the photosensor is configured for sensing which probes within the array of probes generated the fluorescence signal.
• GAS024.2 Preferably, the photosensor is an array of photodiodes positioned between the array of probes and the supporting substrate.
• GAS024.3 Preferably, the photosensor has a field of view such that the fluorescence signal from the probe-target hybrids is incident on the photosensor.
• GAS024.4 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, wherein the array of photodiodes is in registration with the array of probes, the photodiodes being components of the CMOS circuitry; wherein,
• the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS024.5 Preferably, the microfluidic device also has shunt transistors between each of the photodiode anodes and a voltage source, the shunt transistors being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS024.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS024.7 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS024.8 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS024.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS024.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS024.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS024.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS024.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS024.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS024.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS024.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS024.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS024.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS024.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS024.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads.
• GAS025.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a probe with a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid and generate a fluorescence signal in response to an excitation light; and,
• CMOS circuitry on the supporting substrate for operative control of the excitation light.
• GAS025.2 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS025.3 Preferably, the CMOS circuitry has a photodiode for sensing the fluorescence signal and is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS025.4 Preferably, the probe is less than 249 microns from the photodiode.
• GAS025.5 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS025.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS025.7 Preferably, the microfluidic device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes.
• GAS025.8 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS025.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS025.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS025.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS025.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS025.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS025.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS025.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS025.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS025.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS025.18 Preferably, the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS025.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS025.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The integral LED driver on the LOC device, operating from the ubiquitous USB, provides for an easily usable, mass-producible, inexpensive, compact, and light system with a small component count.
• GAS026.1 This aspect of the invention provides a lab-on-a-chip (LOC) device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer with a hybridization section that has an array of fluorescence resonance energy transfer (FRET) probes for hybridization with target nucleic acid sequences in a fluid; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GAS026.2 Preferably, the photosensor is an array of photodiodes incorporated in CMOS circuitry between the MST layer and the supporting substrate.
• GAS026.3 Preferably, the hybridization section has an array of hybridization chambers containing different FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS026.4 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS026.5 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization section by capillary action.
• GAS026.6 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS026.7 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS026.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS026.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS026.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS026.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS026.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS026.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS026.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS026.15 Preferably, the LOC device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GAS026.16 Preferably, the hybridization section has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS026.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS026.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS026.19 Preferably, the photodiodes are less than 249 microns from the FRET probes.
• GAS026.20 Preferably, the LOC device also has a plurality of reagent reservoirs for different reagents required to process the fluid wherein the fluid is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the LOC device.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, identifies the sample's nucleic acid sequences via hybridization with fluorescence resonance energy transfer (FRET) probes using its integral image sensor, and provides the results electronically at its output pads, with the FRET probes providing high specificity and high reliability of detection of the target sequences.
• GAS027.1 This aspect of the invention provides a LOC device comprising:
• a supporting substrate;
• a primer-linked, stem-and-loop probe incorporating a nucleic acid sequence that matches a target nucleic acid sequence, and a primer for elongating against the target nucleic acid sequence to form a complementary sequence such that during use the probe nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence to change a fluorescence emission from the probe in response to an excitation light; and,
• CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
• GAS027.2 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS027.3 Preferably, the CMOS circuitry has a photodiode for sensing the fluorescence signal and is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS027.4 Preferably, the probe is less than 249 microns from the photodiode.
• GAS027.5 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS027.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS027.7 Preferably, the LOC device also has a fluorophore and a quencher configured to be adjacent each other when the stem-and-loop are closed and spaced from each other when the stem-and-loop are open.
• GAS027.8 Preferably, the LOC device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes and an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS027.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS027.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS027.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS027.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS027.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS027.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS027.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS027.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS027.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS027.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS027.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS027.20 Preferably, the LOC device also has a cap overlaying the MST layer, the cap defining a plurality of reagent reservoirs containing reagents for addition to the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with primer-linked stem-and-loop probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked stem-and-loop probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• GAS028.1 This aspect of the invention provides a LOC device comprising:
• a supporting substrate;
• a primer-linked, linear probe with a nucleic acid sequence that matches a target nucleic acid sequence, and a primer for elongating against the target nucleic acid sequence to form a complementary sequence such that during use the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence to change a fluorescence emission from the probe in response to an excitation light; and,
• CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
• GAS028.2 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS028.3 Preferably, the CMOS circuitry has a photodiode for sensing the fluorescence emission and is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS028.4 Preferably, the probe is less than 249 microns from the photodiode.
• GAS028.5 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS028.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS028.7 Preferably, the LOC device also has a fluorophore and a quencher such that the quencher is removed when the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence.
• GAS028.8 Preferably, the LOC device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes and an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS028.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS028.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS028.11 Preferably, the LOC device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS028.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS028.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS028.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS028.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS028.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS028.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS028.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS028.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS028.20 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with primer-linked linear probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked linear probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• GAS030.1 This aspect of the invention provides a LOC device comprising:
• a supporting substrate;
• primer-linked, stem-and-loop probes, each with a nucleic acid sequence that matches one of a plurality of target nucleic acid sequences, and a primer for elongating against the target nucleic acid sequence to form a complementary sequence such that during use the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence to change a fluorescence emission from the probe in response to an excitation light;
• a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes; and,
• CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
• GAS030.2 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS030.3 Preferably, the CMOS circuitry has a photosensor for sensing the fluorescence signal and is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS030.4 Preferably, the probe is less than 249 microns from the photosensor.
• GAS030.5 Preferably, the LOC device also has shunt transistors between each of the photodiode anodes and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS030.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS030.7 Preferably, the LOC device also has a fluorophore and a quencher configured to be adjacent each other when the stem and loop are closed and spaced from each other when the stem and loop are open.
• GAS030.8 Preferably, the LOC device also has an array of hybridization chambers wherein the probes are fluorescence resonance energy transfer (FRET) probes and each of the hybridization chambers contain a type of the FRET probes configured for hybridization with one of the target nucleic acid sequences respectively, each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS030.9 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS030.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS030.11 Preferably, the LOC device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS030.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS030.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS030.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS030.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS030.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS030.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS030.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS030.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS030.20 Preferably, the LOC device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with primer-linked stem-and-loop probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked stem-and-loop probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• GAS031.1 This aspect of the invention provides a LOC device comprising:
• a supporting substrate;
• a primer-linked, linear probe with a nucleic acid sequence that matches a target nucleic acid sequence, and a primer for elongating against the target nucleic acid sequence to form a complementary sequence such that during use the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence to change a fluorescence emission from the probe in response to an excitation light; and,
• CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
• GAS031.2 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS031.3 Preferably, the CMOS circuitry has a photodiode for sensing the fluorescence emission and is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS031.4 Preferably, the probe is less than 249 microns from the photodiode.
• GAS031.5 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS031.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS031.7 Preferably, the LOC device also has a fluorophore and a quencher such that the quencher is removed when the nucleic acid sequence matching the target nucleic acid sequence anneals to the complementary sequence.
• GAS031.8 Preferably, the LOC device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes and an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS031.9 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS031.10 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS031.11 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS031.12 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS031.13 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS031.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS031.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS031.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS031.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS031.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS031.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS031.20 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with primer-linked linear probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked linear probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• GAS032.1 This aspect of the invention provides a LOC device for amplifying nucleic acid sequences and detecting target nucleic acid sequences, the LOC device comprising:
• a supporting substrate;
• restriction enzymes for digesting double stranded nucleic acid sequences at known restriction sites;
• linker primers for ligation to the restricted ends of the double stranded nucleic acid sequences;
• heaters for thermally cycling the nucleic acid sequences through a polymerase chain reaction (PCR) process;
• deoxyribonucleoside triphosphates (dNTPs) for extending the linker primers along the nucleic acid sequences; and,
• probes for hybridizing with target nucleic acid sequences.
• GAS032.2 Preferably, the probes are FRET (fluorescence resonance energy transfer) probes which change their fluorescent response to an excitation light when hybridized into probe-target hybrids.
• GAS032.3 Preferably, the LOC device also has photodiodes for each of the FRET probes respectively.
• GAS032.4 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
• GAS032.5 Preferably, the operative control includes activation and deactivation of the excitation light.
• GAS032.6 Preferably, the CMOS circuitry is configured to deactivate the excitation light and subsequently activate the photodiodes after a time delay.
• GAS032.7 Preferably, the FRET probes are less than 249 microns from the photodiodes.
• GAS032.8 Preferably, the LOC device also has a shunt transistor between each of the photodiode anodes and a voltage source, the shunt transistors being configured to remove carriers generated by absorption of photons of the excitation light in the photodiodes.
• GAS032.9 Preferably, the shunt transistors are configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS032.10 Preferably, the LOC device also has an array of hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS032.11 Preferably, the LOC device also has a polymerase chain reaction (PCR) section in which the heaters are positioned for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS032.12 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS032.13 Preferably, the LOC device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS032.14 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS032.15 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS032.16 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS032.17 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS032.18 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS032.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS032.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads, with the adaptor primers providing the capability for genome-scale amplification and analysis.
• GAS033.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequence;
• a reagent reservoir containing a reagent for addition to the biological sample; and,
• a hybridization chamber containing probes having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form probe-target hybrids; wherein,
• microsystems tech the reagent reservoir has a volume less than 1,000,000,000 cubic microns and the hybridization chamber has a volume less than 900,000 cubic microns.
• GAS033.2 Preferably, the reagent reservoir has a volume less than 300,000,000 cubic microns and the hybridization chamber has a volume less than 200,000 cubic microns.
• GAS033.3 Preferably, the reagent reservoir has a volume less than 70,000,000 cubic microns and the hybridization chamber has a volume less than 40,000 cubic microns.
• GAS033.4 Preferably, the reagent reservoir has a volume less than 20,000,000 cubic microns and the hybridization chamber has a volume less than 9000 cubic microns.
• GAS033.5 Preferably, the probe-target hybrids are configured to emit a fluorescence signal in response to exposure to an excitation light.
• GAS033.6 Preferably, the microfluidic device also has a photodiode for sensing the fluorescent response.
• GAS033.7 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate for operative control of the excitation light, wherein the photodiode is incorporated into the CMOS circuitry.
• GAS033.8 Preferably, the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS033.9 Preferably, the probes are less than 249 microns from the photodiode.
• GAS033.10 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS033.11 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS033.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS033.13 Preferably, the PCR section and the hybridization chambers are incorporated in a microsystems technology (MST) layer having a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS033.14 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS033.15 Preferably, the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
• GAS033.16 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS033.17 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS033.18 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS033.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS033.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The low-volume hybridization chambers and reagent reservoirs, in part, provide for the low probe and reagent volumes, which in turn provide for low probe and reagent costs and the inexpensive assay system.
• GAS034.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of nucleic acid sequences extracted from biological material, the LOC device comprising:
• probes for hybridization with target nucleic acid sequences within the nucleic acid sequences to form probe-target hybrids, the probe-target hybrids each having a reporting fluorophore for emitting a fluorescence signal in response to an excitation light;
• a positive control probe configured to always emit a fluorescence signal; and,
• a negative control probe configured to never emit a fluorescence signal; wherein during use,
• detecting a fluorescence signal from the negative control probe indicates a malfunction; and,
• not detecting a fluorescence signal from the positive control probe indicates a malfunction.
• GAS034.2 Preferably, the LOC device also has an array of detection photodiodes for sensing the fluorescence signal from each of the probe-target hybrids respectively;
• a positive control photodiode for detecting the fluorescence signal from the reporting fluorophores of the positive control probe; and,
• a negative control photodiode for exposure to the negative control probes.
• GAS034.3 Preferably, the negative control probe lacks a reporting fluorophore.
• GAS034.4 Preferably, the LOC device also has an array of hybridization chambers, each of the hybridization chambers containing at least one of the probes, including the positive control probe and the negative control probe.
• GAS034.5 Preferably, the LOC device also has:
• a supporting substrate;
• a hybridization section;
• a microsystems technologies (MST) layer which incorporates the array of hybridization chambers; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry incorporating the array of detection photodiodes, the positive control photodiode and the negative control photodiode; wherein,
• the probes within the probe array are fluorescence resonance energy transfer (FRET) probes.
• GAS034.6 Preferably, the LOC device also has a nucleic acid amplification section for amplifying the nucleic acid sequences.
• GAS034.7 Preferably, the MST layer has a plurality of MST channels configured to draw fluid through the nucleic acid amplification section and into the hybridization section by capillary action.
• GAS034.8 Preferably, the LOC device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to generate a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS034.9 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS034.10 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS034.11 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS034.12 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS034.13 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS034.14 Preferably, the quencher has no native emission in response to the excitation light.
• GAS034.15 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS034.16 Preferably, the hybridization chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
• GAS034.17 Preferably, the hybridization section has a fluid flow-path from the nucleic acid amplification section to a liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS034.18 Preferably, the fluid flow-path is configured to draw the fluid from the nucleic acid amplification section to the liquid sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the liquid sensor indicating that the fluid has reached the liquid sensor.
• GAS034.19 Preferably, the hybridization heater has an annular shape and extends around an optical window in each of the hybridization chambers positioned such that the excitation light is incident on the probe.
• GAS034.20 Preferably, the hybridization photodiode and the probe are spaced apart by less than 249 microns.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS035.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• an array of probes each having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form a probe-target hybrid; wherein,
• the array of probes includes a control probe for hybridization with a target sequence known to be always present in the biological sample.
• GAS035.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the array of probes wherein, the probes are configured to generate a fluorescence signal in response to an excitation light such that the photosensor senses which probes within the array of probes generated the fluorescence signal.
• GAS035.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the array of probes and the supporting substrate.
• GAS035.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the array of probes.
• GAS035.5 Preferably, the array of photodiodes is less than 249 microns from the array of probes.
• GAS035.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense a fluorescence signal from the control probe.
• GAS035.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
• GAS035.8 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS035.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS035.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS035.11 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS035.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS035.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS035.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS035.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS035.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS035.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS035.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS035.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS036.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes that each have a nucleic acid sequence for hybridization with the target nucleic acid sequence to form a probe-target hybrid, a fluorophore and a quencher configured such that the fluorophore emits a fluorescence signal in response to an excitation light and the quencher quenches the fluorescence signal when the probe is not hybridized, but fails to quench the fluorescence signal from the probe-target hybrid; and,
• a control probe with a fluorophore but no quencher; wherein,
• the control probe always emits the fluorescence signal in response to the excitation light.
• GAS036.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the fluorescence signal in response to the excitation light.
• GAS036.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS036.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes.
• GAS036.5 Preferably, the array of photodiodes is less than 249 microns from the probes.
• GAS036.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense a fluorescence signal from the control probe.
• GAS036.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS036.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS036.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS036.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS036.11 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS036.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS036.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS036.14 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS036.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS036.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS036.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS036.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS036.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS037.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequence; probes that each have a nucleic acid sequence for hybridization with the target nucleic acid sequence to form a probe-target hybrid, a fluorophore and a quencher configured such that the fluorophore emits a fluorescence signal in response to an excitation light and the quencher quenches the fluorescence signal when the probe is not hybridized, but fails to quench the fluorescence signal from the probe-target hybrid; and,
• a reporter with a fluorophore positioned for exposure to the excitation light simultaneously with the probes; wherein,
• the reporter always emits the fluorescence signal in response to the excitation light.
• GAS037.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the probes and the reporter for sensing which of the probes generate the fluorescence signal in response to the excitation light, and to sense the fluorescence signal from the reporter.
• GAS037.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS037.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes and the reporter.
• GAS037.5 Preferably, the array of photodiodes is less than 249 microns from the probes and the reporter.
• GAS037.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense a fluorescence signal from the reporter.
• GAS037.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS037.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS037.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS037.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS037.11 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS037.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS037.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS037.14 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS037.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS037.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS037.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS037.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS037.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS038.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• an array of probes each having a nucleic acid sequence for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• a control probe designed to be complementary to sequences known to be absent from the biological sample.
• GAS038.2 Preferably, the LOC device also has a photosensor positioned adjacent the array of probes wherein, the probes are configured to generate a fluorescence signal in response to an excitation light such that the photosensor senses which probes within the array of probes generated the fluorescence signal.
• GAS038.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the array of probes and the supporting substrate as well as the control probe and the supporting substrate.
• GAS038.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the array of probes.
• GAS038.5 Preferably, the array of photodiodes is less than 249 microns from the array of probes.
• GAS038.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a fluorescence signal from the control probe.
• GAS038.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
• GAS038.8 Preferably, the LOC device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS038.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS038.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS038.11 Preferably, the LOC device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS038.12 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS038.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS038.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS038.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS038.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS038.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS038.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS038.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS039.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• probes that each have a nucleic acid sequence for hybridization with the target nucleic acid sequences to form probe-target hybrids, a fluorophore and a quencher configured such that the fluorophore emits a fluorescence signal in response to an excitation light and the quencher quenches the fluorescence signal when the probe is not hybridized, but fails to quench the fluorescence signal from the probe-target hybrid; and,
• a control probe with no fluorophore such that the control probe never emits the fluorescence signal in response to the excitation light.
• GAS039.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the fluorescence signal in response to the excitation light.
• GAS039.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS039.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes.
• GAS039.5 Preferably, the array of photodiodes is less than 249 microns from the probes.
• GAS039.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to sensing a fluorescence signal from the control probe.
• GAS039.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS039.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS039.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS039.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS039.11 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS039.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS039.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS039.14 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS039.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS039.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS039.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS039.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS039.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS040.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• probes that each have a nucleic acid sequence for hybridization with a respective one of the target nucleic acid sequences to form a probe-target hybrid and emit a fluorescence signal in response to an excitation light; and,
• photosensors corresponding to each of the probes respectively; and,
• a control photosensor not corresponding to any of the probes such that the control photosensor does not sense the fluorescence signal of a corresponding probe-target hybrid during correct operation of the microfluidic device.
• GAS040.2 Preferably, the photosensors are photodiodes spaced less than 249 microns from the probes, and the control photosensor is a control photodiode.
• GAS040.3 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the photodiodes being components of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to sensing a fluorescence signal from the control photodiode.
• GAS040.4 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS040.5 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS040.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS040.7 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS040.8 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS040.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS040.10 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS040.11 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS040.12 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS040.13 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS040.14 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS040.15 Preferably, the quencher has no native emission in response to the excitation light.
• GAS040.16 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS040.17 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• GAS041.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a sample, the microfluidic device comprising:
• probes for reaction with the target molecules to form probe-target complexes, the probe-target complexes being configured to emit photons when excited;
• photodiodes for detecting photons emitted by the probe-target complexes and a calibration photodiode for exposure to a calibration source to generate a calibration signal; wherein during use,
• output from the photodiodes for detecting photons emitted by the probe-target complexes is calibrated by the calibration signal.
• GAS041.2 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the target molecules are target nucleic acid sequences for hybridization with complementary oligonucleotides in the probes to form probe-target hybrids, such that the probe-target hybrids emit a fluorescence signal when exposed to an excitation light.
• GAS041.3 Preferably, the calibration source is a calibration chamber that does not contain a reporter fluorophore, the photodiodes being positioned in registration with the hybridization chambers and the calibration photodiode is in registration with the calibration chamber.
• GAS041.4 Preferably, during use, output from any one of the photodiodes in the array is compared to output from the calibration photodiode that is most proximate to that photodiode.
• GAS041.5 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS041.6 Preferably, the microfluidic device also has:
• a supporting substrate;
• a microsystems technologies (MST) layer which incorporates the array of hybridization chambers and the plurality of calibration chambers; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photodiodes and the calibration photodiodes; wherein,
• the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS041.7 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences.
• GAS041.8 Preferably, the MST layer has a plurality of MST channels configured to draw fluid through the PCR section and into the hybridization section by capillary action.
• GAS041.9 Preferably, the microfluidic device also has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to generate a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GAS041.10 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS041.11 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS041.12 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS041.13 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS041.14 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS041.15 Preferably, the quencher has no native emission in response to the excitation light.
• GAS041.16 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS041.17 Preferably, the microfluidic device also has a hybridization heater for providing thermal energy for hybridization, the hybridization heater being operatively controlled by the CMOS circuitry.
• GAS041.18 Preferably, the hybridization section has a fluid flow-path from the PCR section to a liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS041.19 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heaters in response to output from the liquid sensor indicating that the fluid has reached the liquid sensor.
• GAS041.20 Preferably, the photodiodes and the probes are spaced apart by less than 249 microns.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration photosensor improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• GAS042.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing a target nucleic acid sequences;
• a hybridization chamber containing probes each having a nucleic acid sequence for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• a calibration chamber containing a control probe incapable of hybridization with any sequence in the biological sample.
• GAS042.2 Preferably, the microfluidic device also has a plurality of the hybridization chambers and a photosensor positioned adjacent the hybridization chambers wherein, the hybridization chambers and the control chamber have an optical window to expose the probes to an excitation light, the probes being configured to generate a fluorescence signal in response to the excitation light such that the photosensor senses which probes within the array of probes generated the fluorescence signal.
• GAS042.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the hybridization chambers and the supporting substrate.
• GAS042.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS042.5 Preferably, the array of photodiodes is less than 249 microns from the probes.
• GAS042.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a fluorescence signal from the control probe.
• GAS042.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
• GAS042.8 Preferably, the microfluidic device also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS042.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS042.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS042.11 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS042.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS042.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS042.14 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS042.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS042.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS042.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS042.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS042.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• GAS043.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• hybridization chambers containing probes that each have a nucleic acid sequence for hybridization with the target nucleic acid sequences to form probe-target hybrids, a fluorophore and a quencher configured such that the fluorophore emits a fluorescence signal in response to an excitation light and the quencher quenches the fluorescence signal when the probe is not hybridized, but fails to quench the fluorescence signal from the probe-target hybrid; and,
• a calibration chamber with no fluorophore.
• GAS043.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the hybridization chambers and the calibration chamber for sensing which of the probes generate the fluorescence signal in response to the excitation light, wherein the hybridization chambers and the control chamber have an optical window to expose the probes to the excitation light.
• GAS043.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the hybridization chambers and the calibration chamber, and the supporting substrate.
• GAS043.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers and the calibration chamber.
• GAS043.5 Preferably, the array of photodiodes is less than 249 microns from the probes.
• GAS043.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to sensing a fluorescence signal from the control probe.
• GAS043.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS043.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS043.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS043.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS043.11 Preferably, the hybridization chambers contain different types of the FRET probes, each configured for hybridization with different target nucleic acid sequences.
• GAS043.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS043.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS043.14 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS043.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS043.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS043.17 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or
• a europium chelate.
• GAS043.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS043.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• GAS044.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• hybridization chambers in fluid communication with the inlet, the hybridization chambers containing probes that each have a nucleic acid sequence for hybridization with the target nucleic acid sequences to form probe-target hybrids, a fluorophore and a quencher configured such that the fluorophore emits a fluorescence signal in response to an excitation light and the quencher quenches the fluorescence signal when the probe is not hybridized, but fails to quench the fluorescence signal from the probe-target hybrid; and,
• a calibration chamber not in fluid communication the inlet; wherein, the calibration chamber contains a fluorophore.
• GAS044.2 Preferably, the microfluidic device also has a photosensor positioned adjacent the hybridization chambers and the calibration chamber for sensing which of the probes generate the fluorescence signal in response to the excitation light, wherein the hybridization chambers and the control chamber have an optical window to expose the probes to the excitation light.
• GAS044.3 Preferably, the photosensor is a charge coupled device (CCD) array positioned between the hybridization chambers and the calibration chamber, and the supporting substrate.
• GAS044.4 Preferably, the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers and the calibration chamber.
• GAS044.5 Preferably, the array of photodiodes is less than 249 microns from the probes.
• GAS044.6 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to sensing a fluorescence signal from the control probe.
• GAS044.7 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS044.8 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS044.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS044.10 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS044.11 Preferably, the hybridization chambers contain different types of the FRET probes, each configured for hybridization with different target nucleic acid sequences.
• GAS044.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS044.13 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS044.14 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS044.15 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS044.16 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS044.17 Preferably, the fluorophore is selected from
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS044.18 Preferably, the quencher has no native emission in response to the excitation light.
• GAS044.19 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• GAS045.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a biological sample containing target nucleic acid sequences;
• probes that each have a nucleic acid sequence for hybridization with a respective one of the target nucleic acid sequences to form a probe-target hybrid and emit a fluorescence signal in response to an excitation light; and,
• photosensors corresponding to each of the probes respectively; and,
• a calibration photosensor not corresponding to any of the probes such that output from the calibration photosensor is subtracted in a differential circuit from each output from the photosensors.
• GAS045.2 Preferably, the photosensors are photodiodes spaced less than 249 microns from the probes, and the calibration photosensor is a calibration photodiode.
• GAS045.3 Preferably, the microfluidic device also has CMOS circuitry on the supporting substrate, the photodiodes being components of the CMOS circuitry.
• GAS045.4 Preferably, the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiodes.
• GAS045.5 Preferably, the microfluidic device also has a shunt transistor between each photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS045.6 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS045.7 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS045.8 Preferably, the microfluidic device also has an array of the hybridization chambers containing different types of the FRET probes configured for hybridization with different target nucleic acid sequences.
• GAS045.9 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the probes.
• GAS045.10 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS045.11 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS045.12 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS045.13 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS045.14 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS045.15 Preferably, the quencher has no native emission in response to the excitation light.
• GAS045.16 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GAS045.17 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration circuit improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• GAS046.1 This aspect of the invention provides a microfluidic test module comprising:
• an outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• a hybridization chamber mounted in the casing, the hybridization chamber containing probes having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form probe-target hybrids; wherein,
• the hybridization chamber has a volume less than 900,000 cubic microns.
• GAS046.2 Preferably, the hybridization chamber has a volume less than 200,000 cubic microns.
• GAS046.3 Preferably, the hybridization chamber has a volume less than 40,000 cubic microns.
• GAS046.4 Preferably, the hybridization chamber has a volume less than 9000 cubic microns.
• GAS046.5 Preferably, the probe-target hybrids are configured to emit a fluorescence signal in response to exposure to an excitation light and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS046.6 Preferably, the microfluidic test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS046.7 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS046.8 Preferably, the microfluidic test module also has control circuitry for operative control of the excitation LED.
• GAS046.9 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS046.10 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS046.11 Preferably, the microfluidic test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS046.12 Preferably, the microfluidic test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS046.13 Preferably, the microfluidic test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS046.14 Preferably, the microfluidic test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS046.15 Preferably, the microfluidic test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS046.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS046.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS046.18 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS046.19 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS046.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The low-volume hybridization chambers, in part, provide for the low probe volumes, which in turn provide for low probe cost and the inexpensive assay system.
• GAS047.1 This aspect of the invention provides a microfluidic test module comprising:
• an outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• a hybridization chamber mounted in the casing, the hybridization chamber holding a volume of fluid containing probes, the probes each having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form probe-target hybrids; wherein,
• the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS047.2 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS047.3 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• GAS047.4 Preferably, the mass of the probes in each of the hybridization chambers is less than 2.7 picograms.
• GAS047.5 Preferably, the probe-target hybrids are configured to emit a fluorescence signal in response to exposure to an excitation light and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS047.6 Preferably, the microfluidic test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS047.7 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS047.8 Preferably, the microfluidic test module also has control circuitry for operative control of the excitation LED.
• GAS047.9 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS047.10 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS047.11 Preferably, the microfluidic test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS047.12 Preferably, the microfluidic test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS047.13 Preferably, the microfluidic test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS047.14 Preferably, the microfluidic test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS047.15 Preferably, the microfluidic test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS047.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS047.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS047.18 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS047.19 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS047.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The low probe volume provides for low probe cost, in turn, permitting the inexpensive assay system.
• GAS048.1 This aspect of the invention provides a test module for detecting hybridization of probes with target nucleic acid sequences, the test module comprising:
• a receptacle for receiving a biological sample with target nucleic acid sequences;
• an array of probes for hybridization with the target nucleic acid sequences;
• an excitation light emitting diode (LED) for illuminating the array of probes;
• CMOS circuitry having a photosensor for sensing a fluorescence emission from any of the probes in the array that have hybridized with the target nucleic acid sequences such that the CMOS circuitry is configured to use outputs from the photosensor to generate a signal indicative of the probes that have hybridized; wherein,
• the array of probes and the CMOS circuitry are integrated on a lab-on-a-chip (LOC) device contained within the test module.
• GAS048.2 Preferably, the test module also has a nucleic acid amplification section for amplifying the target nucleic acid sequences in the biological sample.
• GAS048.3 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GAS048.4 Preferably, the photosensor is an array of photodiodes and the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS048.5 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS048.6 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS048.7 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS048.8 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS048.9 Preferably, the quencher has no native emission in response to the excitation LED emission.
• GAS048.10 Preferably, the excitation LED has an emission wavelength substantially matching the absorption wavelength of the fluorophores.
• GAS048.11 Preferably, the test module also has a USB (universal serial bus) plug for transmitting the signal to an external device, wherein during use, the circuitry, the nucleic acid amplification section and the excitation LED are powered by the external device via the USB plug.
• GAS048.12 Preferably, the test module also has an outer casing for rigidly retaining the USB plug and the receptacle being in fluid communication with a sample inlet on the LOC device.
• GAS048.13 Preferably, the LOC device has a supporting substrate, and a microsystems technologies (MST) layer with the array of probes, the CMOS circuitry is positioned between the MST layer and the supporting substrate, and the CMOS circuitry incorporates the photosensor.
• GAS048.14 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to the excitation LED.
• GAS048.15 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the nucleic acid amplification section and into the hybridization chambers by capillary action.
• GAS048.16 Preferably, the LOC device has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences.
• GAS048.17 Preferably, the CMOS circuitry has memory for identity data for different FRET probe types.
• GAS048.18 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation LED being extinguished.
• GAS048.19 Preferably, one of the MST channels defines a fluid flow-path from the nucleic acid amplification section to a liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path such that fluid containing the target nucleic acid sequences fills the hybridization chambers as the fluid is drawn to the liquid sensor by capillary action.
• GAS048.20 Preferably, the test module also has at least one valve for preventing the biological sample from entering the nucleic acid amplification section until the USB plug is inserted into the external device.
• The integrated image sensor with the excitation LED obviate the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GAS049.1 This aspect of the invention provides a test module for detecting hybridization of probes with target nucleic acid sequences, the test module comprising:
• a casing configured for hand held portability, the casing having a receptacle for receiving a biological sample with target nucleic acid sequences;
• an array of probes for hybridization with the target nucleic acid sequences;
• an excitation light source for illuminating the array of probes; and
• a photosensor for sensing a fluorescence emission from any of the probes in the array that have hybridized with the target nucleic acid sequences.
• GAS049.2 Preferably, the test module also has circuitry for using outputs from the photosensor to generate a signal indicative of the probes that have hybridized.
• GAS049.3 Preferably, the circuitry is configured for transmitting the signal to an external device.
• GAS049.4 Preferably, the circuitry has a USB (universal serial bus) plug to for transmitting the signal to an external device.
• GAS049.5 Preferably, the test module also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences in the biological sample.
• GAS049.6 Preferably, the photosensor is an array of photodiodes and the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS049.7 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS049.8 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS049.9 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS049.10 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS049.11 Preferably, the quencher has no native emission in response to the excitation light source emission.
• GAS049.12 Preferably, the excitation light source is a light emitting diode (LED) with an emission wavelength substantially matching the absorption wavelength of the fluorophores.
• GAS049.13 Preferably, during use, the circuitry, the PCR section and the excitation light source are powered by the external device via the USB plug.
• GAS049.14 Preferably, the PCR section, the array of probes and the photosensor are provided on a lab-on-a-chip (LOC) device mounted in the casing.
• GAS049.15 Preferably, the LOC device has a supporting substrate, and a microsystems technologies (MST) layer with the array of probes, and the circuitry has CMOS circuitry positioned between the MST layer and the supporting substrate.
• GAS049.16 Preferably, the array of photodiodes are incorporated into the CMOS circuitry.
• GAS049.17 Preferably, the MST layer has an array of hybridization chambers for containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to the excitation LED.
• GAS049.18 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS049.19 Preferably, the LOC device has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences.
• GAS049.20 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• The easily usable, mass-producible, inexpensive, compact, light, and portable test module accepts a biological sample, identifies the sample's nucleic acid sequences via fluorescent probe hybridization using its integral image sensor and excitation light source, and provides the results electronically at its output port.
• GAS050.1 This aspect of the invention provides a test module for detecting hybridization of probes with target nucleic acid sequences, the test module comprising:
• a receptacle for receiving a biological sample with target nucleic acid sequences;
• an array of probes for hybridization with the target nucleic acid sequences;
• an excitation light source for illuminating the array of probes;
• circuitry incorporating a photosensor for sensing a fluorescence emission from any of the probes in the array that have hybridized with the target nucleic acid sequences; wherein,
• the circuitry is configured to use outputs from the photosensor to generate a signal indicative of the probes that have hybridized and transmit the signal to an external device.
• GAS050.2 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the biological sample.
• GAS050.3 Preferably, the photosensor is an array of photodiodes and the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS050.4 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS050.5 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS050.6 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS050.7 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS050.8 Preferably, the quencher has no native emission in response to the excitation light source emission.
• GAS050.9 Preferably, the excitation light source is a light emitting diode (LED) with an emission wavelength substantially matching the absorption wavelength of the fluorophores.
• GAS050.10 Preferably, the test module also has a universal serial bus (USB) plug wherein during use, the circuitry, the PCR section and the excitation light source are powered by the external device via the USB plug.
• GAS050.11 Preferably, the circuitry includes CMOS circuitry fabricated on a lab-on-a-chip (LOC) device mounted in the test module, the LOC device having a sample inlet in fluid communication with the receptacle and incorporating the PCR section and the array of probes, and the CMOS circuitry incorporates the photosensor.
• GAS050.12 Preferably, the test module also has an outer casing for rigidly retaining the USB plug.
• GAS050.13 Preferably, the LOC device has a supporting substrate, and a microsystems technologies (MST) layer incorporating the PCT section and the array of probes, the CMOS circuitry being positioned between the MST layer and the supporting substrate.
• GAS050.14 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to the excitation LED.
• GAS050.15 Preferably, the MST layer has a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS050.16 Preferably, the CMOS circuitry has bond-pads for electrical connection to the external device via the USB plug, wherein the CMOS circuitry is configured use output from the photodiodes to generate a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences.
• GAS050.17 Preferably, the CMOS circuitry has memory for identity data for different FRET probe types.
• GAS050.18 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS050.19 Preferably, one of the MST channels defines a fluid flow-path from the PCR section to a liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path such that fluid containing the target nucleic acid sequences fills the hybridization chambers as the fluid is drawn to the liquid sensor by capillary action.
• GAS050.20 Preferably, the test module also has at least one valve for preventing the biological sample from entering the PCR section until the USB plug is inserted into the external device.
• The easily usable, mass-producible, inexpensive, compact, light, and portable test module accepts a biological sample, identifies the sample's nucleic acid sequences via fluorescent probe hybridization using its integral image sensor and excitation light source, and provides the results electronically at its output port, with the ubiquitous USB port used for the module's power and signaling requirements.
• GAS054.1 This aspect of the invention provides a test module comprising:
• an outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• a reagent reservoir containing a reagent for addition to the biological sample; and,
• a hybridization chamber containing probes having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form probe-target hybrids; wherein,
• the reagent reservoir has a volume less than 20,000,000 cubic microns and the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS054.2 Preferably, the probe-target hybrids are configured to emit a fluorescence signal in response to exposure to an excitation light.
• GAS054.3 Preferably, the test module also has a photodiode for sensing the fluorescent response.
• GAS054.4 Preferably, the test module also has a light source for generating the excitation light and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS054.5 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS054.6 Preferably, the test module also has control circuitry for operative control of the excitation LED.
• GAS054.7 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS054.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS054.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS054.10 Preferably, the probes are less than 249 microns from the photodiode.
• GAS054.11 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers has a respective one of the photodiodes.
• GAS054.12 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes.
• GAS054.13 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS054.14 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS054.15 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS054.16 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS054.17 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS054.18 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS054.19 Preferably, the quencher has no native emission in response to the excitation light.
• GAS054.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The low-volume hybridization chambers and reagent reservoirs, in part, provide for the low probe and reagent volumes, which in turn provide for low probe and reagent costs and the inexpensive assay system.
• GAS055.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• probes in the outer casing for hybridization with the target nucleic acid sequence to form probe-target hybrids, the probes each having a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light; wherein,
• the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS055.2 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS055.3 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS055.4 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS055.5 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS055.6 Preferably, the test module also has control circuitry for operative control of the excitation LED.
• GAS055.7 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS055.8 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS055.9 Preferably, the test module of claim 8 wherein the control circuitry further comprises a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS055.10 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS055.11 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS055.12 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS055.13 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS055.14 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS055.15 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS055.16 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS055.17 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS055.18 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS055.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS055.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• Using the long-lifetime fluorescent probes in conjunction with the time-delayed detection of fluorescence improves the sensitivity and reliability of the assay system and obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS056.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• probes in the outer casing for hybridization with the target nucleic acid sequence to form probe-target hybrids, the probes each having a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light; wherein,
• the fluorophore is a transition metal-ligand complex.
• GAS056.2 Preferably, the fluorophore is a ruthenium chelate.
• GAS056.3 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS056.4 Preferably, the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS056.5 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS056.6 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS056.7 Preferably, the test module also has control circuitry for operative control of the excitation LED.
• GAS056.8 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS056.9 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS056.10 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS056.11 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS056.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS056.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS056.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS056.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS056.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS056.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS056.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS056.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS056.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• Using the long-lifetime transition metal-ligand complex fluorescent probes in conjunction with the time-delayed detection of fluorescence improves the sensitivity and reliability of the assay system and obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS057.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• probes in the outer casing for hybridization with the target nucleic acid sequence to form probe-target hybrids, the probes each having a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light; wherein,
• the fluorophore is a lanthanide metal-ligand complex.
• GAS057.2 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; and,
• a europium chelate.
• GAS057.3 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS057.4 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS057.5 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS057.6 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS057.7 Preferably, the test module also has control circuitry for operative control of the excitation LED.
• GAS057.8 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS057.9 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS057.10 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS057.11 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS057.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS057.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS057.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS057.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS057.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS057.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS057.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS057.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS057.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• Using the long-lifetime lanthanide metal-ligand complex fluorescent probes in conjunction with the time-delayed detection of fluorescence improves the sensitivity and reliability of the assay system and obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS058.1 This aspect of the invention provides a test module comprising:
• an outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• probes in the outer casing for hybridization with the target nucleic acid sequence to form probe-target hybrids, the probes each having a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light; wherein,
• the probes are suspended in a fluid.
• GAS058.2 Preferably, the mass of the probes is less than 270 picograms.
• GAS058.3 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS058.4 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS058.5 Preferably, the test module also has a light source for generating the excitation light and a photodiode for sensing the fluorescent response.
• GAS058.6 Preferably, the light source is an excitation LED mounted in the casing for emitting light through the optical window.
• GAS058.7 Preferably, the test module also has control circuitry for operative control of the excitation LED.
• GAS058.8 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS058.9 Preferably, the control circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
• GAS058.10 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS058.11 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS058.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS058.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS058.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS058.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS058.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS058.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS058.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS058.19 Preferably, the FRET probes have a quencher to suppress the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS058.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The suspended probes with their large excitation and emission optical depth increase the readout sensitivity. The suspended probes are spotted more easily and inexpensively.
• GAS059.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• control circuitry having a photosensor for generating an output signal in response to the fluorescence signal; wherein,
• the control circuitry is configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
• GAS059.2 Preferably, the control circuitry has a trigger photosensor for generating an output in response to the excitation light such that the control circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS059.3 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS059.4 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS059.5 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS059.6 Preferably, the test module also has an excitation LED mounted in the casing for generating the excitation light.
• GAS059.7 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS059.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS059.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS059.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS059.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS059.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS059.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS059.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS059.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS059.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS059.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS059.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS059.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS059.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The time-delayed detection of fluorescence obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS060.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence; and,
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• control circuitry having a photosensor for generating an output signal in response to the fluorescence signal; wherein,
• the control circuitry is configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
• GAS060.2 Preferably, the control circuitry has a trigger photosensor for generating an output in response to the excitation light such that the control circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS060.3 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS060.4 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS060.5 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS060.6 Preferably, the test module also has an excitation LED mounted in the casing for generating the excitation light.
• GAS060.7 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS060.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS060.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS060.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS060.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS060.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS060.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS060.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS060.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS060.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS060.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS060.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS060.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS060.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The controlled exposure of fluorophores to excitation light obviates the need for any wavelength dependent filter components, making the design inexpensive, small, and light.
• GAS061.1 This aspect of the invention provides a single-use test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• a photosensor to detect the fluorescence signal.
• GAS061.2 Preferably, the single-use test module also has control circuitry configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
• GAS061.3 Preferably, the control circuitry has a trigger photosensor for generating an output in response to the excitation light such that the control circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS061.4 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS061.5 Preferably, the single-use test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS061.6 Preferably, and the hybridization chamber has an optical window for exposing the probes to the excitation light.
• GAS061.7 Preferably, the single-use test module also has an excitation LED mounted in the casing for generating the excitation light.
• GAS061.8 Preferably, the control circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS061.9 Preferably, the single-use test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS061.10 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS061.11 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS061.12 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS061.13 Preferably, the single-use test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS061.14 Preferably, the single-use test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through from the inlet to the hybridization chambers by capillary action.
• GAS061.15 Preferably, the single-use test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS061.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS061.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS061.18 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS061.19 Preferably, the FRET probes have a quencher suppress the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS061.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output port.
• GAS062.1 This aspect of the invention provides a single-use test module for detecting hybridization of probes with nucleic acid sequences, the test module comprising:
• a sample input for receiving a biological sample with target nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the biological sample;
• an array of probes for hybridization with target nucleic acid sequences in the biological sample; and,
• circuitry having a photosensor for sensing hybridization between the probes and target nucleic acid sequences in the biological sample; wherein,
• the circuitry is configured to use outputs from the photosensor to generate a signal indicative of the probes that have hybridized.
• GAS062.2 Preferably, the test module also has an excitation light source for illuminating the array of probes wherein the photo sensor is configured to sense a fluorescence emission from any of the probes in the array that have hybridized with the target nucleic acid sequences.
• GAS062.3 Preferably, the test module also has a USB (universal serial bus) plug for transmitting the signal to an external device.
• GAS062.4 Preferably, the photosensor is spaced less than 249 microns from the probes.
• GAS062.5 Preferably, the photosensor is an array of photodiodes positioned in registration with the probes and the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS062.6 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS062.7 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS062.8 Preferably, the quencher has no native emission in response to the excitation light source emission.
• GAS062.9 Preferably, the excitation light source is a light emitting diode (LED) with an emission wavelength substantially matching the absorption wavelength of the fluorophores.
• GAS062.10 Preferably, the test module also has a dialysis section wherein the biological sample includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GAS062.11 Preferably, the circuitry includes CMOS circuitry fabricated on a lab-on-a-chip (LOC) device mounted in the test module, the LOC device having a sample inlet and incorporates the dialysis section.
• GAS062.12 Preferably, the test module also has an outer casing for rigidly retaining the USB plug and having a receptacle in fluid communication with the sample inlet on the LOC device.
• GAS062.13 Preferably, the LOC device has a supporting substrate, and a microsystems technologies (MST) layer that incorporates the array of probes, the CMOS circuitry being positioned between the supporting substrate and the MST layer, and the CMOS circuitry incorporating the photosensor.
• GAS062.14 Preferably, the MST layer has an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to the excitation LED.
• GAS062.15 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the target nucleic acids and a plurality of MST channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS062.16 Preferably, the CMOS circuitry has bond-pads for electrical connection to the external device via the USB plug, wherein the CMOS circuitry is configured use output from the photodiodes to generate a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences.
• GAS062.17 Preferably, the CMOS circuitry has memory for identity data for different FRET probe types.
• GAS062.18 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS062.19 Preferably, one of the MST channels defines a fluid flow-path from the PCR section to a liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path such that fluid containing the target nucleic acid sequences fills the hybridization chambers as the fluid is drawn to the liquid sensor by capillary action.
• GAS062.20 Preferably, the test module also has at least one valve for preventing the biological sample from entering the PCR section until the USB plug is inserted into the external device.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output port.
• GAS063.1 This aspect of the invention provides a single-use test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• an excitation light source.
• GAS063.2 Preferably, the excitation light source is a noble gas flash tube mounted in the outer casing.
• GAS063.3 Preferably, the single-use test module also has a light filter wherein the noble gas flash tube illuminates the probes through the light filter.
• GAS063.4 Preferably, the excitation light source is an excitation LED mounted in the outer casing for generating the excitation light.
• GAS063.5 Preferably, the single-use test module also has a photosensor to detect the fluorescence signal.
• GAS063.6 Preferably, the single-use test module also has control circuitry configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
• GAS063.7 Preferably, the control circuitry has a trigger photosensor for generating an output in response to the excitation light such that the control circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS063.8 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS063.9 Preferably, the single-use test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS063.10 Preferably, the control circuitry controls activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS063.11 Preferably, the single-use test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS063.12 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS063.13 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS063.14 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS063.15 Preferably, the single-use test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS063.16 Preferably, the single-use test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through from the inlet to the hybridization chambers by capillary action.
• GAS063.17 Preferably, the single-use test module also has an electrical connection for communication to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS063.18 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS063.19 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS063.20 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor with excitation light source, and provides the results electronically at its output port.
• GAS065.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light; and,
• an excitation LED for generating the excitation light, the LED positioned in the outer casing for simultaneously illuminating all the probes.
• GAS065.2 Preferably, the test module also has circuitry configured to initiate a time delay upon deactivation of the excitation LED before activating the photosensor.
• GAS065.3 Preferably, the circuitry has a trigger photosensor for generating an output in response to the excitation light such that the circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS065.4 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS065.5 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS065.6 Preferably, the hybridization chamber has an optical window for simultaneously exposing the probes to the excitation LED.
• GAS065.7 Preferably, the circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS065.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS065.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS065.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS065.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS065.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS065.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS065.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS065.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the circuitry incorporates the CMOS circuitry and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS065.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS065.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS065.18 Preferably, the test module also has a humidifier for controlling humidity within the outer casing.
• GAS065.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS065.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor with excitation LED, and provides the results electronically at its output port. Utilizing the LED without an optical train provides for a more inexpensive and more easily manufacturable module with lower component-count.
• GAS066.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light;
• an excitation light for generating the excitation light; and,
• a lens positioned in the outer casing for directing light from the excitation light to simultaneously illuminate the probes.
• GAS066.2 Preferably, the test module also has circuitry configured to initiate a time delay upon deactivation of the excitation LED before activating the photosensor.
• GAS066.3 Preferably, the circuitry has a trigger photosensor for generating an output in response to the excitation light such that the circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS066.4 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS066.5 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS066.6 Preferably, the hybridization chamber has an optical window for simultaneously exposing the probes to the excitation light.
• GAS066.7 Preferably, the excitation light is an LED and the circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS066.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS066.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS066.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS066.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS066.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS066.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS066.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS066.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the circuitry incorporates the CMOS circuitry and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS066.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS066.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS066.18 Preferably, the test module also has a humidifier for controlling humidity within the outer casing.
• GAS066.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS066.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor and excitation LED with lens, and provides the results electronically at its output port. Utilizing the LED with lens improves the distribution of the excitation light, which in turn increases the sensitivity and reliability of the assay system.
• GAS067.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light;
• an excitation light for generating the excitation light; and,
• prisms positioned in the outer casing for redirecting light from the excitation light to simultaneously illuminate the probes.
• GAS067.2 Preferably, the test module also has circuitry configured to initiate a time delay upon deactivation of the excitation LED before activating the photosensor.
• GAS067.3 Preferably, the circuitry has a trigger photosensor for generating an output in response to the excitation light such that the circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS067.4 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS067.5 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS067.6 Preferably, the hybridization chamber has an optical window for simultaneously exposing the probes to the excitation light.
• GAS067.7 Preferably, the excitation light is an LED and the circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS067.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS067.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS067.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS067.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS067.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS067.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS067.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS067.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the circuitry incorporates the CMOS circuitry and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS067.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS067.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS067.18 Preferably, the test module also has a humidifier for controlling humidity within the outer casing.
• GAS067.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS067.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor and excitation LED with lens and prisms, and provides the results electronically at its output port. Utilizing the LED with lens and prisms improves the distribution of the excitation light, which in turn increases the sensitivity and reliability of the assay system. Incorporation of prisms in the optical train increases the compactness of the test module.
• GAS068.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization having a nucleic acid sequence for hybridization with a target nucleic acid sequence to form a probe-target hybrid, the probe-target hybrid being configured to generate a fluorescence signal in response to an excitation light;
• an excitation light for generating the excitation light; and,
• mirrors positioned in the outer casing for redirecting light from the excitation light to simultaneously illuminate the probes.
• GAS068.2 Preferably, the test module also has circuitry configured to initiate a time delay upon deactivation of the excitation LED before activating the photosensor.
• GAS068.3 Preferably, the circuitry has a trigger photosensor for generating an output in response to the excitation light such that the circuitry initiates the time delay when the trigger photosensor indicates the excitation light has deactivated.
• GAS068.4 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to an excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS068.5 Preferably, the test module also has a hybridization chamber mounted in the casing for containing probes wherein the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS068.6 Preferably, the hybridization chamber has an optical window for simultaneously exposing the probes to the excitation light.
• GAS068.7 Preferably, the excitation light is an LED and the circuitry controls activation and deactivation of the excitation LED, and conditioning of power supplied to the excitation light.
• GAS068.8 Preferably, the test module also has a shunt transistor between the photodiode anode and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiode.
• GAS068.9 Preferably, the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
• GAS068.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS068.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS068.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS068.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS068.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation LED, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS068.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the circuitry incorporates the CMOS circuitry and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS068.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS068.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS068.18 Preferably, the test module also has a humidifier for controlling humidity within the outer casing.
• GAS068.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS068.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor and excitation LED with lens and mirrors, and provides the results electronically at its output port. Utilizing the LED with lens and mirrors improves the distribution of the excitation light, which in turn increases the sensitivity and reliability of the assay system. Incorporation of minors in the optical train increases the compactness of the test module.
• GAS069.1 This aspect of the invention provides a test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having an inlet for receiving a biological sample containing a target nucleic acid sequence;
• probes in the outer casing for hybridization with a target nucleic acid sequence to form probe-target hybrids, the probe-target hybrids being configured to generate a fluorescence signal in response to an excitation light;
• a laser positioned in the outer casing for generating the excitation light.
• GAS069.2 Preferably, the test module also has minors for redirecting light from the laser to simultaneously illuminate the probes.
• GAS069.3 Preferably, the test module also has prisms for redirecting light from the laser to illuminate the probes.
• GAS069.4 Preferably, the test module also has circuitry configured to initiate a time delay upon deactivation of the excitation laser before activating the photosensor.
• GAS069.5 Preferably, the circuitry controls activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
• GAS069.6 Preferably, the probes each have a fluorophore such that the probe-target hybrids emit a fluorescence signal in response to exposure to the excitation light, and, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• GAS069.7 Preferably, the test module also has an array of hybridization chambers, each of the hybridization chambers containing some of the probes, each of the hybridization chambers has an optical window for exposing the probes to the excitation light.
• GAS069.8 Preferably, the test module also has a shunt transistor between each of the photodiode anodes and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiodes.
• GAS069.9 Preferably, the shunt transistor is configured to activate when the laser activates and deactivate when the laser deactivates.
• GAS069.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS069.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS069.12 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GAS069.13 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample prior to hybridization with the FRET probes.
• GAS069.14 Preferably, the test module also has a microfluidic device mounted in the outer casing adjacent the excitation laser, the microfluidic device having a supporting substrate, CMOS circuitry on the supporting substrate and a microsystems technology (MST) layer on the CMOS circuitry wherein the PCR section and the hybridization chambers are incorporated in the MST layer which has a plurality of channels configured to draw the fluid through the PCR section and into the hybridization chambers by capillary action.
• GAS069.15 Preferably, the test module also has an electrical connection for communication to an external device wherein the circuitry incorporates the CMOS circuitry and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the electrical connection for transmission to the external device.
• GAS069.16 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GAS069.17 Preferably, the CMOS circuitry includes the array of photodiodes such that the array of photodiodes is less than 249 microns from the FRET probes.
• GAS069.18 Preferably, the test module also has a humidifier for controlling humidity within the outer casing.
• GAS069.19 Preferably, the FRET probes have a quencher to suppress most of the fluorescence signal from the fluorophore when the probe is in a non-hybridized configuration, the quencher having no native emission in response to the excitation light.
• GAS069.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor and excitation laser, and provides the results electronically at its output port. Utilizing the laser as the excitation source increases the intensity and wavelength-specificity of the excitation light, which in turn increases the sensitivity and reliability of the assay system.
• GAS070.1 This aspect of the invention provides a test module comprising:
• an outer casing having a receptacle for receiving a biological sample;
• an excitation source mounted in the outer casing for sequentially illuminating the biological sample with different wavelengths of light; and,
• a photosensor positioned to detect the different wavelengths of light transmitted through the biological sample; wherein during use,
• the photosensor output signal is used to generate a spectrogram for analysis of a characteristic of the biological sample.
• GAS070.2 Preferably, the excitation source is an array of LEDs for emitting the different wavelengths of light, the LEDs being configured for sequential activation.
• GAS070.3 Preferably, the outer casing is configured for hand held portability.
• GAS070.4 Preferably, the test module also has a data connection for transmitting the photosensor output signal to an external device.
• GAS070.5 Preferably, the data connection is an electrical connection to the external device, the test module being configured to draw power from the external device via the electrical connection.
• GAS070.6 Preferably, the electrical connection is a universal serial bus (USB) plug for insertion in a USB port on the external device.
• GAS070.7 Preferably, the test module also has a lab-on-a-chip (LOC) device in fluid communication with the receptacle, the LOC device having an array of chambers configured to fill with the biological sample by capillary driven flow from the receptacle.
• GAS070.8 Preferably, the photosensor is incorporated into the LOC device and positioned adjacent the array of chambers.
• GAS070.9 Preferably, the LOC device has a supporting substrate and CMOS circuitry on the supporting substrate, the CMOS circuitry incorporating the photosensor and a series of bond-pads for connection to the USB plug.
• GAS070.10 Preferably, the CMOS circuitry has an LED driver for controlling activation of the array of LEDs via the bond-pads.
• GAS070.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the array of chambers.
• GAS070.12 Preferably, each of the chambers has an optical window for exposing the biological sample to the array of LEDs.
• GAS070.13 Preferably, the biological sample is blood.
• GAS070.14 Preferably, the characteristic analysed is glucose content within the blood.
• GAS070.15 Preferably, the test module also has a shunt transistor between each of the photodiode anodes and a voltage source, the shunt transistor being configured to remove carriers generated by absorption of photons of the excitation light in the photodiodes.
• GAS070.16 Preferably, the shunt transistor is configured to activate when each of the LEDs deactivate.
• GAS070.17 Preferably, the CMOS circuitry has memory storing identity data for identifying the test module to the external device.
• GAS070.18 Preferably, the array of photodiodes is less than 249 microns from the array of hybridization chambers.
• The easily usable, mass-producible, inexpensive, compact, and light microfluidic test module accepts a sample, analyzes the sample utilizing an integral discrete spectrometer with its integral image sensor and multiple-LED light source, and provides the results electronically at its output port.
• GAS080.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GAS080.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS080.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS080.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS080.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS080.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS080.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS080.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS080.9 Preferably, the luminophore is a metalorganic complex.
• GAS080.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS080.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS080.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS080.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS080.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS080.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS080.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS080.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the surface area optically coupled to the active surface area is greater than 50% of the active surface area of the photodiode.
• GAS080.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS080.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS080.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This enables more sensitive, and more specific, detection of target DNA.
• GAS081.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• • a supporting substrate;
  • probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) metalorganic complex; and,
  • electrodes for generating an excited state in the metalorganic complex in which the metalorganic complex emits photons of light; and,
  • CMOS circuitry between the supporting substrate and the probes for applying a voltage across the electrodes.
• GAS081.2 Preferably, the probes each have a functional moiety for quenching photon emission from the metalorganic complex by resonant energy transfer.
• GAS081.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the metalorganic complex is further from the metalorganic complex when the probe forms a probe-target hybrid.
• GAS081.4 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS081.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS081.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS081.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS081.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS081.9 Preferably, the metalorganic complex is a ruthenium organic complex.
• GAS081.10 Preferably, the LOC device also has an electrochemical coreactant that is present with the metalorganic complex during electrochemiluminescence.
• GAS081.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the metalorganic complex.
• GAS081.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS081.13 Preferably, the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS081.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS081.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the metalorganic complex, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS081.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the metalorganic complex to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS081.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the metalorganic complex, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS081.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS081.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS081.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. An advantage of this chemistry is that the reporter complex is not consumed during photon emission, which increases the signal level for a given reporter concentration.
• GAS082.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• a supporting substrate;
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and a ruthenium organic complex;
• electrodes for generating an excited state in the ruthenium organic complex in which the ruthenium organic complex emits photons of light; and,
• CMOS circuitry for applying a voltage across the electrodes.
• GAS082.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ruthenium organic complex by resonant energy transfer.
• GAS082.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ruthenium organic complex is further from the ruthenium organic complex when the probe forms a probe-target hybrid.
• GAS082.4 Preferably, the CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS082.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS082.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS082.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS082.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS082.9 Preferably, the photosensor has a planar active surface area for receiving the light from the ruthenium organic complex and the electrodes are thicker than 2 microns in a direction normal to the active surface area.
• GAS082.10 Preferably, the LOC device also has an electrochemical coreactant that is present with the ruthenium organic complex molecule during electrochemiluminescence.
• GAS082.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ruthenium organic complex molecule.
• GAS082.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS082.13 Preferably, the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS082.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS082.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the ruthenium organic complex, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS082.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the ruthenium organic complex molecule to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS082.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the ruthenium organic complex molecule, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS082.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS082.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS082.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. An advantage of this chemistry is that the reporter complex is not consumed during photon emission, which increases the signal level for a given reporter concentration.
• GAS083.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• an electrochemical coreactant that is present with the luminophore during electrochemiluminescence.
• GAS083.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS083.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS083.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS083.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS083.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS083.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS083.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS083.9 Preferably, the luminophore is a metalorganic complex.
• GAS083.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS083.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS083.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS083.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS083.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS083.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS083.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS083.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS083.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS083.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS083.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. Use of a coreactant increases the signal strength and decreases the voltage required to generate the electrochemiluminescent signal.
• GAS084.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• a supporting substrate;
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• CMOS circuitry between the supporting substrate and the probes for applying a voltage across the electrodes.
• GAS084.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS084.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS084.4 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS084.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS084.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS084.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS084.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS084.9 Preferably, the luminophore is a metalorganic complex.
• GAS084.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS084.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS084.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS084.13 Preferably, the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS084.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS084.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS084.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS084.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS084.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS084.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS084.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This increases the specificity of the detection of target molecules.
• GAS085.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• a supporting substrate;
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, an electrochemiluminescent (ECL) luminophore, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• CMOS circuitry between the supporting substrate and the probes for applying a voltage across the electrodes.
• GAS085.2 Preferably, the LOC device also has an electrochemical coreactant that is present with the luminophore during electrochemiluminescence.
• GAS085.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS085.4 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes. The LOC device according to claim
• GAS085.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS085.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS085.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS085.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS085.9 Preferably, the luminophore is a metalorganic complex.
• GAS085.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS085.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS085.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS085.13 Preferably, the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS085.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS085.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS085.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS085.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS085.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS085.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS085.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. An advantage conferred by the signal change based on complementarity is the ability to obtain a signal in a homogeneous format. No washing, additional sensitisation, or development steps are required to produce a signal whose level changes in the presence of the target.
• GAS086.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, an electrochemiluminescent (ECL) luminophore, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; wherein during use,
• the functional moiety changes proximity to the luminophore upon formation of a probe-target hybrid.
• GAS086.2 Preferably, the LOC device also has an electrochemical coreactant that is present with the luminophore during electrochemiluminescence.
• GAS086.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS086.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS086.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS086.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS086.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS086.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS086.9 Preferably, the luminophore is a metalorganic complex.
• GAS086.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS086.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS086.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS086.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS086.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS086.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS086.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS086.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS086.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS086.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS086.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This increases the specificity of the detection of target molecules. An advantage conferred by the signal change based on complementarity is the ability to obtain a signal in a homogeneous format. No washing, additional sensitisation, or development steps are required to produce a signal whose level changes in the presence of the target.
• GAS087.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• an array of hybridization chambers, each of the hybridization chambers having a pair of the electrodes respectively and containing a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS087.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS087.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS087.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS087.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS087.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS087.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS087.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS087.9 Preferably, the luminophore is a metalorganic complex.
• GAS087.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS087.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS087.12 Preferably, the LOC device also has an electrochemical coreactant that is present with the luminophore during electrochemiluminescence.
• GAS087.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS087.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS087.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS087.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS087.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS087.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS087.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS087.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This increases the specificity of the detection of target molecules. LOC device designs with parallel reaction sites enable parallel diagnostic tests to be performed on a very small sample volume, which increases the range and quality of diagnostic data obtained.
• GAS088.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS088.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS088.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS088.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS088.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS088.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS088.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS088.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS088.9 Preferably, the luminophore is a metalorganic complex.
• GAS088.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS088.11 Preferably, the CMOS circuitry incorporates the photosensor such that the photosensor is immediately adjacent the hybridization chambers.
• GAS088.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS088.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS088.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS088.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS088.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS088.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS088.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS088.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS088.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This enables more sensitive, and more specific, detection of target DNA.
• GAS089.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore; wherein,
• the electrodes are plates of conductive material, the plates having peripheral edges optically coupled to the photosensor.
• GAS089.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS089.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS089.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS089.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS089.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS089.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS089.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.9 microns and 2 microns wide.
• GAS089.9 Preferably, the luminophore is a metalorganic complex.
• GAS089.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS089.11 Preferably, the CMOS circuitry incorporates the photosensor such that the photosensor is immediately adjacent the hybridization chambers.
• GAS089.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS089.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS089.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS089.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS089.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS089.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS089.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS089.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS089.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS090.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore; wherein,
• the electrodes have at least one working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits the photons, the working electrode being positioned immediately adjacent the photosensor.
• GAS090.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS090.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS090.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS090.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS090.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS090.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS090.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS090.9 Preferably, the luminophore is a metalorganic complex.
• GAS090.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS090.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS090.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS090.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS090.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS090.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS090.16 Preferably, the working electrodes are plates of conductive material, the plates defining a series of fingers to increase the length of peripheral edge of the plate optically coupled to the photosensor.
• GAS090.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS090.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS090.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS090.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS091.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore; wherein,
• the photosensor has a planar active surface area for receiving the light from the ECL luminophore and the electrodes are between 0.25 micron and 2 microns thick in a direction normal to the planar active surface area of the photodiodes.
• GAS091.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS091.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS091.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS091.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS091.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS091.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS091.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS091.9 Preferably, the luminophore is a metalorganic complex.
• GAS091.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS091.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS091.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS091.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS091.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS091.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS091.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS091.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS091.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS091.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS091.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS092.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore; wherein,
• the electrodes are plates of conductive material, the plates having edge profiles configured such that the length of peripheral edge of each of the plates is greater than 128 microns.
• GAS092.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS092.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS092.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS092.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS092.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS092.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS092.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS092.9 Preferably, the luminophore is a metalorganic complex.
• GAS092.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS092.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS092.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS092.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS092.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS092.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS092.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS092.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS092.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS092.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS092.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS093.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; wherein,
• the electrodes have an anode and a cathode, each having fingers configured for mutual interdigitation.
• GAS093.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS093.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS093.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS093.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS093.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS093.7 Preferably, the LOC device also has an electrochemical coreactant that is present with the ECL luminophore during electrochemiluminescence.
• GAS093.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS093.9 Preferably, the luminophore is a metalorganic complex.
• GAS093.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS093.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS093.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS093.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS093.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS093.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS093.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS093.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS093.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS093.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS093.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS094.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrode pairs in which one electrode in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon; and,
• photodiodes corresponding to each of the electrode pairs respectively; wherein,
• the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS094.2 Preferably, both electrodes in one of the electrode pairs have fingers configured for mutual interdigitation.
• GAS094.3 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS094.4 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS094.5 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS094.6 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS094.7 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS094.8 Preferably, the working electrode is configured such that the optically coupled surface area is greater than 90% of the active surface area of the photodiode.
• GAS094.9 Preferably, the electrodes in each of the electrode pairs are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS094.10 Preferably, the luminophore is a metalorganic complex.
• GAS094.11 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS094.12 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS094.13 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers one of the electrode pairs respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS094.14 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photodiodes are adjacent the hybridization chambers.
• GAS094.15 Preferably, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS094.16 Preferably, each of the active surface areas are coplanar, and the electrodes are a layer of conductive material patterned to form separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS094.17 Preferably, the electrodes are between 0.25 micron and 2 microns thick in a direction normal to the planar active surface area of the photodiodes.
• GAS094.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS094.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS094.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS095.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; wherein,
• the electrodes are arranged in pairs, one of the electrodes in each electrode pair being a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photosensor and the working electrode.
• GAS095.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS095.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS095.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS095.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS095.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS095.7 Preferably, the electrode pairs have an anode and a cathode each having fingers configured for mutual interdigitation.
• GAS095.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS095.9 Preferably, the luminophore is a metalorganic complex.
• GAS095.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS095.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS095.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has one of the electrode pairs respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS095.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS095.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS095.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS095.16 Preferably, the LOC device also has an electrochemical coreactant that is present with the luminophore during electrochemiluminescence.
• GAS095.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS095.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS095.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS095.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS096.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting a target nucleic acid sequence in a sample, the LOC device comprising:
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; wherein,
• the electrodes are arranged in pairs, one of the electrodes in each electrode pair being a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being transparent to light at the wavelength of the photons emitted by the ECL luminophores.
• GAS096.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS096.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS096.4 Preferably, the LOC device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS096.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS096.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS096.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS096.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS096.9 Preferably, the luminophore is a metalorganic complex.
• GAS096.10 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS096.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS096.12 Preferably, the LOC device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS096.13 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS096.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS096.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS096.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS096.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the surface area optically coupled to the active surface area is greater than 50% of the active surface area of the photodiode.
• GAS096.18 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS096.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS096.20 Preferably, the LOC device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• An integrated photosensor has the advantage of higher optical efficiency than an off-chip sensor scheme. An integrated photosensor has the advantage of increased ease of synchronisation with other system events. An integrated photosensor has the advantage of decreasing the number of discrete components. Electrochemiluminescence has the advantage of efficient light generation at controlled locations in microfluidic environments. Furthermore, synchronisation with sensors is facilitated in comparison to techniques such as fluorescence. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device design has the advantage of increased coupling between the light emitting region and the photosensor.
• GAS097.1 This aspect of the invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• reagent reservoirs for adding reagents to the sample prior to detection of the target nucleic acid sequences;
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GAS097.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS097.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS097.4 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS097.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS097.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS097.7 Preferably, the electrodes have an anode and a cathode each having fingers configured for mutual interdigitation.
• GAS097.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS097.9 Preferably, the microfluidic device also has a supporting substrate for the CMOS circuitry, and a cap in which the reagent reservoirs are defined, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS097.10 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS097.11 Preferably, the CMOS circuitry incorporates a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS097.12 Preferably, the microfluidic device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS097.13 Preferably, the microfluidic device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS097.14 Preferably, the photosensor is an array of photodiodes in registration with the array of hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS097.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS097.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS097.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS097.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS097.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS097.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS098.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a fluid, the microfluidic device comprising:
• reagent reservoirs for adding reagents to the fluid prior to detection of the target molecules;
• probes for reaction with the target molecules to form probe-target hybrids, and an electrochemiluminescent (ECL) luminophore;
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; and,
• a photosensor for sensing the photons emitted from the ECL luminophore.
• GAS098.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS098.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target complex.
• GAS098.4 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS098.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS098.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS098.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS098.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS098.9 Preferably, the microfluidic device also has a supporting substrate for the CMOS circuitry, and a cap in which the reagent reservoirs are defined, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS098.10 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the fluid is required.
• GAS098.11 Preferably, the CMOS circuitry incorporates the photosensor such that the photosensor is immediately adjacent the hybridization chambers.
• GAS098.12 Preferably, the microfluidic device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, wherein the fluid is a biological sample and the targets are target nucleic acid sequences, the probes each having a nucleic acid sequence complementary to a respective one of the targets.
• GAS098.13 Preferably, the microfluidic device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS098.14 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS098.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS098.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS098.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS098.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS098.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS098.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS099.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer with a hybridization section that has an array of electrochemiluminescent (ECL) probes for hybridization with target nucleic acid sequences in a fluid, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes; and,
• a photosensor for detecting photons of light from the ECL probes that have hybridized.
• GAS099.2 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS099.3 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS099.4 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS099.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS099.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS099.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS099.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS099.9 Preferably, the microfluidic device also has a supporting substrate for the CMOS circuitry, and a cap in which the reagent reservoirs are defined, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS099.10 Preferably, the cap has reagent reservoirs for adding reagents to the sample prior to detection of the target nucleic acid sequences, the reagent reservoirs each having an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS099.11 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS099.12 Preferably, the microfluidic device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS099.13 Preferably, the microfluidic device also has a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS099.14 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS099.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS099.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS099.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS099.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS099.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS099.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The sample inlet permits the introduction of the sample into the microfluidic test module, delivering small sample quantities with high volumetric efficiency to the required sections of the microfluidic device. The probe hybridization section provides for analysis of the targets via hybridization.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS100.1 This aspect of the invention provides a microfluidic device for detecting a target nucleic acid sequence in a sample, the microfluidic device comprising:
• a sample inlet for receiving the sample;
• probes with a nucleic acid sequence complementary to the target nucleic acid sequence for forming probe-target hybrids, and an electrochemiluminescent (ECL) luminophore; and,
• electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light; wherein,
• the sample inlet draws the sample along a fluid flow-path leading to the probes by capillary action.
• GAS100.2 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS100.3 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS100.4 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS100.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS100.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS100.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS100.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS100.9 Preferably, the microfluidic device also has a supporting substrate for the CMOS circuitry, and a cap in which the reagent reservoirs are defined, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS100.10 Preferably, the cap has reagent reservoirs for adding reagents to the sample prior to detection of the target nucleic acid sequences, the reagent reservoirs each having an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS100.11 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS100.12 Preferably, the microfluidic device also has an array of hybridization chambers wherein each of the hybridization chambers has a pair of the electrodes respectively and contains a plurality of the probes, the nucleic acid sequence in the probes in each of the hybridization chambers being different to the nucleic acid sequence in at least one other hybridization chamber in the array such that a plurality of target nucleic acid sequences are detectable.
• GAS100.13 Preferably, the microfluidic device also has a photosensor for sensing the photons emitted from the ECL luminophore and a supporting substrate wherein the CMOS circuitry is positioned between the hybridization chambers and the supporting substrate such that the photosensor is adjacent the hybridization chambers.
• GAS100.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS100.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS100.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS100.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS100.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS100.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS100.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS101.1 This aspect of the invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer overlying the supporting substrate for processing the sample, the MST layer having an array of hybridization chambers, each containing electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes;
• an array of temperature sensors positioned such that at least one of the temperature sensors corresponds to each of the hybridization chambers respectively; and,
• heaters for heating each of the hybridization chambers; such that,
• output from the temperature sensors is used for feedback control of the heaters.
• GAS101.2 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating a photosensor.
• GAS101.3 Preferably, the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
• GAS101.4 Preferably, the CMOS circuitry has a digital memory for storing data relating to processing of the sample, the data including the probe details and location of each of the probes in the array.
• GAS101.5 Preferably, the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the sample.
• GAS101.6 Preferably, each of the hybridization chambers has a heater controlled by the CMOS circuitry for maintaining the probes and target nucleic acid sequences at a hybridization temperature.
• GAS101.7 Preferably, the photosensor is less than 1600 microns from the corresponding hybridization chamber.
• GAS101.8 Preferably, the hybridization chamber has a volume less than 900,000 cubic microns.
• GAS101.9 Preferably, the hybridization chamber has a volume less than 200,000 cubic microns.
• GAS101.10 Preferably, the hybridization chamber has a volume less than 40,000 cubic microns.
• GAS101.11 Preferably, the hybridization chamber has a volume less than 9000 cubic microns.
• GAS101.12 Preferably, the CMOS circuitry derives a single result from the photodiodes corresponding to the hybridization chambers that contain identical probes.
• GAS101.13 Preferably, the CMOS circuitry is configured to provide the electrode pairs with an excitation pulse having a duration less than 0.69 seconds.
• GAS101.14 Preferably, the photosensor is an array of photodiodes positioned such that each of the photodiodes corresponds to one of the hybridization chambers respectively.
• GAS101.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS101.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS101.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS101.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS101.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS101.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The probe hybridization section provides for analysis of the targets via hybridization. The temperature feedback control assures control of the temperature in the hybridization chambers for optimal hybridization temperature and the subsequent optimal detection temperature.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS102.1 This aspect of the invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• an array of hybridization chambers, each containing electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes; wherein,
• the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.
• GAS102.2 Preferably, the microfluidic device also has a photosensor for detecting the light emitted by the ECL probes, wherein the wall section is positioned between the ECL probes and the photosensor.
• GAS102.3 Preferably, the microfluidic device also has a supporting substrate for the photosensor and the array of hybridization chambers wherein the photosensor is between the hybridization chambers and the supporting substrate, and the wall section is a layer incorporating silicon dioxide.
• GAS102.4 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS102.5 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS102.6 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS102.7 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS102.8 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS102.9 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS102.10 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS102.11 Preferably, the microfluidic device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS102.12 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS102.13 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS102.14 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS102.15 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS102.16 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS102.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, and the working electrode has a surface area optically coupled to the active surface area of the photodiode, the working electrode being configured such that the optically coupled surface area is greater than 50% of the active surface area of the photodiode.
• GAS102.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS102.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS102.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The probe hybridization section provides for analysis of the targets via hybridization. The optically transparent hybridization chambers provide for the transmission of the electrochemiluminescence signals used for the detection of hybridization of the targets to the probes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS103.1 This aspect of the invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• an array of hybridization chambers, each of the hybridization chambers containing electrode pairs for receiving an electrical pulse and electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and excited by current between the electrodes; wherein,
• the mass of the probes in each of the hybridization chambers is less than 270 picograms.
• GAS103.2 Preferably, the mass of the probes in each of the hybridization chambers is less than 60 picograms.
• GAS103.3 Preferably, the mass of the probes in each of the hybridization chambers is less than 12 picograms.
• GAS103.4 Preferably, the mass of the probes in each of the hybridization chambers is less than 2.7 picograms.
• GAS103.5 Preferably, each of the hybridization chambers contains one of the electrode pairs respectively.
• GAS103.6 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.
• GAS103.7 Preferably, the microfluidic device also has a photosensor for detecting the light emitted by the ECL probes.
• GAS103.8 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS103.9 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS103.10 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS103.11 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS103.12 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS103.13 Preferably, the electrode pairs have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS103.14 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS103.15 Preferably, the microfluidic device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS103.16 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS103.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS103.18 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• GAS103.19 Preferably, the PCR section has a heater element for thermal cycling the target nucleic acid sequences with polymerase, the heater element being configured for operative control by the CMOS circuitry.
• GAS103.20 Preferably, the microfluidic device also has a plurality of sensors connected to the CMOS circuitry for feedback control of the electrodes and the heater element.
• The low probe volume provides for low probe cost, in turn, permitting the inexpensive assay system. The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS104.1 This aspect of the invention provides a microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• an array of hybridization chambers, each containing electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes; wherein,
• the hybridization chambers each have a volume less than 900000 cubic microns.
• GAS104.2 Preferably, the hybridization chambers each have a volume less than 200000 cubic microns.
• GAS104.3 Preferably, the hybridization chambers each have a volume less than 40000 cubic microns.
• GAS104.4 Preferably, the hybridization chambers each have a volume less than 9000 cubic microns.
• GAS104.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.
• GAS104.6 Preferably, the microfluidic device also has a photosensor for detecting the light emitted by the ECL probes, wherein the wall section is positioned between the ECL probes and the photosensor.
• GAS104.7 Preferably, the microfluidic device also has a supporting substrate for the photosensor and the array of hybridization chambers wherein the photosensor is between the hybridization chambers and the supporting substrate, and the wall section is a layer incorporating silicon dioxide.
• GAS104.8 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS104.9 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS104.10 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS104.11 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS104.12 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS104.13 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS104.14 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS104.15 Preferably, the microfluidic device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS104.16 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS104.17 Preferably, the layer is less than 1600 microns thick.
• GAS104.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS104.19 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS104.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The low-volume hybridization chambers, in part, provide for the low probe volumes, which in turn provide for low probe cost and the inexpensive assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS105.1 This aspect of the invention provides a microfluidic device for amplifying and detecting target nucleic acid sequences in a sample, the microfluidic device comprising:
• a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences; and,
• a hybridization section that has an array of electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, and a plurality of electrodes for receiving an electrical pulse such that the probe-target hybrids emit a photon of light.
• GAS105.2 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS105.3 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS105.4 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS105.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS105.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS105.7 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS105.8 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.
• GAS105.9 Preferably, the microfluidic device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS105.10 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS105.11 Preferably, the microfluidic device also has an array of hybridization chambers for containing the ECL probes and a pair of the electrodes.
• GAS105.12 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.
• GAS105.13 Preferably, the microfluidic device also has a photosensor for detecting the light emitted by the ECL probes, wherein the wall section is positioned between the ECL probes and the photosensor.
• GAS105.14 Preferably, the microfluidic device also has a supporting substrate for the photosensor and the array of hybridization chambers wherein the photosensor is between the hybridization chambers and the supporting substrate, and the wall section is a layer incorporating silicon dioxide.
• GAS105.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS105.16 Preferably, the array of photodiodes is less than 1600 microns from the hybridization chambers.
• GAS105.17 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS105.18 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS105.19 Preferably, the PCR section has a microchannel extending between a PCR inlet and a PCR outlet, the microchannel being configured to draw the sample the PCR inlet to the PCR outlet by capillary action.
• GAS105.20 Preferably, the microchannel has a plurality of elongate heaters, each of the plurality of elongate heaters being independently operable.
• The PCR section, via the amplification of the target, provides the requisite sensitivity for target detection. The probe hybridization section provides for analysis of the targets via hybridization. The integrated PCR and probe hybridization sections substantially reduce the possibility of the introduction of contaminants into the assay, simplify the analysis stages, and provide for a small, light, and inexpensive single-device analytical solution.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS106.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a fluid, the microfluidic device comprising:
• an array of chambers, each containing electrochemiluminescent (ECL) probes for reaction with the target molecules to form a probe-target complex, and electrodes positioned in each of the chambers for receiving an electrical pulse, the probe-target complexes being configured to emit a photon of light when excited by current between the electrodes;
• a flow-path for the fluid containing the targets; wherein,
• each of the chambers have a chamber inlet for fluid communication between the sample flow-path and the probes within the chamber, the chamber inlet being configured as a diffusion barrier to prevent diffusion of the probe-target complexes between the chambers to an extent that causes erroneous detection results.
• GAS106.2 Preferably, the chamber inlet defines a tortuous flow-path.
• GAS106.3 Preferably, the tortuous flow-path has a serpentine configuration.
• GAS106.4 Preferably, the fluid is a biological sample and the targets are nucleic acid sequences, the probes each having a nucleic acid sequence complementary to one of the targets and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids
• GAS106.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS106.6 Preferably, the microfluidic device also has a photosensor for detecting the light emitted by the probes, wherein the wall section is positioned between the probes and the photosensor.
• GAS106.7 Preferably, the microfluidic device also has a supporting substrate for the photosensor and the array of hybridization chambers wherein the photosensor is between the hybridization chambers and the supporting substrate, and the wall section is a layer incorporating silicon dioxide.
• GAS106.8 Preferably, the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS106.9 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS106.10 Preferably, the microfluidic device also has CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS106.11 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS106.12 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS106.13 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS106.14 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS106.15 Preferably, the microfluidic device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• GAS106.16 Preferably, the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.
• GAS106.17 Preferably, the layer is less than 1600 microns thick.
• GAS106.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS106.19 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS106.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The probe hybridization section provides for analysis of the targets via hybridization. The diffusion barrier virtually eliminates a backflow of the probes, both before and after hybridization, preventing the loss of signal and providing high assay sensitivity.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS108.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a fluid, the microfluidic device comprising:
• an array of chambers containing electrochemiluminescent (ECL) probes for reaction with the target molecules to form a probe-target complexes;
• electrodes positioned in each of the chambers for receiving an electrical pulse, the probe-target complexes being configured to emit a photon of light when excited by current between the electrodes; and,
• a photosensor for detecting the light emitted by the probes; wherein,
• the photosensor is less than 1600 microns from the probes.
• GAS108.2 Preferably, the mass of the probes in each of the chambers is less than 270 picograms.
• GAS108.3 Preferably, the mass of the probes in each of the chambers is less than 60 picograms.
• GAS108.4 Preferably, the mass of the probes in each of the chambers is less than 12 picograms.
• GAS108.5 Preferably, the microfluidic device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse; and,
• a flow-path for the fluid containing the targets; wherein,
• the CMOS is between the chambers and the supporting substrate and the flow-path draws the fluid to each of the chambers by capillary action.
• GAS108.6 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids.
• GAS108.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS108.8 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS108.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS108.10 Preferably, the probes each have an ECL luminophore that emits a photon when excited by current between the electrodes, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS108.11 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS108.12 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS108.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS108.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS108.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS108.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS108.17 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS108.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS108.19 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS108.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The nonimaging optics provide for a mass-producible inexpensive integrated solution with low system component-count and provides for a compact, light, and highly portable system. The nonimaging optics increase the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS109.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a fluid, the microfluidic device comprising:
• a hybridization chamber containing electrodes and probes for hybridization with target molecules to form probe-target complexes and generate an electrochemiluminescence (ECL) signal in response to an electrical current between the electrodes; and,
• a photodiode with an active area and an optical axis extending normal to the active area and through the hybridization chamber; wherein,
• the hybridization chamber has a floor surface positioned parallel to the active area of the photodiode, the floor surface having a centroid and the active area being encompassed within a cone having the centroid at its vertex, and a vertex angle less than 174°.
• GAS109.2 Preferably, the vertex angle is less than 29 degrees.
• GAS109.3 Preferably, the vertex angle is less than 4.8 degrees.
• GAS109.4 Preferably, the vertex angle is less than 0.8 degrees.
• GAS109.5 Preferably, the microfluidic device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse;
• an array of the hybridization chambers; and,
• a flow-path for the fluid containing the target molecules; wherein,
• the CMOS circuitry is between the chambers and the supporting substrate and the flow-path draws the fluid to each of the chambers by capillary action.
• GAS109.6 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids
• GAS109.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS109.8 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS109.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS109.10 Preferably, the probes each have an ECL luminophore that emits a photon when excited by current between the electrodes, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS109.11 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS109.12 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS109.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS109.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS109.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS109.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS109.17 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS109.18 Preferably, the photosensor is an array of the photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS109.19 Preferably, each of the active areas are coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active areas of the photodiodes.
• GAS109.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The large emission light angle of collection provides for a mass-producible inexpensive integrated solution with low system component-count and provides for a compact, light, and highly portable system. The large emission light angle of collection increases the readout sensitivity that obviates the need for optical components in the optical collection train.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS110.1 This aspect of the invention provides a microfluidic device for detecting target molecules in a fluid, the microfluidic device comprising:
• an array of electrochemiluminescent (ECL) probes for reaction with the target molecules to form a probe-target complexes;
• electrodes for receiving an electrical pulse, the probe-target complexes being configured to emit a photon of light when excited by current between the electrodes; and,
• a photosensor for detecting the light emitted by the probes; wherein during use,
• addition of the fluid to the probes prevents subsequent addition of other fluid to the probes.
• GAS110.2 Preferably, the microfluidic device also has an array of chambers wherein each of the chambers contains a pair of the electrodes and the probes for one of the targets respectively, wherein the fluid fills each of the chambers by capillary action.
• GAS110.3 Preferably, the chambers each have a volume less than 900000 cubic microns.
• GAS110.4 Preferably, the photosensor is less than 1600 microns from the probes.
• GAS110.5 Preferably, the microfluidic device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse; and,
• a flow-path for the fluid containing the targets; wherein,
• the CMOS is between the chambers and the supporting substrate and the flow-path draws the fluid to each of the chambers by capillary action.
• GAS110.6 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids
• GAS110.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS110.8 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS110.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS110.10 Preferably, the probes each have an ECL luminophore that emits a photon when excited by current between the electrodes, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS110.11 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS110.12 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS110.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS110.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS110.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS110.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS110.17 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS110.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS110.19 Preferably, the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS110.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The easily usable, mass-producible, inexpensive, compact, and light microfluidic device accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS111.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target molecules in a fluid, the LOC device comprising:
• an array of electrochemiluminescent (ECL) probes for reaction with the target molecules to form probe-target complexes;
• electrodes for receiving an electrical pulse, the probe-target complexes being configured to emit a photon of light when excited by current between the electrodes; and,
• a photosensor for detecting light emitted by the probe-target complexes.
• GAS111.2 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse;
• an array of chambers, each containing the probes for reaction with the target molecules to form a probe-target complex, and a pair of the electrodes; and,
• a flow-path for the fluid containing the targets; wherein,
• the CMOS is between the chambers and the supporting substrate and the flow-path draws the fluid to each of the chambers by capillary action.
• GAS111.3 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids
• GAS111.4 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS111.5 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS111.6 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS111.7 Preferably, the probes each have an ECL luminophore that emits a photon when excited by current between the electrodes, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS111.8 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS111.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS111.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS111.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS111.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS111.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS111.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS111.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS111.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS111.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS111.18 Preferably, the luminophore is a metalorganic complex.
• GAS111.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS111.20 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS112.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for exciting electrochemiluminescent luminophores, the LOC device comprising:
• a supporting substrate;
• an array of electrochemiluminescent (ECL) probes for reaction with the target molecules to form probe-target complexes, the probes each having a luminophore for emitting photons of light when in an excited state;
• electrodes for receiving an electrical pulse to excite the luminophores with current between the electrodes; and,
• CMOS circuitry for controlling the electrical pulse transmitted to the electrodes; wherein,
• the CMOS circuitry is between the supporting substrate and the array of probes.
• GAS112.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the probe-target complexes;
• an array of chambers, each containing the probes for reaction with the target molecules to form a probe-target complex, and a pair of the electrodes; and,
• a flow-path for the fluid containing the targets; wherein,
• the flow-path draws the fluid to each of the chambers by capillary action.
• GAS112.3 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids
• GAS112.4 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS112.5 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS112.6 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS112.7 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS112.8 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS112.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS112.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS112.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS112.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS112.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS112.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS112.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS112.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS112.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS112.18 Preferably, the luminophore is a metalorganic complex.
• GAS112.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS112.20 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• The integral driver for excitation of electrochemiluminescent luminophores on the LOC device, operating from the ubiquitous USB, provides for an easily usable, mass-producible, inexpensive, compact, and light system with a small component count.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS113.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target molecules in a fluid, the LOC device comprising:
• an array of electrochemiluminescent (ECL) probes for reaction with the target molecules to form probe-target complexes; and,
• electrodes for receiving an electrical pulse; wherein,
• the probes each have an ECL luminophore that emits a photon when excited by current between the electrodes, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GAS113.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the probe-target complexes.
• GAS113.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse;
• an array of chambers, each containing the probes for reaction with the target molecules to form a probe-target complex, and a pair of the electrodes; and,
• a flow-path for the fluid containing the targets; wherein,
• the CMOS is between the chambers and the supporting substrate, and the flow-path draws the fluid to each of the chambers by capillary action.
• GAS113.4 Preferably, the fluid is a biological sample and the target is a nucleic acid sequence, the probes each having a nucleic acid sequence complementary to the target and the chambers are hybridization chambers for hybridization of the probes to form probe-target hybrids.
• GAS113.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS113.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS113.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS113.8 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS113.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS113.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS113.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS113.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS113.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS113.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS113.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS113.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS113.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS113.18 Preferably, the luminophore is a metalorganic complex.
• GAS113.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS113.20 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, identifies the sample's nucleic acid sequences via hybridization with electrochemiluminescence resonance energy transfer probes using its integral image sensor, and provides the results electronically at its output pads, with the electrochemiluminescence resonance energy transfer probes providing high specificity and high reliability of detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS114.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting hybridization of target nucleic acid sequences, the LOC device comprising:
• electrochemiluminescent (ECL), resonant energy transfer, primer-linked, stem-and-loop probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, each of the probes having a loop portion containing the target nucleic acid sequence, a primer for extension along the target nucleic acid sequence to form a nucleic acid sequence complementary to the target, an ECL luminophore for emitting photons when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein during use,
• forming the complementary nucleic acid sequence causes the loop portion to open such the target nucleic acid sequence therein hybridizes to the complementary nucleic acid sequence and the ECL luminophore is moved away from the functional moiety.
• GAS114.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores when in the probe-target hybrid configuration.
• GAS114.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse;
• hybridization chambers containing the probes for hybridization with the targets, and a pair of the electrodes; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS114.4 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS114.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS114.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS114.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS114.8 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS114.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS114.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS114.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS114.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS114.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS114.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS114.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS114.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS114.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS114.18 Preferably, the luminophore is a metalorganic complex.
• GAS114.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS114.20 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with electrochemiluminescence resonance energy transfer primer-linked stem-and-loop probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked stem-and-loop probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS115.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting hybridization of target nucleic acid sequences, the LOC device comprising:
• electrochemiluminescent (ECL), resonant energy transfer, primer-linked, linear probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, each of the probes having a linear portion containing the target nucleic acid sequence, a primer for extension along the target nucleic acid sequence to form a nucleic acid sequence complementary to the target, an ECL luminophore for emitting photons when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein during use,
• replicating the target nucleic acid sequence causes the linear portion to dissociate from the functional moiety such that the complementary nucleic acid sequence therein hybridizes to the target nucleic acid sequence and photons emitted by the ECL luminophore are not quenched.
• GAS115.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores.
• GAS115.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse;
• hybridization chambers containing the probes for hybridization with the targets, and a pair of the electrodes; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS115.4 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS115.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS115.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS115.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS115.8 Preferably, the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GAS115.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS115.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS115.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS115.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS115.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS115.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS115.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS115.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS115.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS115.18 Preferably, the luminophore is a metalorganic complex.
• GAS115.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS115.20 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the sample.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with electrochemiluminescence resonance energy transfer primer-linked linear probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked linear probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS117.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for amplifying and detecting target nucleic acid sequences, the LOC device comprising:
• electrochemiluminescent (ECL), resonant energy transfer, stem-and-loop probes for hybridization with the target nucleic acid sequences, each of the probes having a loop portion containing a sequence complementary to the target nucleic acid sequence, an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, and a covalently attached primer for extension along a complementary sequence denatured from the target nucleic acid sequence to replicate the target nucleic acid sequence;
• heaters for thermally cycling the target nucleic acid sequences through a polymerase chain reaction (PCR), in which the covalently attached primers anneal to oligonucleotides containing the target nucleic acid sequences; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein during use,
• replicating the target nucleic acid sequence causes the loop portion to open such the complementary nucleic acid sequence therein hybridizes to the target nucleic acid sequence and the ECL luminophore is moved away from the functional moiety.
• GAS117.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores.
• GAS117.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for activating the heaters and providing the electrodes with an electrical pulse;
• hybridization chambers containing the probes for hybridization with the targets, at least one of the heaters and a pair of the electrodes; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS117.4 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS117.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS117.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS117.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS117.8 Preferably, the covalently attached primer is attached to the functional moiety for quenching photon emission from the ECL luminophore and the loop portion is between the ECL luminophore and the functional moiety.
• GAS117.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS117.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS117.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS117.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS117.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS117.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS117.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS117.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS117.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS117.18 Preferably, the luminophore is a metalorganic complex.
• GAS117.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS117.20 Preferably, the LOC device also has a dialysis section for diverting the cells smaller than a predetermined size threshold into a separate stream.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with electrochemiluminescence resonance energy transfer primer-linked stem-and-loop probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked stem-and-loop probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS118.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for amplifying and detecting target nucleic acid sequences, the LOC device comprising:
• electrochemiluminescent (ECL), resonant energy transfer, linear probes for hybridization with the target nucleic acid sequences, each of the probes having a linear portion containing a sequence complementary to the target nucleic acid sequence, an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, and a covalently attached primer for extension along a complementary sequence denatured from the target nucleic acid sequence to replicate the target nucleic acid sequence;
• heaters for thermally cycling the target nucleic acid sequences through a polymerase chain reaction (PCR), in which the covalently attached primers anneal to oligonucleotides containing the target nucleic acid sequences; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein during use,
• replicating the target nucleic acid sequence causes the linear portion to dissociate from the functional moiety such that the complementary nucleic acid sequence therein hybridizes to the target nucleic acid sequence and photons emitted by the ECL luminophore are not quenched.
• GAS118.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores.
• GAS118.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for activating the heaters and providing the electrodes with an electrical pulse;
• hybridization chambers containing the probes for hybridization with the targets, at least one of the heaters and a pair of the electrodes; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS118.4 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS118.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS118.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS118.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS118.8 Preferably, the probe has a PCR blocker moiety between the covalently attached primer and the linear portion.
• GAS118.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS118.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS118.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS118.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS118.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS118.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS118.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS118.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS118.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS118.18 Preferably, the luminophore is a metalorganic complex.
• GAS118.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS118.20 Preferably, the LOC device also has a dialysis section for diverting the cells smaller than a predetermined size threshold into a separate stream.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via hybridization with electrochemiluminescence resonance energy transfer primer-linked linear probes using its integral image sensor, and provides the results electronically at its output pads, with the primer-linked linear probes providing for a large number of optimal parallel amplification reactions to be run, also providing for high specificity, sensitivity, and reliability of detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS119.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for amplifying and detecting target nucleic acid sequences, the LOC device comprising:
• restriction enzymes for restricting double stranded oligonucleotides at known ligation sites;
• linker molecules for attaching to the ligated ends of the double stranded oligonucleotides;
• heaters for thermally cycling the oligonucleotides through a polymerase chain reaction (PCR) process;
• primers for annealing to the linker molecules on single strands of the oligonucleotides after denaturation;
• deoxyribonucleoside triphosphates (dNTPs) for extending the primers along the single strand oligonucleotides; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS119.2 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores.
• GAS119.3 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for activating the heaters and providing the electrodes with an electrical pulse;
• hybridization chambers containing the probes for hybridization with the targets, at least one of the heaters and a pair of the electrodes; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS119.4 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS119.5 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS119.6 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS119.7 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS119.8 Preferably, the probe has a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS119.9 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS119.10 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS119.11 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS119.12 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS119.13 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS119.14 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS119.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS119.16 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS119.17 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• GAS119.18 Preferably, the luminophore is a metalorganic complex.
• GAS119.19 Preferably, the metalorganic complex is a ruthenium organic complex molecule.
• GAS119.20 Preferably, the LOC device also has a dialysis section for diverting the cells smaller than a predetermined size threshold into a separate stream.
• The easily usable, mass-producible, inexpensive, compact, and light LOC device accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output pads, with the adaptor primers providing the capability for genome-scale amplification and analysis.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS120.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL), resonance energy transfer probes for hybridization with the target nucleic acid sequences, each of the probes containing a sequence complementary to the target nucleic acid sequence, and an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores; hybridization chambers containing the probes for hybridization with the targets, at least one of the heaters and a pair of the electrodes; and,
• a reagent reservoir containing a reagent for addition to the fluid; wherein,
• the hybridization chambers each have a volume less than 900,000 cubic microns and the reagent reservoir has a volume less than 1,000,000,000 cubic microns.
• GAS120.2 Preferably, the hybridization chambers each have a volume less than 200,000 cubic microns and the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GAS120.3 Preferably, the hybridization chambers each have a volume less than 40,000 cubic microns and the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GAS120.4 Preferably, the hybridization chambers each have a volume less than 9000 cubic microns and the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GAS120.5 Preferably, the LOC device also has:
• a photosensor for detecting light emitted by the ECL luminophores.
• GAS120.6 Preferably, the LOC device also has:
• a supporting substrate;
• CMOS circuitry for providing the electrodes with an electrical pulse; and,
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS120.7 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS120.8 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS120.9 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS120.10 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS120.11 Preferably, the probe has a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS120.12 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS120.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS120.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS120.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS120.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS120.17 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS120.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS120.19 Preferably, the photodiodes each have a planar active surface area such that the planar active surface areas collectively provide the collection surface, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.
• GAS120.20 Preferably, one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.
• The low-volume hybridization chambers and reagent reservoirs, in part, provide for the low probe and reagent volumes, which in turn provide for low probe and reagent costs and the inexpensive assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS121.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one positive control probe that has one of the ECL luminophores but not the functional moiety for quenching photon emission;
• at least one negative control probe that does not have one of the ECL luminophores; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS121.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the photons in response to the electrical pulse.
• GAS121.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS121.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes.
• GAS121.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS121.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense an ECL signal from the positive control probe or in response to sensing a signal from the negative control probe.
• GAS121.7 Preferably, the LOC device also has hybridization chambers containing the probes and a pair of the electrodes.
• GAS121.8 Preferably, the LOC device also has:
• • a flow-path for fluid containing the targets; wherein,
  • the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS121.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS121.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS121.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS121.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS121.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS121.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS121.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS121.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS121.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS121.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS121.19 Preferably, the transparent wall section is less than 1600 microns thick.
• GAS121.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS122.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one positive control probe for detecting a nucleic acid sequence known to be always present in the fluid; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS122.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS122.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS122.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes.
• GAS122.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS122.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to ECL emissions not being detected from the positive control probe.
• GAS122.7 Preferably, the LOC device also has hybridization chambers containing the probes and a pair of the electrodes.
• GAS122.8 Preferably, the LOC device also has:
• • a flow-path for fluid containing the targets; wherein,
  • the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS122.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS122.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS122.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS122.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS122.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS122.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS122.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS122.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS122.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS122.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS122.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences.
• GAS122.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS123.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one positive control probe that has the ECL luminophore but not the functional moiety for quenching photon emission; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS123.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS123.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS123.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the probes.
• GAS123.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS123.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to ECL emissions being detected from the negative control probe.
• GAS123.7 Preferably, the LOC device also has hybridization chambers containing the probes and a pair of the electrodes.
• GAS123.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS123.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS123.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS123.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS123.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS123.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS123.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS123.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS123.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS123.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS123.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS123.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS123.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS124.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• hybridization chambers containing the probes for detection of the targets, and a pair of the electrodes; and,
• at least one positive control chamber containing positive control probes that have the ECL luminophore but not the functional moiety for quenching photon emission.
• GAS124.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS124.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS124.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS124.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS124.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense ECL emissions from the positive control probe.
• GAS124.7 Preferably, the LOC device also has at least one negative control chamber containing negative control probes that are incapable of hybridization with any nucleic acid sequences in the fluid.
• GAS124.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS124.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS124.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS124.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS124.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS124.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS124.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS124.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS124.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS124.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS124.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS124.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS124.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS125.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• hybridization chambers containing the probes for detection of the targets, and a pair of the electrodes; and,
• at least one negative control chamber containing negative control probes that are incapable of hybridization with any nucleic acid sequences in the fluid.
• GAS125.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS125.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS125.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS125.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS125.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to ECL emissions being detected from the negative control chamber.
• GAS125.7 Preferably, the LOC device also has at least one positive control chamber containing positive control probes that have the ECL luminophore but not the functional moiety for quenching photon emission.
• GAS125.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS125.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS125.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS125.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS125.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS125.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS125.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS125.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS125.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS125.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS125.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS125.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS125.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS126.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• hybridization chambers containing the probes for detection of the targets, and a pair of the electrodes; and,
• at least one negative control chamber containing negative control probes without an ECL luminophore.
• GAS126.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS126.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS126.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS126.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS126.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to ECL emissions being detected from the negative control chamber.
• GAS126.7 Preferably, the LOC device also has at least one positive control chamber containing positive control probes that have the ECL luminophore but not the functional moiety for quenching photon emission.
• GAS126.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS126.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS126.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS126.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS126.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS126.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS126.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS126.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS126.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS126.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS126.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS126.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS126.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS127.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• hybridization chambers containing the probes for detection of the targets, and a pair of the electrodes; and,
• at least one negative control chamber without the ECL probes.
• GAS127.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the ECL photons in response to the electrical pulse.
• GAS127.3 Preferably, the LOC device also has a supporting substrate wherein the photosensor is a charge coupled device (CCD) array positioned between the probes and the supporting substrate.
• GAS127.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS127.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS127.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to ECL emissions being detected from the negative control chamber.
• GAS127.7 Preferably, the LOC device also has at least one positive control chamber containing positive control probes that have the ECL luminophore but not the functional moiety for quenching photon emission.
• GAS127.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS127.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS127.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS127.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS127.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS127.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS127.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS127.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS127.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS127.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS127.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS127.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS127.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the control probes improving the reliability of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS128.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• detection photodiodes for exposure to the photons emitted by the ECL luminophores; and,
• at least one calibration photodiode for exposure to ambient light; wherein during use,
• a difference between the output from any of the detection photodiode and the output from the calibration photodiode is compared to a predetermined threshold difference such that output differences greater than the predetermined threshold indicate the target is present.
• GAS128.2 Preferably, the LOC device also has:
• hybridization chambers containing the probes for detection of the targets, and a pair of the electrodes, the detection photodiodes being positioned in registration with each of the hybridization chambers respectively; and,
• a calibration chamber positioned adjacent the calibration photodiode.
• GAS128.3 Preferably, the LOC device also has a plurality of the calibration chambers and a corresponding plurality of the calibration photodiodes distributed throughout the hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS128.4 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS128.5 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS128.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the detection and calibration photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry has a comparator circuit for determining the output difference between each of the detection photodiodes and the calibration photodiode most proximate.
• GAS128.7 Preferably, the calibration chamber has probes lacking the ECL luminophore but including the functional moiety for quenching photon emission.
• GAS128.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS128.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS128.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS128.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS128.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS128.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS128.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS128.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS128.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS128.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS128.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS128.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS128.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration photosensor improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS129.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• hybridization chambers containing electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one calibration chamber containing ECL probes designed to be non-complementary to any nucleic acid sequence in the fluid; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS129.2 Preferably, the LOC device also has:
• detection photodiodes for exposure to the photons emitted by the ECL luminophores; and,
• at least one calibration photodiode for exposure to ambient light; wherein during use,
• a difference between the output from any of the detection photodiode and the output from the calibration photodiode is compared to a predetermined threshold difference such that output differences greater than the predetermined threshold indicate the target is present.
• GAS129.3 Preferably, the LOC device also has a plurality of the calibration chambers and a corresponding plurality of the calibration photodiodes distributed throughout the hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS129.4 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS129.5 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS129.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the detection and calibration photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry has a comparator circuit for determining the output difference between each of the detection photodiodes and the calibration photodiode most proximate.
• GAS129.7 Preferably, the calibration chamber has probes lacking the ECL luminophore but including the functional moiety for quenching photon emission.
• GAS129.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS129.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS129.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS129.11 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS129.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS129.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS129.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS129.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS129.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS129.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS129.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS129.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS129.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS130.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• calibration probes without an ECL luminophore; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS130.2 Preferably, the LOC device also has:
• detection photodiodes for exposure to the photons emitted by the ECL luminophores; and,
• at least one calibration photodiode for exposure to the calibration probes; wherein during use,
• a difference between the output from any of the detection photodiode and the output from the calibration photodiode is compared to a predetermined threshold difference such that output differences greater than the predetermined threshold indicate the target is present.
• GAS130.3 Preferably, the LOC device also has an array of calibration chambers for containing the ECL probes and a plurality of calibration chambers distributed throughout the array of hybridization chambers, each of the calibration chambers containing the calibration probes and each of the calibration chambers having a corresponding calibration photodiode wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS130.4 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS130.5 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS130.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the detection and calibration photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry has a comparator circuit for determining the output difference between each of the detection photodiodes and the calibration photodiode most proximate.
• GAS130.7 Preferably, the calibration probes include the functional moiety for quenching photon emission.
• GAS130.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS130.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS130.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS130.11 Preferably, the CMOS circuitry incorporates the photodiode wherein the wall section is positioned between the probes and the photodiode.
• GAS130.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS130.13 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS130.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS130.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS130.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS130.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS130.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS130.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS130.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS131.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• hybridization chambers containing electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one calibration chamber containing probes sealed from contact with the fluid; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores.
• GAS131.2 Preferably, the LOC device also has:
• detection photodiodes for exposure to the photons emitted by the ECL luminophores; and,
• at least one calibration photodiode for exposure to the calibration chamber; wherein during use,
• a difference between the output from any of the detection photodiode and the output from the calibration photodiode is compared to a predetermined threshold difference such that output differences greater than the predetermined threshold indicate the target is present.
• GAS131.3 Preferably, the LOC device also has a plurality of the calibration chambers distributed throughout the array of hybridization chambers, each of the calibration chambers having a corresponding calibration photodiode wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS131.4 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS131.5 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS131.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the detection and calibration photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry has a comparator circuit for determining the output difference between each of the detection photodiodes and the calibration photodiode most proximate.
• GAS131.7 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets wherein the hybridization chambers each have an inlet for fluid communication between the flow-path and the probes and the at least one calibration chamber is sealed from the flow-path.
• GAS131.8 Preferably,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS131.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS131.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS131.11 Preferably, the CMOS circuitry incorporates the photodiode wherein the wall section is positioned between the probes and the photodiode.
• GAS131.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS131.13 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS131.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS131.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS131.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS131.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS131.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS131.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS131.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration probes improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS132.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• at least one calibration source configured to generate a calibration emission;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• at least one detection photodiode for sensing photon emissions from the ECL luminophores and generating a detection output;
• at least one calibration photodiode for sensing the calibration emission and generating a calibration output; and,
• CMOS circuitry having a differential circuit for subtracting the calibration output from the detection output.
• GAS132.2 Preferably, the LOC device also has a plurality of the calibration sources and a corresponding plurality of the calibration photodiodes, and a plurality of the detection photodiodes in registration with each of the ECL probes respectively.
• GAS132.3 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS132.4 Preferably, the LOC device also has an array of hybridization chambers containing the ECL probes and a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS132.5 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS132.6 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS132.7 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS132.8 Preferably, the calibration probes include the functional moiety for quenching photon emission.
• GAS132.9 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS132.10 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cellular membranes to release any genetic material therein.
• GAS132.11 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.
• GAS132.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS132.13 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS132.14 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS132.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS132.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS132.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS132.18 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS132.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS132.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the fluid prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.
• The hybridization array provides for analysis of the targets via hybridization, with the calibration circuit improving the reliability, sensitivity, and dynamic range of the analytical outcomes.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS133.1 This aspect of the invention provides a microfluidic test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing configured for hand-held portability, the outer casing having an inlet for receiving the fluid containing the target nucleic acid sequences;
• a hybridization chamber mounted in the casing, the hybridization chamber containing electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, and electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein,
• the hybridization chamber has a volume less than 900,000 cubic microns.
• GAS133.2 Preferably, the hybridization chamber has a volume less than 200,000 cubic microns.
• GAS133.3 Preferably, the hybridization chamber has a volume less than 40,000 cubic microns.
• GAS133.4 Preferably, the hybridization chamber has a volume less than 9,000 cubic microns.
• GAS133.5 Preferably, the microfluidic test module also has:
• a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry for providing the electrical pulse to the electrodes.
• GAS133.6 Preferably, the microfluidic test module also has:
• • a communication interface for the control circuitry to transmit data to an external device.
• GAS133.7 Preferably, the microfluidic test module also has an array of the hybridization chambers containing ECL probes for different target nucleic acid sequences wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS133.8 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS133.9 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS133.10 Preferably, the microfluidic test module also has:
• at least one calibration source for providing a calibration emission, and a calibration photosensor for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photosensor output from the detection photosensor output.
• GAS133.11 Preferably, the microfluidic test module also has a plurality of the calibration sources wherein the detection photosensor is an array of photodiodes in registration with each of the ECL probes respectively and the calibration photosensor is a plurality of calibration photodiodes in registration with the calibration sources respectively.
• GAS133.12 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS133.13 Preferably, the microfluidic test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS133.14 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS133.15 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS133.16 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS133.17 Preferably, the calibration probes include the functional moiety for quenching photon emission.
• GAS133.18 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS133.19 Preferably, the electrical pulse to the electrodes is a DC pulse and has a duration less than 0.69 seconds.
• GAS133.20 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• The low-volume hybridization chambers, in part, provide for the low probe volumes, which in turn provide for low probe cost and the inexpensive assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS134.1 This aspect of the invention provides a microfluidic test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing having an inlet for receiving the fluid containing the target nucleic acid sequences;
• electrode pairs for receiving an electrical pulse;
• electrochemiluminescent (ECL) probe spots in contact with each of the electrode pairs respectively, the ECL probe spots containing ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, such that the electrical pulse to the electrode pair excites the ECL luminophores; wherein,
• the mass of the ECL probes in each of the probe spots is less than 270 picograms.
• GAS134.2 Preferably, the mass of the probes in each of the probe spots is less than 60 picograms.
• GAS134.3 Preferably, the mass of the probes in each of the probe spots is less than 12 picograms.
• GAS134.4 Preferably, the mass of the probes in each of the probe spots is less than 2.7 picograms.
• GAS134.5 Preferably, the ECL luminophore has a transition metal-ligand complex.
• GAS134.6 Preferably, the microfluidic test module also has:
• a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry for providing the electrical pulse to the electrodes.
• GAS134.7 Preferably, the microfluidic test module also has:
• a communication interface for the control circuitry to transmit data to an external device.
• GAS134.8 Preferably, the microfluidic test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS134.9 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS134.10 Preferably, the hybridization chambers have a volume less than 200,000 cubic microns.
• GAS134.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS134.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS134.13 Preferably, the microfluidic test module also has:
• at least one calibration source for providing a calibration emission, and at least one calibration photodiode for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photodiode output from each of the detection photodiode outputs.
• GAS134.14 Preferably, the microfluidic test module also has a plurality of the calibration sources and a corresponding plurality of the calibration photodiodes in registration with the calibration sources respectively.
• GAS134.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS134.16 Preferably, the microfluidic test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS134.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS134.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS134.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS134.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• The low probe volume provides for low probe cost, in turn, permitting the inexpensive assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS135.1 This aspect of the invention provides a test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing having a receptacle for receiving the fluid containing the target nucleic acid sequences;
• electrochemiluminescent (ECL) probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores; and,
• a detection photosensor for exposure to the photons emitted by the ECL luminophores.
• GAS135.2 Preferably, the test module also has a lab-on-a-chip (LOC) device with circuitry for providing the electrical pulse to the electrodes wherein the detection photosensor is incorporated into the circuitry and the ECL probes, the electrodes, and the control circuitry are integrated into the LOC device.
• GAS135.3 Preferably, the LOC device has a supporting substrate for supporting the circuitry which in turn supports the electrodes and the ECL probes.
• GAS135.4 Preferably, the circuitry has layers of CMOS circuitry deposited on the supporting substrate for providing the electrodes with an electrical pulse.
• GAS135.5 Preferably, the electrical pulse has duration less than 0.69 seconds.
• GAS135.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS135.7 Preferably, the test module also has:
• a communication interface for the circuitry to transmit data to an external device.
• GAS135.8 Preferably, the test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the CMOS circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS135.9 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS135.10 Preferably, the hybridization chambers have a volume less than 200,000 cubic microns.
• GAS135.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS135.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS135.13 Preferably, the test module also has at least one calibration source for providing a calibration emission, and at least one calibration photodiode for sensing the calibration emission wherein the CMOS circuitry has a differential circuit for subtracting the calibration photodiode output from the output of one or more of the detection photodiodes.
• GAS135.14 Preferably, the test module also has a plurality of the calibration sources and a corresponding plurality of the calibration photodiodes in registration with the calibration sources respectively.
• GAS135.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS135.16 Preferably, the test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS135.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS135.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS135.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS135.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• The integrated image sensor with the driver for excitation of electrochemiluminescence luminophores obviate the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS136.1 This aspect of the invention provides a portable test module for excitation of electrochemiluminescent probes configured to detect target nucleic acid sequences, the test module comprising:
• an outer casing configured for hand-held portability, the outer casing having a receptacle for receiving a fluid containing the target nucleic acid sequences;
• electrochemiluminescent (ECL) probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein during use,
• the ECL probes that have detected one of the target nucleic acid sequences reconfigure such that the functional moiety does not quench the photon emission from the ECL luminophore when excited by the electrodes.
• GAS136.2 Preferably, the portable test module also has circuitry for providing the electrical pulse to the electrodes, the circuitry incorporating a detection photosensor for exposure to the photons emitted by the ECL luminophores.
• GAS136.3 Preferably, the portable test module also has a lab-on-a-chip (LOC) device with a supporting substrate for supporting the circuitry which in turn supports the electrodes and the ECL probes.
• GAS136.4 Preferably, the circuitry has layers of CMOS circuitry deposited on the supporting substrate for providing the electrodes with an electrical pulse.
• GAS136.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS136.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS136.7 Preferably, the portable test module also has:
• a communication interface for the circuitry to transmit data to an external device.
• GAS136.8 Preferably, the portable test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the CMOS circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS136.9 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS136.10 Preferably, the hybridization chambers have a volume less than 200,000 cubic microns.
• GAS136.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS136.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS136.13 Preferably, the portable test module also has at least one calibration source for providing a calibration emission, and at least one calibration photodiode for sensing the calibration emission wherein the CMOS circuitry has a differential circuit for subtracting the calibration photodiode output from the output of one or more of the detection photodiodes.
• GAS136.14 Preferably, the portable test module also has a plurality of the calibration sources and a corresponding plurality of the calibration photodiodes in registration with the calibration sources respectively.
• GAS136.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS136.16 Preferably, the portable test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS136.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS136.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS136.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS136.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• The easily usable, mass-producible, inexpensive, compact, light, and portable test module accepts a biological sample, identifies the sample's nucleic acid sequences via electrochemiluminescent probe hybridization using its integral image sensor and integral driver for excitation of electrochemiluminescence luminophores, and provides the results electronically at its output port.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS137.1 This aspect of the invention provides a test module for excitation of electrochemiluminescent probes configured to detect target nucleic acid sequences, the test module comprising:
• an outer casing having a receptacle for receiving a fluid containing the target nucleic acid sequences;
• electrochemiluminescent (ECL) probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores;
• a detection photosensor for exposure to the photons emitted by the ECL luminophores;
• control circuitry providing the electrical pulse to the electrodes; and,
• a universal serial bus (USB) connection such that the outer casing is configured as a USB drive for transmitting data regarding detection of the targets in the fluid to an external device; wherein during use,
• the ECL probes that have detected one of the target nucleic acid sequences reconfigure such that the functional moiety does not quench the photon emission from the ECL luminophore when excited by the electrodes.
• GAS137.2 Preferably, the electrodes are plates of conductive material, the plates having edge profiles configured such that the length of peripheral edge of each of the plates is greater than 128 microns.
• GAS137.3 Preferably, the test module also has a lab-on-a-chip (LOC) device wherein the ECL probes, the electrodes, the detection photosensor and the control circuitry are integrated into the LOC device wherein the LOC device has a supporting substrate for supporting the control circuitry which in turn supports the detection photosensor, the electrodes and the ECL probes.
• GAS137.4 Preferably, the control circuitry is layers of CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS137.5 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS137.6 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS137.7 Preferably, the test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences and a pair of the electrodes wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS137.8 Preferably, the CMOS circuitry is configured to apply a voltage across the electrode pair in each of the hybridization chambers, the voltage being in the range 1.7 Volts to 2.8 Volts.
• GAS137.9 Preferably, the voltage is in the range 1.9 Volts to 2.6 Volts.
• GAS137.10 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS137.11 Preferably, the hybridization chambers have a volume less than 200,000 cubic microns.
• GAS137.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS137.13 Preferably, the test module also has:
• at least one calibration source for providing a calibration emission, and a calibration photosensor for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photosensor output from the detection photosensor output.
• GAS137.14 Preferably, the test module also has a plurality of the calibration sources wherein the detection photosensor is an array of photodiodes in registration with each of the ECL probes respectively and the calibration photosensor is a plurality of calibration photodiodes in registration with the calibration sources respectively.
• GAS137.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS137.16 Preferably, the test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS137.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS137.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS137.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS137.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• The easily usable, mass-producible, inexpensive, compact, light, and portable test module accepts a biological sample, identifies the sample's nucleic acid sequences via electrochemiluminescent probe hybridization using its integral image sensor and integral driver for excitation of electrochemiluminescence luminophores, and provides the results electronically at its output port, with the ubiquitous USB port used for the module's power and signaling requirements.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS138.1 This aspect of the invention provides a test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing having an inlet for receiving the fluid containing the target nucleic acid sequences;
• a hybridization chamber mounted in the casing, the hybridization chamber containing electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer;
• electrodes for receiving an electrical pulse to excite the ECL luminophores; and,
• a reagent reservoir containing a reagent for addition to the fluid prior to detection of the target nucleic acid sequences; wherein,
• the hybridization chamber has a volume less than 900,000 cubic microns; and,
• the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GAS138.2 Preferably, the hybridization chamber has a volume less than 200,000 cubic microns, and the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GAS138.3 Preferably, the hybridization chamber has a volume less than 40,000 cubic microns, and the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GAS138.4 Preferably, the hybridization chamber has a volume less than 9000 cubic microns, and the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GAS138.5 Preferably, the test module also has:
• a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry providing the electrical pulse to the electrodes.
• GAS138.6 Preferably, the test module also has:
• a communication interface for the control circuitry to transmit data to an external device.
• GAS138.7 Preferably, the test module also has an array of the hybridization chambers containing the ECL probes for different target nucleic acid sequences and a pair of the electrodes wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS138.8 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS138.9 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS138.10 Preferably, the test module also has a lab-on-a-chip (LOC) device which incorporates the hybridization chambers, the reagent reservoir, the detection photodiodes, the electrodes and the ECL probes, the LOC device having a supporting substrate for layers of CMOS circuitry forming part of the control circuitry such that the detection photodiodes are between the hybridization chambers and the CMOS circuitry.
• GAS138.11 Preferably, the test module also has a plurality of the reagent reservoirs and a polymerase chain reaction (PCR) section incorporated into the LOC device, wherein the PCR section is configured to amplify the target nucleic acid sequences and the reagent reservoirs contain polymerase, dNTPs, and primers.
• GAS138.12 Preferably, the LOC device has a cap in which the reagent reservoirs are defined.
• GAS138.13 Preferably, each of the reagent reservoirs have a surface tension valve, each of the surface tension valve having a meniscus anchor for pinning a meniscus to retaining reagents therein.
• GAS138.14 Preferably, the electrode pairs in each of the hybridization chambers have an anode and a cathode each with fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS138.15 Preferably, the CMOS circuitry is configured to provide each of the electrode pairs with an electrical pulse to generate a voltage across the electrode pairs, the voltage being between 1.7 Volts to 2.8 Volts.
• GAS138.16 Preferably, the voltage is between 1.9 Volts to 2.6 Volts.
• GAS138.17 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers and the calibration probes include the functional moiety for quenching photon emission.
• GAS138.18 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS138.19 Preferably, the electrical pulse to the electrodes is a DC pulse and has a duration less than 0.69 seconds.
• GAS138.20 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• The low-volume hybridization chambers and reagent reservoirs, in part, provide for the low probe and reagent volumes, which in turn provide for low probe and reagent costs and the inexpensive assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS139.1 This aspect of the invention provides a test module for excitation of electrochemiluminescent probes configured to detect target nucleic acid sequences, the test module comprising:
• an outer casing with an inlet for receiving a fluid containing the target nucleic acid sequences;
• electrochemiluminescent (ECL) probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores, such that the ECL probes that have detected one of the target nucleic acid sequences reconfigure such that the functional moiety does not quench the photon emission from the ECL luminophore when excited by the electrodes; wherein,
• the ECL luminophore has a transition metal-ligand complex.
• GAS139.2 Preferably, the transition metal-ligand complex is a ruthenium chelate.
• GAS139.3 Preferably, the test module also has a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry providing the electrical pulse to the electrodes.
• GAS139.4 Preferably, the test module also has a lab-on-a-chip (LOC) device wherein the ECL probes, the electrodes, the detection photosensor and the control circuitry are integrated into the LOC device wherein the LOC device has a supporting substrate for supporting the control circuitry which in turn supports the detection photosensor, the electrodes and the ECL probes.
• GAS139.5 Preferably, the control circuitry is layers of CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS139.6 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS139.7 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS139.8 Preferably, the test module also has:
• a communication interface for the control circuitry to transmit data to an external device.
• GAS139.9 Preferably, the test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS139.10 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS139.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS139.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS139.13 Preferably, the test module also has:
• at least one calibration source for providing a calibration emission, and a calibration photosensor for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photosensor output from the detection photosensor output.
• GAS139.14 Preferably, the test module also has a plurality of the calibration sources wherein the detection photosensor is an array of photodiodes in registration with each of the ECL probes respectively and the calibration photosensor is a plurality of calibration photodiodes in registration with the calibration sources respectively.
• GAS139.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS139.16 Preferably, the test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS139.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS139.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS139.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS139.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• Using the long-lifetime transition metal-ligand complex electrochemiluminescence-based probes improves the sensitivity and reliability of the assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS140.1 This aspect of the invention provides a test module for excitation of electrochemiluminescent probes configured to detect target nucleic acid sequences, the test module comprising:
• an outer casing with an inlet for receiving a fluid containing the target nucleic acid sequences;
• electrochemiluminescent (ECL) probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores, such that the ECL probes that have detected one of the target nucleic acid sequences reconfigure such that the functional moiety does not quench the photon emission from the ECL luminophore when excited by the electrodes; wherein,
• the ECL luminophore has a lanthanide metal-ligand complex.
• GAS140.2 Preferably, the lanthanide metal-ligand complex is selected from:
• a ruthenium chelate;
• a terbium chelate; and,
• a europium chelate.
• GAS140.3 Preferably, the test module also has a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry providing the electrical pulse to the electrodes.
• GAS140.4 Preferably, the test module also has a lab-on-a-chip (LOC) device wherein the ECL probes, the electrodes, the detection photosensor and the control circuitry are integrated into the LOC device wherein the LOC device has a supporting substrate for supporting the control circuitry which in turn supports the detection photosensor, the electrodes and the ECL probes.
• GAS140.5 Preferably, the control circuitry is layers of CMOS circuitry configured to provide an electrical pulse to the electrodes.
• GAS140.6 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS140.7 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS140.8 Preferably, the test module also has:
• a communication interface for the control circuitry to transmit data to an external device.
• GAS140.9 Preferably, the test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS140.10 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS140.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS140.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS140.13 Preferably, the test module also has:
• at least one calibration source for providing a calibration emission, and a calibration photosensor for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photosensor output from the detection photosensor output.
• GAS140.14 Preferably, the test module also has a plurality of the calibration sources wherein the detection photosensor is an array of photodiodes in registration with each of the ECL probes respectively and the calibration photosensor is a plurality of calibration photodiodes in registration with the calibration sources respectively.
• GAS140.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS140.16 Preferably, the test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS140.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS140.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS140.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS140.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• Using the long-lifetime lanthanide metal-ligand complex electrochemiluminescence-based probes improves the sensitivity and reliability of the assay system.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS141.1 This aspect of the invention provides a test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing having an inlet for receiving the fluid containing the target nucleic acid sequences;
• electrode pairs for receiving an electrical pulse;
• electrochemiluminescent (ECL) probes adjacent each of the electrode pairs respectively, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, such that the electrical pulse to the electrode pair excites the ECL luminophores; wherein,
• the ECL probes are not anchored to a surface and form a suspension in the fluid containing the target nucleic acid sequences during detection.
• GAS141.2 Preferably, the spots of liquid each have a volume less than 200000 cubic microns.
• GAS141.3 Preferably, the spots of liquid each have a volume less than 30000 cubic microns.
• GAS141.4 Preferably, the spots of liquid each have a volume less than 2000 cubic microns.
• GAS141.5 Preferably, the ECL luminophore has a transition metal-ligand complex.
• GAS141.6 Preferably, the test module also has:
• a detection photosensor for exposure to the photons emitted by the ECL luminophores; and,
• control circuitry providing the electrical pulse to the electrodes.
• GAS141.7 Preferably, the test module also has:
• a communication interface for the control circuitry to transmit data to an external device.
• GAS141.8 Preferably, the test module also has an array of hybridization chambers containing the ECL probes for different target nucleic acid sequences wherein the control circuitry has memory for storing the identity data relating to the ECL probes in each of the hybridization chambers.
• GAS141.9 Preferably, the hybridization chambers have a volume less than 900,000 cubic microns.
• GAS141.10 Preferably, the ECL luminophore has a lanthanide metal-ligand complex.
• GAS141.11 Preferably, the communication interface is a universal serial bus (USB) connection such that the outer casing is configured as a USB drive.
• GAS141.12 Preferably, the detection photosensor is an array of detection photodiodes positioned in registration with the hybridization chambers.
• GAS141.13 Preferably, the test module also has:
• at least one calibration source for providing a calibration emission, and a calibration photosensor for sensing the calibration emission wherein the control circuitry has a differential circuit for subtracting the calibration photosensor output from the detection photosensor output.
• GAS141.14 Preferably, the test module also has a plurality of the calibration sources wherein the detection photosensor is an array of photodiodes in registration with each of the ECL probes respectively and the calibration photosensor is a plurality of calibration photodiodes in registration with the calibration sources respectively.
• GAS141.15 Preferably, the calibration sources are calibration probes without an ECL luminophore.
• GAS141.16 Preferably, the test module also has a plurality of calibration chambers containing the calibration sources distributed throughout the array of hybridization chambers, wherein during use, output from any one of the detection photodiodes is compared to output from the calibration photodiode most proximate to that detection photodiode.
• GAS141.17 Preferably, the calibration sources are calibration probes and the calibration chambers are configured to seal the calibration probes from the fluid containing the target nucleic acid sequences.
• GAS141.18 Preferably, each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
• GAS141.19 Preferably, the detection photodiodes are less than 1600 microns from the hybridization chambers.
• GAS141.20 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• The suspended probes with their large electrical excitation depth and large emission optical depth increase the readout sensitivity. The suspended probes are spotted more easily and inexpensively.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS142.1 This aspect of the invention provides a test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing with an inlet for receiving a fluid containing the target nucleic acid sequences;
• an array of electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• electrodes positioned for receiving an electrical pulse, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes; and,
• a photosensor for detecting the light emitted by the probes; wherein during use,
• addition of the fluid to the probes prevents subsequent addition of other fluid to the probes.
• GAS142.2 Preferably, the test module also has an array of hybridization chambers wherein each of the chambers contains a pair the electrode pairs and the probes for one of the targets respectively, wherein the fluid fills each of the chambers by capillary action.
• GAS142.3 Preferably, the hybridization chambers each have a volume less than 900000 cubic microns.
• GAS142.4 Preferably, the test module also has a lab-on-a-chip (LOC) device wherein the ECL probes, the electrodes, and the detection photosensor are integrated into the LOC device wherein the LOC device has CMOS circuitry, a supporting substrate for supporting the CMOS circuitry which in turn supports the detection photosensor, the electrodes and the ECL probes.
• GAS142.5 Preferably, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, such that the electrical pulse to the electrode pair excites the ECL luminophores.
• GAS142.6 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS142.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS142.8 Preferably, the CMOS circuitry incorporates the photosensor wherein the wall section is positioned between the probes and the photosensor.
• GAS142.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS142.10 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS142.11 Preferably, the CMOS circuitry is configured to apply a voltage across the electrodes in each of the hybridization chambers, the voltage being in the range 1.7 Volts to 2.8 Volts.
• GAS142.12 Preferably, the voltage is in the range 1.9 Volts to 2.6 Volts.
• GAS142.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS142.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS142.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS142.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS142.17 Preferably, the LOC device has a flow-path configured to draw the fluid containing the targets into all the hybridization chambers by capillary action.
• GAS142.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS142.19 Preferably, the ECL luminophores each have a lanthanide metal-ligand complex.
• GAS142.20 Preferably, the ECL luminophores each have a transition metal-ligand complex.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output port.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS143.1 This aspect of the invention provides a test module for amplifying and detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing with a receptacle for receiving a fluid containing the target nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences;
• an array of electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• electrodes positioned for receiving an electrical pulse, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes; and,
• a photosensor for detecting the light emitted by the probes; wherein during use, addition of the fluid to the probes prevents subsequent addition of other fluid to the probes.
• GAS143.2 Preferably, the test module also has an array of hybridization chambers wherein each of the chambers contains one of the electrode pairs and the probes for one of the targets respectively, wherein the fluid fills each of the chambers by capillary action.
• GAS143.3 Preferably, the test module also has a plurality of reagent reservoirs in fluid communication with the PCR section wherein the reagent reservoirs contain polymerase, dNTPs, and primers.
• GAS143.4 Preferably, the test module also has a lab-on-a-chip (LOC) device having a supporting substrate and CMOS circuitry wherein the LOC device incorporates the ECL probes and the electrodes such that the supporting substrate supports the CMOS circuitry and the CMOS circuitry incorporates the detection photosensor adjacent the electrodes and the ECL probes.
• GAS143.5 Preferably, each of the ECL probes having an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer, such that the electrical pulse to the electrode pair excites the ECL luminophores.
• GAS143.6 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS143.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the light emitted by the probes.
• GAS143.8 Preferably, the wall section is positioned between the probes and the photosensor.
• GAS143.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS143.10 Preferably, the CMOS circuitry is configured to provide the electrical pulse to the electrodes.
• GAS143.11 Preferably, the electrical pulse has a voltage between 1.7 Volts to 2.8 Volts.
• GAS143.12 Preferably, the voltage is in the range 1.9 Volts to 2.6 Volts.
• GAS143.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS143.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS143.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS143.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS143.17 Preferably, the LOC device has a flow-path configured to draw the fluid containing the targets into all the hybridization chambers by capillary action.
• GAS143.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS143.19 Preferably, the ECL luminophores each have a lanthanide metal-ligand complex.
• GAS143.20 Preferably, the ECL luminophores each have a transition metal-ligand complex.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, amplifies the nucleic acid targets in the sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor, and provides the results electronically at its output port.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS144.1 This aspect of the invention provides a test module for detecting target nucleic acid sequences in a fluid, the test module comprising:
• an outer casing with a receptacle for receiving a fluid containing the target nucleic acid sequences;
• an array of electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• electrodes positioned for receiving an electrical pulse, the probe-target hybrids being configured to emit photons when excited by current between the electrodes; and,
• control circuitry for providing the electrodes with the electrical pulse; wherein during use,
• addition of the fluid to the probes prevents subsequent addition of other fluid to the probes.
• GAS144.2 Preferably, the test module also has:
• a photosensor for detecting the photons emitted by the probes; and,
• an array of hybridization chambers wherein each of the chambers contains a pair of the electrodes and the probes for one of the targets respectively; wherein,
• the fluid fills each of the chambers by capillary action.
• GAS144.3 Preferably, the hybridization chambers each have a volume less than 900,000 cubic microns.
• GAS144.4 Preferably, the test module also has a lab-on-a-chip (LOC) device, the LOC device having a supporting substrate, CMOS circuitry, the ECL probes, the electrodes, and the photosensor, wherein the supporting substrate supports the CMOS circuitry which incorporates the detection photosensor, which in turn supports the electrodes and the ECL probes.
• GAS144.5 Preferably, each of the ECL probes have an ECL luminophore for emitting photons when in an excited state and a functional moiety for quenching photon emissions from the ECL luminophore by resonant energy transfer, such that the electrical pulse to the electrode pair excites the ECL luminophores.
• GAS144.6 Preferably, the ECL probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS144.7 Preferably, the hybridization chambers each have a wall section that is optically transparent to the photons emitted by the probes.
• GAS144.8 Preferably, the CMOS circuitry incorporates the photosensor, the wall section being positioned between the probes and the photosensor.
• GAS144.9 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS144.10 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes.
• GAS144.11 Preferably, the electrical pulse has a voltage in the range 1.7 Volts to 2.8 Volts.
• GAS144.12 Preferably, the voltage is in the range 1.9 Volts to 2.6 Volts.
• GAS144.13 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS144.14 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS144.15 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS144.16 Preferably, the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS144.17 Preferably, the LOC device has a flow-path configured to draw the fluid containing the targets into all the hybridization chambers by capillary action.
• GAS144.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.
• GAS144.19 Preferably, the ECL luminophores each have a lanthanide metal-ligand complex.
• GAS144.20 Preferably, the ECL luminophores each have a transition metal-ligand complex.
• The easily usable, mass-producible, inexpensive, compact, and light genetic test module accepts a biological sample, identifies the sample's nucleic acid sequences via probe hybridization using its integral image sensor with driver for excitation of electrochemiluminescent luminophores, and provides the results electronically at its output port.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GAS146.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting target nucleic acid sequences in a fluid, the LOC device comprising:
• electrochemiluminescent (ECL) probes for detecting the target nucleic acid sequences, each of the probes having an ECL luminophore for emitting photons when in an excited state, a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer; and,
• electrodes for receiving an electrical pulse to excite the ECL luminophores; wherein,
• the electrodes are arranged into pairs, each having an anode and a cathode, the anode and the cathode being separated by a dielectric gap between 0.4 microns and 2 microns wide.
• GAS146.2 Preferably, the LOC device also has a photosensor positioned adjacent the probes for sensing which of the probes generate the photons in response to the electrical pulse.
• GAS146.3 Preferably, the LOC device also has an array of hybridization chambers wherein each of the chambers contains one of the electrode pairs and the probes for one of the targets respectively, wherein the fluid fills each of the chambers by capillary action.
• GAS146.4 Preferably, the LOC device also has a supporting substrate wherein the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the hybridization chambers.
• GAS146.5 Preferably, the array of photodiodes is less than 1600 microns from the probes.
• GAS146.6 Preferably, the LOC device also has CMOS circuitry on the supporting substrate, the array of photodiodes being a component of the CMOS circuitry wherein during use, the CMOS circuitry initiates an error signal in response to a failure to sense ECL photons from a positive control probe.
• GAS146.7 Preferably, the LOC device also has at least one negative control chamber containing negative control probes that are incapable of hybridization with any nucleic acid sequences in the fluid.
• GAS146.8 Preferably, the LOC device also has:
• a flow-path for fluid containing the targets; wherein,
• the CMOS circuitry is between the hybridization chambers and the supporting substrate, and the flow-path draws the fluid to each of the hybridization chambers by capillary action.
• GAS146.9 Preferably, the LOC device also has a lysis section wherein the fluid is a biological sample containing cells and the lysis section disrupts the cell membranes to release any genetic material therein.
• GAS146.10 Preferably, the hybridization chambers each have a wall section that is optically transparent to the photons emitted by the probes.
• GAS146.11 Preferably, the wall section is positioned between the probes and the array of photodiodes.
• GAS146.12 Preferably, the wall section is a layer incorporating silicon dioxide.
• GAS146.13 Preferably, the probes have a stem-and-loop structure with a loop portion containing the sequence complementary to the target nucleic acid sequence, the loop portion being positioned between the functional moiety for quenching photon emission from the ECL luminophore, and the ECL luminophore, such that hybridization with the target nucleic acid sequence opens the loop portion and moves the ECL luminophore away from the functional moiety.
• GAS146.14 Preferably, the CMOS circuitry is configured to an electrical pulse to the electrodes.
• GAS146.15 Preferably, the electrical pulse has a duration less than 0.69 seconds.
• GAS146.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GAS146.17 Preferably, the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.
• GAS146.18 Preferably, the ECL luminophores each have a transition metal-ligand complex.
• GAS146.19 Preferably, the LOC device also has a PCR section for amplifying the target nucleic acid sequences prior to detection by the probes.
• GAS146.20 Preferably, the LOC device also has a cap having reagent reservoirs for addition to the fluid prior to detection of the target sequences.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• The absence of the requirement for an electrochemical coreactant makes the assay system simpler and less expensive and facilitates the utilization of a larger number of assay chemistry options.
• GAS147.1 This aspect of the invention provides a test module for analyzing a sample fluid containing target molecules, the test module comprising:
• an outer casing with a receptacle for receiving the sample fluid;
• probes for reaction with the target molecules to form probe-target complexes, the probe-target complexes being configured to emit photons when excited; and,
• a photosensor for detecting photons emitted by the probe-target complexes.
• GAS147.2 Preferably, the test module also has a microfluidic device with a sample inlet in fluid communication with the receptacle, the microfluidic device incorporating the probes and the photosensor.
• GAS147.3 Preferably, the target molecules are target nucleic acid sequences and the probes are configured to form probe-target hybrids such that the photosensor detects the probe-target hybrids within the array of probes.
• GAS147.4 Preferably, the microfluidic device has CMOS circuitry which incorporates the photosensor and the probes are fluorescence resonance energy transfer (FRET) probes.
• GAS147.5 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid prior to hybridization with the FRET probes, and an array of hybridization chambers containing the FRET probes, the hybridization chambers each having an optical window to expose the FRET probes to an excitation light.
• GAS147.6 Preferably, the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
• GAS147.7 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and memory for storing identity data identifying each FRET probe type, the CMOS circuitry being configured to use output from the photodiodes to generate a signal indicative of the FRET probes that have formed probe-target hybrids, and provide the signal to the bond-pads for transmission to the external device.
• GAS147.8 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GAS147.9 Preferably, the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GAS147.10 Preferably, the fluorophore is a transition metal-ligand complex.
• GAS147.11 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GAS147.12 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GAS147.13 Preferably, the quencher has no native emission in response to the excitation light.
• GAS147.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
• GAS147.15 Preferably, the test module also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GAS147.16 Preferably, the hybridization section has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GAS147.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GAS147.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GAS147.19 Preferably, the photodiodes are less than 249 microns from the FRET probes.
• GAS147.20 Preferably, the microfluidic device has a sample inlet for receiving the fluid sample, and a plurality of reagent reservoirs for different reagents required to process the fluid sample wherein the fluid sample is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the microfluidic device.
• The photosensor provides for a mass-producible, inexpensive, compact, light, and highly portable integrated solution with low system component-count. The photosensor incorporated in the module increases the readout sensitivity by benefiting from large angle of light collection. The integrated image sensor obviates the need for optical components in the optical collection train.
• GRR001.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving biochemical liquid; and,
• a reagent reservoir containing a reagent for addition to the biochemical liquid for use in chemical analysis of the biochemical liquid; wherein,
• the reagent reservoir has an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR001.2 Preferably, the MST layer has a flow-path extending from the inlet to the surface tension valve, the flow-path configured to draw the biochemical liquid from the inlet to the surface tension valve by capillary action.
• GRR001.3 Preferably, the reagent reservoir has a volume less than 1,000,000,000 cubic microns.
• GRR001.4 Preferably, the reagent reservoir is supported on the MST layer, such that the MST layer is between the reagent reservoir and the supporting substrate.
• GRR001.5 Preferably, the reagent reservoir is defined in a cap, the cap comprising a layer of material with a cavity to define the reagent reservoir.
• GRR001.6 Preferably, the cap defines a plurality of the reagent reservoirs for containing all the reagents required for the biochemical processing and analysis to be performed.
• GRR001.7 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR001.8 Preferably, the flow-path extends through the MST layer and the cap.
• GRR001.9 Preferably, the surface tension valve has a meniscus anchor configured to pin the meniscus of the reagent such that the meniscus contacts the biochemical liquid flowing along the flow-path.
• GRR001.10 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR001.11 Preferably, the microfluidic device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequences.
• GRR001.12 Preferably, the microfluidic device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR001.13 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR001.14 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• buffer;
• primers; and,
• ligase.
• GRR001.15 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR001.16 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR001.17 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photo sensor being an array of the photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR001.18 Preferably, the flow-path is configured to draw the sample from the inlet to all the hybridization chambers by capillary action.
• GRR001.19 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GRR001.20 Preferably, the array of photodiodes is less than 249 microns from the FRET probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the biochemical mixture, as required, by surface tension actuated valves. The surface tension actuated valves are highly reliable and easily manufacturable, in turn providing for the highly reliable, mass-producible, portable and inexpensive assay system.
• The reagent reservoirs, being integral to the device, provide for self-contained reagent storage, which in turn provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GRR002.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving biochemical liquid; and,
• a plurality of reagent reservoirs containing all reagents necessary for the biochemical processing; wherein,
• each of the reagent reservoirs have an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR002.2 Preferably, the MST layer has a flow-path extending from the inlet to the surface tension valves of each of the reagent reservoirs, the flow-path being configured to draw the biochemical liquid from the inlet to the surface tension valves by capillary action.
• GRR002.3 Preferably, each of the reagent reservoirs has a volume less than 1000,000,000 cubic microns.
• GRR002.4 Preferably, the reagent reservoirs are supported on the MST layer, such that the MST layer is between the reagent reservoirs and the supporting substrate.
• GRR002.5 Preferably, the reagent reservoirs are defined in a cap, the cap comprising a layer of material with cavities to define the reagent reservoirs.
• GRR002.6 Preferably, the MST layer has heaters for use in processing the biochemical liquid.
• GRR002.7 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR002.8 Preferably, the flow-path extends through the MST layer and the cap.
• GRR002.9 Preferably, the surface tension valve has a meniscus anchor configured to pin the meniscus of the reagent such that the meniscus contacts the biochemical liquid flowing along the flow-path.
• GRR002.10 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR002.11 Preferably, the microfluidic device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR002.12 Preferably, the microfluidic device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR002.13 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR002.14 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR002.15 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR002.16 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR002.17 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photo sensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR002.18 Preferably, the flow-path is configured to draw the sample from the inlet to all the hybridization chambers by capillary action.
• GRR002.19 Preferably, the CMOS circuitry has memory storing identity data for the different FRET probe types.
• GRR002.20 Preferably, the array of photodiodes is less than 249 microns from the FRET probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the biochemical mixture, as required, by surface tension actuated valves. The surface tension actuated valves are highly reliable and easily manufacturable, in turn providing for the highly reliable, mass-producible, portable and inexpensive assay system.
• The reagent reservoirs, being integral to the device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GRR003.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving biochemical liquid; and,
• a reagent reservoir containing a reagent necessary for the biochemical processing; wherein,
• the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GRR003.2 Preferably, the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GRR003.3 Preferably, the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GRR003.4 Preferably, the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GRR003.5 Preferably, the microfluidic device also has a plurality of the reagent reservoirs, the plurality of reagent reservoirs containing all reagents necessary for the biochemical processing, and each of the reagent reservoirs having an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR003.6 Preferably, the MST layer has a flow-path extending from the inlet to the surface tension valves of each of the reagent reservoirs, the flow-path configured to draw the biochemical liquid from the inlet to the surface tension valves by capillary action.
• GRR003.7 Preferably, the reagent reservoirs are supported on the MST layer, such that the MST layer is between the reagent reservoirs and the supporting substrate.
• GRR003.8 Preferably, the reagent reservoirs are defined in a cap, the cap comprising a layer of material with cavities to define the reagent reservoirs.
• GRR003.9 Preferably, the MST layer has heaters for use in processing the biochemical liquid.
• GRR003.10 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR003.11 Preferably, the flow-path extends through the MST layer and the cap.
• GRR003.12 Preferably, the surface tension valve has a meniscus anchor configured to pin the meniscus of the reagent such that the meniscus contacts the biochemical liquid flowing along the flow-path.
• GRR003.13 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR003.14 Preferably, the microfluidic device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR003.15 Preferably, the microfluidic device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR003.16 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR003.17 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• buffer;
• primers; and,
• ligase.
• GRR003.18 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR003.19 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR003.20 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photo sensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the device's reagent reservoirs. The reagent reservoirs, being integral to the device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system. The low-volume reagent reservoirs also, in part, provide for the low reagent volumes, which in turn provide for the low reagent costs and further reducing the cost of the assay system.
• GRR004.1 This aspect of the invention provides a microfluidic device for biochemical processing and analysis, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving biochemical liquid; and,
• a reagent reservoir containing a reagent necessary for the biochemical processing; wherein,
• the reagent reservoir contains less than 1,000,000,000 cubic microns of reagent.
• GRR004.2 Preferably, the microfluidic device also has a plurality of the reagent reservoirs, the plurality of reagent reservoirs containing all reagents necessary for the biochemical processing, and each of the reagent reservoirs having an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR004.3 Preferably, the MST layer has a flow-path extending from the inlet to the surface tension valves of each of the reagent reservoirs, the flow-path being configured to draw the biochemical liquid from the inlet to the surface tension valves by capillary action.
• GRR004.4 Preferably, the reagent reservoirs are supported on the MST layer, such that the MST layer is between the reagent reservoirs and the supporting substrate.
• GRR004.5 Preferably, the reagent reservoirs are defined in a cap, the cap comprising a layer of material with cavities to define the reagent reservoirs.
• GRR004.6 Preferably, the MST layer has heaters for use in processing the biochemical liquid.
• GRR004.7 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR004.8 Preferably, the flow-path extends through the MST layer and the cap.
• GRR004.9 Preferably, the surface tension valve has a meniscus anchor configured to pin the meniscus of the reagent such that the meniscus contacts the biochemical liquid flowing along the flow-path.
• GRR004.10 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR004.11 Preferably, the microfluidic device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR004.12 Preferably, the microfluidic device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR004.13 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR004.14 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• buffer;
• primers; and,
• ligase.
• GRR004.15 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR004.16 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR004.17 Preferably, the microfluidic device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photo sensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR004.18 Preferably, the flow-path is configured to draw the sample from the inlet to all the hybridization chambers by capillary action.
• GRR004.19 Preferably, the CMOS circuitry has memory storing identity data for the different FRET probe types.
• GRR004.20 Preferably, the array of photodiodes is less than 249 microns from the FRET probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the device's reagent reservoirs. The low reagent volumes provide for the low reagent costs and the inexpensive assay system.
• GRR005.1 This aspect of the invention provides a LOC device for performing a genetic diagnostic assay, the LOC device comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving a biological sample containing target nucleic acid sequences;
• probes for hybridization with the target nucleic acid sequences to form probe-target hybrids;
• a flow-path extending from the inlet to the probes; and,
• a reagent reservoir containing a reagent for addition to the sample in the flow-path upstream of the probes.
• GRR005.2 Preferably, the reagent reservoir has an outlet in fluid communication with the flow-path, the outlet having a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with flow of the sample along the flow-path removes the reagent meniscus.
• GRR005.3 Preferably, the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GRR005.4 Preferably, the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GRR005.5 Preferably, the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GRR005.6 Preferably, the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GRR005.7 Preferably, the reagent reservoir is supported on the MST layer, such that the MST layer is between the reagent reservoir and the supporting substrate.
• GRR005.8 Preferably, the reagent reservoir is defined in a cap, the cap comprising a layer of material with a cavity to define the reagent reservoir.
• GRR005.9 Preferably, the cap defines a plurality of the reagent reservoirs for containing all the reagents required for the genetic diagnostic assay to be performed.
• GRR005.10 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR005.11 Preferably, the flow-path extends through the MST layer and the cap.
• GRR005.12 Preferably, the reagent reservoir has a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the LOC device, and allow airflow into the reagent reservoir as the reagent flow out of the outlet.
• GRR005.13 Preferably, the LOC device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequences.
• GRR005.14 Preferably, the LOC device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR005.15 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR005.16 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR005.17 Preferably, the LOC device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR005.18 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR005.19 Preferably, the LOC device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR005.20 Preferably, the flow-path is configured to draw the sample from the inlet to all the hybridization chambers by capillary action.
• The easily usable, mass-producible, and inexpensive genetic analysis LOC device accepts a biological sample and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes, utilizing reagents stored in the LOC device's reagent reservoirs. The reagent reservoirs, being integral to the device, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GRR006.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving a biological sample containing target nucleic acid sequences;
• probes for hybridization with the target nucleic acid sequence to form probe-target hybrids; and,
• a plurality of reagent reservoirs containing all reagents necessary for performing the diagnostic assay.
• GRR006.2 Preferably, the LOC device also has a flow-path extending from the inlet to the probes wherein the reagent reservoirs are positioned along the flow-path for addition to the sample in the flow-path upstream of the probes.
• GRR006.3 Preferably, the reagent reservoirs each have an outlet in fluid communication with the flow-path, the outlet having a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with flow of the sample along the flow-path removes the reagent meniscus.
• GRR006.4 Preferably, the reagent reservoirs each have a volume less than 1,000,000,000 cubic microns.
• GRR006.5 Preferably, each of the reagent reservoirs are supported on the MST layer, such that the MST layer is between the reagent reservoirs and the supporting substrate.
• GRR006.6 Preferably, the reagent reservoirs are defined in a cap, the cap comprising a layer of material with cavities to define the reagent reservoirs.
• GRR006.7 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR006.8 Preferably, the flow-path extends through the MST layer and the cap.
• GRR006.9 Preferably, the reagent reservoirs each have a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the LOC device, and allow airflow into the reagent reservoir as the reagent is added to the sample.
• GRR006.10 Preferably, the LOC device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR006.11 Preferably, the LOC device also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR006.12 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR006.13 Preferably, the reagents contained in the reagent reservoirs include:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR006.14 Preferably, the LOC device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR006.15 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR006.16 Preferably, the LOC device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR006.17 Preferably, the flow-path is configured to draw the sample from the inlet to all the hybridization chambers by capillary action.
• GRR006.18 Preferably, the CMOS circuitry has memory for identity data for the different FRET probe types.
• GRR006.19 Preferably, the array of photodiodes is less than 249 microns from the FRET probes.
• GRR006.20 Preferably, the fluorophore has a fluorescence lifetime greater than 100 nanoseconds.
• The easily usable, mass-producible, and inexpensive genetic analysis LOC device accepts a biological sample and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes, utilizing reagents stored in the LOC device's reagent reservoirs. The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GRR007.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis comprising:
• a supporting substrate;
• a microsystems technology (MST) layer supported on the substrate, the MST layer having an inlet for receiving a biological sample containing target nucleic acid sequences;
• a hybridization section comprising an array of chambers with probes for hybridization with the target nucleic acid sequences to form probe-target hybrids; and,
• a reagent reservoir containing a reagent necessary for processing the biological sample prior to hybridization; wherein,
• the hybridization section contains less than 270 picograms of each probe per chamber and the reagent reservoir containing less than 1000,000,000 cubic microns of the reagent.
• GRR007.2 Preferably, the hybridization section contains less than 60 picograms of each probe per chamber and the reagent reservoir containing less than 300,000,000 cubic microns of the reagent.
• GRR007.3 Preferably, the hybridization section contains less than 12 picograms of each probe per chamber and the reagent reservoir containing less than 70,000,000 cubic microns of the reagent.
• GRR007.4 Preferably, the hybridization section contains less than 2.7 picograms of each probe per chamber and the reagent reservoir containing less than 20,000,000 cubic microns of the reagent.
• GRR007.5 Preferably, the LOC device also has a plurality of reagent reservoirs containing all reagents necessary for performing the diagnostic assay wherein each of the reagent reservoirs have an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biological sample removes the reagent meniscus.
• GRR007.6 Preferably, the MST layer has a flow-path extending from the inlet to the surface tension valves of each of the reagent reservoirs, the flow-path configured to draw the biological sample to the surface tension valves by capillary action.
• GRR007.7 Preferably, the reagent reservoirs are supported on the MST layer, such that the MST layer is between the reagent reservoirs and the supporting substrate.
• GRR007.8 Preferably, the reagent reservoirs are defined in a cap, the cap comprising a layer of material with cavities to define the reagent reservoirs.
• GRR007.9 Preferably, the MST layer has heaters for use in processing the biochemical liquid.
• GRR007.10 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR007.11 Preferably, the flow-path extends through the MST layer and the cap.
• GRR007.12 Preferably, the reagent reservoirs each have a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the LOC device, and allow airflow into the reagent reservoir as the reagent flow out of the outlet.
• GRR007.13 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR007.14 Preferably, the LOC device also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR007.15 Preferably, each hybridization chamber has an optical window for exposing the probes to the excitation light.
• GRR007.16 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR007.17 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR007.18 Preferably, the LOC device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR007.19 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR007.20 Preferably, the LOC device also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• The easily usable, mass-producible, and inexpensive genetic analysis LOC device accepts a biological sample and analyzes the sample's nucleic acid sequences via hybridization with low volumes of oligonucleotide probes, utilizing low volumes of reagents stored in the LOC device's reagent reservoirs. The low oligonucleotide probe and reagent volumes provide for the low probe and reagent costs and the inexpensive assay system.
• GRR008.1 This aspect of the invention provides a test module for biochemical processing and analysis, the test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having a receptacle for receiving biochemical liquid; and,
• a reagent reservoir containing a reagent for addition to the biochemical liquid for use in chemical analysis of the biochemical liquid; wherein,
• the reagent reservoir has an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR008.2 Preferably, the test module also has a flow-path extending from the receptacle to the surface tension valve, the flow-path being configured to draw the biochemical liquid to the surface tension valve by capillary action.
• GRR008.3 Preferably, the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GRR008.4 Preferably, the test module also has a microfluidic device supported in the casing, the microfluidic device having a supporting substrate and a MST layer formed on the substrate, wherein the reagent reservoir is supported on the MST layer, such that the MST layer is between the reagent reservoir and the supporting substrate.
• GRR008.5 Preferably, the reagent reservoir is defined in a cap, the cap comprising a layer of material with a cavity to define the reagent reservoir.
• GRR008.6 Preferably, the cap defines a plurality of the reagent reservoirs for containing all the reagents required for the biochemical processing and analysis to be performed.
• GRR008.7 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR008.8 Preferably, the flow-path extends through the MST layer and the cap.
• GRR008.9 Preferably, the reagent reservoir has a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the microfluidic device, and allow airflow into the reagent reservoir as the reagent flow out of the outlet.
• GRR008.10 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR008.11 Preferably, the test module also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR008.12 Preferably, the test module also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR008.13 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR008.14 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR008.15 Preferably, the test module also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR008.16 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR008.17 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• GRR008.18 Preferably, the flow-path is configured to draw the sample from the receptacle to all the hybridization chambers by capillary action.
• GRR008.19 Preferably, the CMOS circuitry has memory storing identity data for the different FRET probe types.
• GRR008.20 Preferably, the array of photodiodes is less than 249 microns from the FRET probes.
• The easily usable, mass-producible, inexpensive, and portable microfluidic test module accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the module's reagent reservoirs, with the reagents being added to the biochemical mixture, as required, by surface tension actuated valves. The surface tension actuated valves are highly reliable and easily manufacturable, in turn providing for the highly reliable, mass-producible, portable and inexpensive assay system.
• GRR009.1 This aspect of the invention provides a test module for biochemical processing and analysis, the test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having a receptacle for receiving biochemical liquid; and,
• a reagent reservoir containing a reagent for addition to the biochemical liquid for use in chemical analysis of the biochemical liquid; wherein,
• the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GRR009.2 Preferably, the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GRR009.3 Preferably, the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GRR009.4 Preferably, the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GRR009.5 Preferably, the reagent reservoir has an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biochemical liquid removes the reagent meniscus.
• GRR009.6 Preferably, the test module also has a flow-path extending from the receptacle to the surface tension valve, the flow-path being configured to draw the biochemical liquid to the surface tension valve by capillary action.
• GRR009.7 Preferably, the test module also has a microfluidic device supported in the casing, the microfluidic device having a supporting substrate and a MST layer formed on the substrate, wherein the reagent reservoir is supported on the MST layer, such that the MST layer is between the reagent reservoir and the supporting substrate.
• GRR009.8 Preferably, the reagent reservoir is defined in a cap, the cap comprising a layer of material with a cavity to define the reagent reservoir.
• GRR009.9 Preferably, the cap defines a plurality of the reagent reservoirs for containing all the reagents required for the biochemical processing and analysis to be performed.
• GRR009.10 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR009.11 Preferably, the flow-path extends through the MST layer and the cap.
• GRR009.12 Preferably, the reagent reservoir has a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the microfluidic device, and allow airflow into the reagent reservoir as the reagent flow out of the outlet.
• GRR009.13 Preferably, the biochemical liquid is a biological sample and the biochemical processing is a genetic diagnostic assay, the biological sample containing target nucleic acid sequences, the MST layer having probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GRR009.14 Preferably, the test module also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR009.15 Preferably, the test module also has a hybridization chamber containing the probes, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR009.16 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR009.17 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR009.18 Preferably, the test module also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR009.19 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR009.20 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• The easily usable, mass-producible, inexpensive, and portable microfluidic test module accepts a biochemical sample, processes, and analyzes the sample, utilizing the reagents stored in the module's reagent reservoirs. The low-volume reagent reservoirs, in part, provide for the low reagent volumes, which in turn provide for the low reagent costs and the inexpensive assay system.
• GRR010.1 This aspect of the invention provides a test module for performing a genetic diagnostic assay, the test module comprising:
• an outer casing dimensioned for hand-held portability, the outer casing having a receptacle for a biological sample containing target nucleic acid sequences;
• an array of chambers containing probes for hybridization with the target nucleic acid sequences to form probe-target hybrids;
• a flow-path extending from the inlet to the probes; and,
• a reagent reservoir containing a reagent for addition to the sample in the flow-path upstream of the probes; wherein,
• each of the chambers contains less than 270 picograms of probe and the reagent reservoir has a volume less than 1000,000,000 cubic microns.
• GRR010.2 Preferably, each of the chambers contains less than 60 picograms of probe and the reagent reservoir has a volume less than 300,000,000 cubic microns.
• GRR010.3 Preferably, each of the chambers contains less than 12 picograms of probe and the reagent reservoir has a volume less than 70,000,000 cubic microns.
• GRR010.4 Preferably, each of the chambers contains less than 2.7 picograms of probe and the reagent reservoir has a volume less than 20,000,000 cubic microns.
• GRR010.5 Preferably, the reagent reservoir has an outlet with a surface tension valve for retaining the reagent in the reservoir with a reagent meniscus until contact with the biological sample removes the reagent meniscus.
• GRR010.6 Preferably, the test module also has a flow-path extending from the receptacle to the surface tension valve, the flow-path being configured to draw the biological sample to the surface tension valve by capillary action.
• GRR010.7 Preferably, the test module also has a microfluidic device supported in the casing, the microfluidic device having a supporting substrate and a MST layer formed on the substrate, wherein the reagent reservoir is supported on the MST layer, such that the MST layer is between the reagent reservoir and the supporting substrate.
• GRR010.8 Preferably, the reagent reservoir is defined in a cap, the cap comprising a layer of material with a cavity to define the reagent reservoir.
• GRR010.9 Preferably, the cap defines a plurality of the reagent reservoirs for containing all the reagents required for the genetic analysis to be performed.
• GRR010.10 Preferably, at least one of the reagent reservoirs has a plurality of the outlets to increase the reagent flow rate out of the reservoir.
• GRR010.11 Preferably, the flow-path extends through the MST layer and the cap.
• GRR010.12 Preferably, the reagent reservoir has a vent to atmosphere sized to prevent leakage of the reagent during storage and handling of the microfluidic device, and allow airflow into the reagent reservoir as the reagent flow out of the outlet.
• GRR010.13 Preferably, the test module also has a photosensor wherein the probe-target hybrids each have a fluorophore for emitting a fluorescence signal in response to an excitation light such that the photosensor senses the fluorescence signal to generate an output indicating hybridization of the probes with the target nucleic acid sequence.
• GRR010.14 Preferably, the test module also has an LED for generating the excitation light.
• GRR010.15 Preferably, each of the chambers is a hybridization chamber, the hybridization chamber having an optical window for exposing the probes to the excitation light.
• GRR010.16 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying the oligonucleotides in the sample of biological material.
• GRR010.17 Preferably, the reagent contained in the reagent reservoir has one or more of:
• lysis reagent;
• anticoagulant;
• polymerase;
• dNTP's;
• primers; or,
• ligase.
• GRR010.18 Preferably, the test module also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating the photosensor and bond-pads for communication with an external device.
• GRR010.19 Preferably, the CMOS circuitry controls activation and deactivation of an external light source configured to generate the excitation light.
• GRR010.20 Preferably, the test module also has an array of hybridization chambers containing different types of the probes, the probes being fluorescence resonance energy transfer (FRET) probes configured for hybridization with different target nucleic acid sequences, and the photosensor being an array of photodiodes such that each of the hybridization chambers corresponds to a respective one of the photodiodes.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a biological sample and analyzes the sample's nucleic acid sequences via hybridization with low volumes of oligonucleotide probes, utilizing low volumes of reagents stored in the module's reagent reservoirs. The low oligonucleotide probe and reagent volumes provide for the low probe and reagent costs and the inexpensive assay system.
• GVA001.1 This aspect of the invention provides a microfluidic device comprising:
• a channel having an inlet, an outlet and a meniscus anchor between the inlet and the outlet such that liquid flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• an actuator valve with a movable member for contacting the liquid, and a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus such that the liquid flow towards the outlet resumes.
• GVA001.2 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action.
• GVA001.3 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA001.4 Preferably, the movable member at least partially defines the aperture.
• GVA001.5 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA001.6 Preferably, the actuator valve reciprocates the movable member between the quiescent and displaced positions until the channel immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GVA001.7 Preferably, the microfluidic device of claim 6 further comprising a supporting substrate for the channel and CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve; and,
• at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve; wherein,
• the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA001.8 Preferably, the aperture is a nozzle in the movable member.
• GVA001.9 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• The mass-producible and inexpensive microfluidic device accepts a liquid for processing and/or analysis, with the requisite fluidic propulsion being provided via capillary action, and the requisite valve functionality being provided via reliable, easily manufacturable thermal bend actuated pressure pulse valves. The thermal bend actuated pressure pulse valves exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA002.1 This aspect of the invention provides a microfluidic device comprising:
• a channel having an inlet, an outlet and a meniscus anchor between the inlet and the outlet such that liquid flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• an actuator valve with a movable member for contacting the liquid, and an actuator for displacing the movable member from a quiescent position to an actuated position where the meniscus is extended into contact with a surface downstream of the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA002.2 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action.
• GVA002.3 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA002.4 Preferably, the movable member partially defines the aperture.
• GVA002.5 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA002.6 Preferably, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA002.7 Preferably, the microfluidic device of claim 6 further comprising a supporting substrate for the channel and CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve.
• GVA002.8 Preferably, the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve.
• GVA002.9 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA002.10 Preferably, the aperture is a nozzle in the movable member.
• GVA002.11 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• The mass-producible and inexpensive microfluidic device accepts a liquid for processing and/or analysis, with the requisite fluidic propulsion being provided via capillary action, and the requisite valve functionality being provided via reliable, easily manufacturable thermal bend actuated surface tension valves. The thermal bend actuated surface tension valves exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA004.1 This aspect of the invention provides a microfluidic device comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet; and,
• a fault-tolerant multiple valve assembly having a plurality of flow-paths extending from the inlet to the outlet and a plurality of valves positioned along each of the flow-paths respectively.
• GVA004.2 Preferably, each of the flow-paths is configured for capillary driven flow of the liquid towards the outlet, and each of the valves are configured to arrest flow towards the outlet until opened.
• GVA004.3 Preferably, the microfluidic device also has a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA004.4 Preferably, the valves are bend actuated valves, each having a movable member configured for movement between a quiescent position and an actuated position displaced from the quiescent position and, an aperture at least partially defined by the movable member, the aperture configured to arrest the capillary driven flow by pinning a meniscus at the aperture wherein during use, displacement of the movable member to the actuated position unpins the meniscus from the aperture such that the capillary driven flow towards the outlet resumes.
• GVA004.5 Preferably, the bend actuated valve has a resistive element for causing differential thermal expansion to move the movable member.
• GVA004.6 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA004.7 Preferably, the valves are thermally actuated valves, each having an aperture for anchoring a meniscus to arrest the capillary drive flow, and a valve heater for heating the meniscus such that the meniscus unpins from the aperture such that the capillary driven flow towards the outlet resumes.
• GVA004.8 Preferably, the valve heater extends about the periphery of the aperture.
• GVA004.9 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA004.10 Preferably, the microfluidic device also has two of the flow-paths.
• GVA004.11 Preferably, the microfluidic device also has CMOS circuitry connected to the sensors for operative control of the valves and detecting faults including failure of either of the upstream valves to arrest the liquid flow.
• GVA004.12 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences by thermally cycling the nucleic acid sequences and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature; wherein,
• the fault-tolerant multiple valve assembly retains the nucleic acid sequences and a PCR mix of reagents in the PCR section during the thermal cycling.
• GVA004.13 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids;
• the array of probes being downstream of the fault-tolerant multiple valve assembly such that opening the fault-tolerant multiple valve assembly allows amplicon from the PCR section to contact the probes.
• GVA004.14 Preferably, the PCR section is configured to produce sufficient amplicon for hybridization with more than 1000 of the probes in less than 10 minutes of thermal cycling.
• GVA004.15 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA004.16 Preferably, the microfluidic device also has a temperature sensor and wherein the PCR section has at least one heater for thermally cycling the nucleic acid sequences and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA004.17 Preferably, the CMOS circuitry opens the fault-tolerant valve assembly after a predetermined number of thermal cycles.
• GVA004.18 Preferably, the PCR section has a plurality of elongate PCR chambers having a longitudinal extent much greater than their lateral dimensions, and a plurality of heaters, each of the heaters being elongate and parallel with the longitudinal extent of the PCR chambers.
• GVA004.19 Preferably, the plurality of elongate heaters are independently operable.
• GVA004.20 Preferably, the microfluidic device also has an array of photodiodes for detecting hybridization of probes within the array of probes.
• The mass-producible and inexpensive microfluidic device accepts a liquid for processing and/or analysis, with the requisite fluidic propulsion being provided via capillary action, and the requisite valve functionality being provided via reliable, easily manufacturable fault-tolerant multiple-valve assemblies.
• Fault-tolerant multiple-valve assemblies provide the requisite level of reliability via individual valve fault tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA005.1 This aspect of the invention provides a microfluidic device for processing a fluid sample, the microfluidic device comprising:
• a reservoir for containing a reagent; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus such that the reagent flows out of the reagent reservoir.
• GVA005.2 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GVA005.3 Preferably, the microfluidic device also has a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate; and,
• a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer; wherein,
• at least one of the fluidic connections between the cap and the MST layer is the surface tension valve.
• GVA005.4 Preferably, the cap has a cap channel connecting at least two of the fluidic connections, the cap channel being configured to draw fluid flow between the fluidic connections by capillary action.
• GVA005.5 Preferably, the reagent reservoir is in fluid communication with the cap channel via the MST layer and at least two of the fluidic connections, one of the fluidic connections being the surface tension valve.
• GVA005.6 Preferably, the cap channel and the reagent reservoir are formed in a unitary layer of material.
• GVA005.7 Preferably, the cap channel is formed in one surface of the cap such that an outer surface of the MST layer encloses the cap channel.
• GVA005.8 Preferably, the cap has an exterior surface opposite said one surface, the exterior surface having a sample inlet for receiving the fluid sample and feeding the fluid sample to the cap channel.
• GVA005.9 Preferably, the fluid sample contains a biological sample including cells of different sizes, and at least one of the fluidic connections is an array of holes sized to prevent passage of cells larger than a predetermined threshold.
• GVA005.10 Preferably, the array of holes is part of a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GVA005.11 Preferably, the biological sample is blood and the holes are configured such that cells smaller than the predetermined threshold include pathogens.
• GVA005.12 Preferably, the reagent in the reservoir is an anticoagulant and the cap is configured such that the anticoagulant is mixed with the blood prior to entering the dialysis section.
• GVA005.13 Preferably, the microfluidic device also has a lysis section in fluid communication with a lysis reagent reservoir containing a lysis reagent, the lysis section being configured to lyse pathogens and release genetic material within.
• GVA005.14 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid; wherein the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplifying the nucleic acid sequences.
• GVA005.15 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GVA005.16 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GVA005.17 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences amplified by the PCR section.
• GVA005.18 Preferably, the array has more than 1000 probes.
• GVA005.19 Preferably, the microfluidic device also has an array of photodiodes for detecting hybridization of probes within the array of probes.
• GVA005.20 Preferably, the microfluidic device also has a plurality of heaters for controlling the temperature of the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a liquid for processing and analysis, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the liquid, as required, by surface tension valves. The surface tension valves are highly reliable and easily manufacturable, in turn providing for the highly reliable, mass-producible, portable and inexpensive assay system.
• The reagent reservoirs, being integral to the device, provide for self-contained reagent storage, which in turn provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GVA006.1 This aspect of the invention provides a microfluidic device comprising:
• a reservoir for containing a reagent;
• an outlet valve in fluid communication with the reservoir, the outlet valve having a meniscus anchor configured to form a meniscus that retains the reagent in the reservoir, and an actuator for receiving an activation signal and actuating in response to the activation signal such that the meniscus unpins from the meniscus anchor and the reagent flows out of the reservoir.
• GVA006.2 Preferably, the outlet valve has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member to generate a pulse in the reagent to dislodge the meniscus from the meniscus anchor.
• GVA006.3 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the reagent flow out of the reservoir by pinning the meniscus at the aperture.
• GVA006.4 Preferably, the movable member at least partially defines the aperture.
• GVA006.5 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA006.6 Preferably, during use, the activation signal is a series of electrical pulses and in response the actuator valve reciprocates the movable member between the quiescent and displaced positions.
• GVA006.7 Preferably, the movable member is configured to displace from a quiescent position to an actuated position such that displacement of the movable member to the actuated position extends the meniscus into contact with a surface downstream of the outlet valve such that capillary action drives the reagent flow out of the reservoir.
• GVA006.8 Preferably, the surface downstream of the outlet valve is a capillary initiation feature for reconfiguring the meniscus and moving the meniscus away from the outlet valve.
• GVA006.9 Preferably, the thermal expansion actuator is configured to cause differential thermal expansion to move the movable member.
• GVA006.10 Preferably, the actuator has a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor.
• GVA006.11 Preferably, the meniscus anchor is an aperture configured to arrest the reagent flow out of the reservoir by pinning the meniscus at the aperture and the valve heater extends about the periphery of the aperture.
• GVA006.12 Preferably, the valve heater is configured to boil the reagent at the meniscus to unpin the meniscus from the meniscus anchor.
• GVA006.13 Preferably, the microfluidic device of claim 1 further comprising a liquid sensor for sensing the presence or absence of liquid adjacent the outlet valve.
• GVA006.14 Preferably, the microfluidic device of claim 13 further comprising a supporting substrate and CMOS circuitry on the supporting substrate for operatively controlling the outlet valve.
• GVA006.15 Preferably, the sensor provides feedback to the CMOS circuitry for use in the operative control of the outlet valve.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a liquid for processing and analysis, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the liquid, as required, by active valves. The reagent reservoirs, being integral to the device, provide for self-contained reagent storage, which in turn provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GVA007.1 This aspect of the invention provides a microfluidic device for testing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• a reservoir containing a reagent;
• a flow-path extending from the inlet;
• a valve assembly for establishing a fluid connection between the flow-path and the reservoir, the valve assembly having a plurality of outlet valves and a plurality of channels from the reservoir to the flow-path; wherein during use,
• a number of the outlet valves open such that the reagent flows through the valve assembly to the flow-path to combine with the fluid from the inlet to produce a combined flow having a proportion of the reagent, the proportion of the reagent in the combined flow being determined by the number of the outlet valves opened.
• GVA007.2 Preferably, the valve assembly has one of the outlet valves in each of the channels respectively.
• GVA007.3 Preferably, the outlet valves are surface tension valves having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GVA007.4 Preferably, more than one of the outlet valves are in each of the channels.
• GVA007.5 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GVA007.6 Preferably, the microfluidic device of claim 5 further comprising a plurality of the reservoirs, each containing a different reagent and a plurality of the valve assemblies between the reservoirs and the flow-path respectively, wherein the proportion of any of the different reagents in the combined flow relates to the number of outlet valves that are opened in the corresponding valve arrangement.
• GVA007.7 Preferably, the microfluidic device of claim 5 further comprises a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate; and,
• a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer; and,
• at least one of the fluidic connections between the cap and the MST layer is the surface tension valve, the surface tension valve being part of a valve assembly.
• GVA007.8 Preferably, the flow-path extends through the MST layer and the cap connecting at least some of the fluidic connections, the flow-path being configured to draw fluid flow between the fluidic connections by capillary action.
• GVA007.9 Preferably, the fluid contains a biological sample including cells of different sizes, and at least one of the fluidic connections is an array of holes sized to prevent passage of cells larger than a predetermined threshold.
• GVA007.10 Preferably, the array of holes is part of a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GVA007.11 Preferably, the biological sample is blood and the holes are configured such that cells smaller than the predetermined threshold include pathogens.
• GVA007.12 Preferably, one of the reagent reservoirs is an anticoagulant reservoir in fluid communication with the flow-path via the surface tension valve of a valve assembly corresponding to the anticoagulant reservoir such that anticoagulant is mixed with the blood prior to entering the dialysis section.
• GVA007.13 Preferably, the microfluidic device of claim 12 further comprising a lysis section in fluid communication with the flow-path, the lysis section being configured to lyse pathogens and release genetic material within.
• GVA007.14 Preferably, the microfluidic device of claim 13 further comprising a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid; wherein the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the sample prior to amplifying the nucleic acid sequences.
• GVA007.15 Preferably, the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
• GVA007.16 Preferably, the microfluidic device of claim 15 further comprising CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GVA007.17 Preferably, the microfluidic device of claim 16 further comprising a hybridization section that has an array of probes for hybridization with target nucleic acid sequences amplified by the PCR section.
• GVA007.18 Preferably, the array has more than 1000 probes.
• GVA007.19 Preferably, the microfluidic device of claim 18 wherein the probes are fluorescent resonant energy transfer (FRET) probes and the CMOS circuitry further comprises an array of photodiodes for detecting hybridization of probes within the array of probes.
• GVA007.20 Preferably, the microfluidic device of claim 19 further comprising a plurality of heaters for controlling the temperature of the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a liquid sample for processing and analysis, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the liquid, as required, by a number of valves.
• The requisite mixing ratio in between the reagents and other liquid components is determined by the number of the open valves, thus manufacturably and reliably achieving this difficult control goal in the microfluidic context.
• GVA008.1 This aspect of the invention provides a microfluidic device for testing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• a reservoir containing a reagent;
• a flow-path extending from the inlet;
• a plurality of outlet valves for fluid communication between the flow-path and the reservoir, each of the outlet valves having an actuator for opening the outlet valve in response to an activation signal; wherein during use,
• a number of the outlet valves are selectively opened such that the reagent flows into the flow-path to combine with the fluid from the inlet to produce a combined flow having a proportion of the reagent, the proportion of the reagent in the combined flow being determined by the number of the outlet valves opened.
• GVA008.2 Preferably, the microfluidic device also has a plurality of channels extending between the reservoir and the flow-path, the outlet valves being positioned in each of the channels, the channels being configured to draw the reagent from the reservoir to the flow-path by capillary action, wherein the outlet valves each have a meniscus anchor where capillary driven reagent flow towards the flow-path is stopped and a meniscus forms.
• GVA008.3 Preferably, more than one of the outlet valves are in each of the channels.
• GVA008.4 Preferably, the actuators are heaters to unpin the meniscus from the meniscus anchor in response to the activation signal.
• GVA008.5 Preferably, each of the outlet valves has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member to generate a pulse in the reagent to dislodge the meniscus such that the reagent flow towards the flow-path resumes.
• GVA008.6 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the reagent flow by pinning the meniscus at the aperture, the thermal actuator being configured to reciprocate the movable member between the quiescent position and the actuated position to force the reagent through the aperture.
• GVA008.7 Preferably, the aperture is defined in the movable member.
• GVA008.8 Preferably, the meniscus anchor is an aperture configured to arrest the reagent flow by pinning the meniscus at the aperture, and the thermal actuator is configured to boil some of the reagent at the aperture to unpin the meniscus from the meniscus anchor.
• GVA008.9 Preferably, each of the outlet valves has a movable member for contacting the reagent, and the actuator is a thermal expansion actuator for displacing the movable member to move the meniscus into contact with a surface downstream of the meniscus anchor thereby unpinning the meniscus such that capillary driven flow towards the flow-path resumes.
• GVA008.10 Preferably, the surface downstream of the meniscus anchor is a capillary initiation feature configured to direct the meniscus to the channel walls.
• GVA008.11 Preferably, the microfluidic device of claim 1 further comprising a supporting substrate for the reservoir, the outlet valves, the inlet and the flow-path, and CMOS circuitry for operatively controlling the outlet valves.
• GVA008.12 Preferably, the microfluidic device of claim 11 further comprising at least one sensor responsive to liquid for providing feedback to the CMOS circuitry for use in the operative control of the separate valves.
• GVA008.13 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in one of the channels.
• GVA008.14 Preferably, the microfluidic device of claim 1 further comprising a plurality of the reservoirs, each containing a different reagent and each having a plurality of the outlet valves between the reservoirs and the flow-path respectively, wherein the proportion of any of the different reagents in the combined flow relates to the number of separate valves that are opened in the corresponding valve arrangement.
• GVA008.15 Preferably, the CMOS circuitry selects how many of the outlet valves open for each of the reservoirs in accordance with the different reagent types and their desired proportions in the combined flow.
• GVA008.16 Preferably, the microfluidic device of claim 15 further comprising a polymerase chain reaction (PCR) section for amplifying target nucleic acid sequences in the fluid.
• GVA008.17 Preferably, the different reagents in the reservoirs include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GVA008.18 Preferably, the microfluidic device of claim 15 further comprising a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the fluid wherein the CMOS circuitry has an array of sensors for detecting any hybridization of probes within the array of probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a liquid sample for processing and analysis, utilizing the reagents stored in the device's reagent reservoirs, with the reagents being added to the liquid, as required, by a number of active valves.
• The requisite mixing ratio in between the reagents and other liquid components is determined, during device operation, by the number of the open valves, thus manufacturably and reliably achieving this difficult control goal in the microfluidic context.
• GVA009.1 This aspect of the invention provides a microfluidic device for amplifying nucleic acid sequences, the microfluidic device comprising:
• a polymerase chain reaction (PCR) section for thermally cycling the nucleic acid sequences and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature; and,
• a PCR outlet valve to retain the nucleic acid sequences and a PCR mix of reagents in the PCR section during the thermal cycling; wherein,
• the PCR outlet valve is configured to open in response to an activation signal such that amplicon can flow from the PCR section and once open, the PCR outlet valve is unable to close.
• GVA009.2 Preferably, the PCR outlet valve has a meniscus anchor configured to form a meniscus that retains the nucleic acid sequences and a PCR mix of reagents in the PCR section, and an actuator for receiving the activation signal and actuating in response to the activation signal such that the meniscus unpins from the meniscus anchor and the amplicon flows out of the PCR section.
• GVA009.3 Preferably, the PCR outlet valve has a movable member for contacting the amplicon, and the actuator is a thermal expansion actuator for displacing the movable member to generate a pulse in the amplicon to dislodge the meniscus from the meniscus anchor.
• GVA009.4 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest any liquid flow out of the PCR section by pinning the meniscus at the aperture.
• GVA009.5 Preferably, the movable member at least partially defines the aperture.
• GVA009.6 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA009.7 Preferably, during use, the activation signal is a series of electrical pulses and in response the actuator reciprocates the movable member between the quiescent and displaced positions.
• GVA009.8 Preferably, the PCR outlet valve has a movable member for contacting the amplicon, and the movable member is configured to displace from a quiescent position to an actuated position such that displacement of the movable member to the actuated position extends the meniscus into contact with a surface downstream of the PCR outlet valve such that capillary action drives the amplicon flow out of the PCR section.
• GVA009.9 Preferably, the surface downstream of the PCR outlet valve is a capillary initiation feature for reconfiguring the meniscus and moving the meniscus away from the PCR outlet valve.
• GVA009.10 Preferably, the thermal expansion actuator is configured to cause differential thermal expansion to move the movable member.
• GVA009.11 Preferably, the actuator has a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor.
• GVA009.12 Preferably, the meniscus anchor is an aperture configured to arrest liquid flow out of the PCR section by pinning the meniscus at the aperture and the valve heater extends about the periphery of the aperture.
• GVA009.13 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA009.14 Preferably, the microfluidic device also has a supporting substrate and CMOS circuitry on the supporting substrate for operatively controlling the PCR outlet valve.
• GVA009.15 Preferably, the PCR section has a sample inlet for receiving a sample of biological material containing the nucleic acid sequences and is configured to produce sufficient amplicon for hybridization with more than 1000 probes in less than 10 minutes after the sample enters the sample inlet.
• GVA009.16 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA009.17 Preferably, the microfluidic device also has a temperature sensor and wherein the PCR section has at least one heater for thermally cycling the nucleic acid sequences and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA009.18 Preferably, the CMOS circuitry activates the PCR outlet valve after a predetermined number of thermal cycles.
• GVA009.19 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the amplicon.
• GVA009.20 Preferably, the PCR section is configured to generate enough amplicon for hybridization with the probes in less than 220 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then amplifies the nucleic acid targets in the sample, with the one requisite valving action being provided by a one-time active valve incorporated on the device, thus manufacturably and reliably achieving this difficult fluid control goal in the microfluidic context.
• GVA010.1 This aspect of the invention provides a lab-on-a-chip device (LOC) for genetic analysis comprising:
• a supporting substrate;
• an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids;
• a channel for directing a flow of liquid containing the target nucleic acid sequences, the channel having an inlet, an outlet and a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• an actuator valve with a movable member for contacting the liquid, and a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus such that the liquid flow towards the outlet resumes.
• GVA010.2 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action.
• GVA010.3 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA010.4 Preferably, the movable member at least partially defines the aperture.
• GVA010.5 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA010.6 Preferably, the actuator valve reciprocates the movable member between the quiescent and displaced positions until the channel immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GVA010.7 Preferably, the LOC device also has CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve; and,
• at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve; wherein,
• the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA010.8 Preferably, the aperture is a nozzle in the movable member.
• GVA010.9 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA010.10 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for thermally cycling oligonucleotides and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature wherein the actuator valve is a PCR section outlet valve for retaining the oligonucleotides and a PCR mix of reagents in the PCR section during the thermal cycling, and the PCR outlet valve being configured to open in response to an activation signal such that amplicon can flow to the array of probes.
• GVA010.11 Preferably, the PCR section is configured to produce sufficient amplicon for more than 1000 of the probes in less than 10 minutes after the sample enters the sample inlet.
• GVA010.12 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA010.13 Preferably, the LOC device also has a temperature sensor and wherein the PCR section has at least one heater for thermally cycling the oligonucleotides and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA010.14 Preferably, the CMOS circuitry activates the PCR outlet valve after a predetermined number of thermal cycles.
• GVA010.15 Preferably, the PCR section has a plurality of elongate PCR chambers having a longitudinal extent much greater than its lateral dimensions, and a plurality of heaters, each of the heaters being elongate and parallel with the longitudinal extent of the PCR chambers.
• GVA010.16 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GVA010.17 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GVA010.18 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GVA010.19 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GVA010.20 Preferably, the plurality of elongate heaters are independently operable.
• The mass-producible and inexpensive genetic analysis LOC device accepts a biological sample for analysis of its nucleic acid content via hybridization with an array of nucleic acid probes, with the requisite valve functionality being provided via reliable, easily manufacturable thermal bend actuated pressure pulse valves. The thermal bend actuated pressure pulse valves exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA011.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis comprising:
• a supporting substrate;
• an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids;
• a channel for directing a flow of liquid containing the target nucleic acid sequences, the channel having an inlet, an outlet and a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• an actuator valve with a movable member for contacting the liquid, and an actuator for displacing the movable member from a quiescent position to an actuated position where the meniscus is extended into contact with a surface downstream of the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA011.2 Preferably, the meniscus anchor is an aperture at least partially defined by the movable member.
• GVA011.3 Preferably, the actuator is a resistive element for causing differential thermal expansion to move the movable member.
• GVA011.4 Preferably, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA011.5 Preferably, the LOC device also has CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve.
• GVA011.6 Preferably, the LOC device also has at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve.
• GVA011.7 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA011.8 Preferably, the aperture is a nozzle in the movable member.
• GVA011.9 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA011.10 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for thermally cycling nucleic acid sequences and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature wherein the actuator valve is a PCR section outlet valve for retaining the nucleic acid sequences and a PCR mix of reagents in the PCR section during the thermal cycling, and the PCR outlet valve being configured to open in response to an activation signal such that amplicon can flow to the array of probes.
• GVA011.11 Preferably, the PCR section is configured to produce sufficient amplicon for more than 1000 of the probes in less than 10 minutes after the sample enters the sample inlet.
• GVA011.12 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA011.13 Preferably, the LOC device also has a temperature sensor, and wherein the PCR section has at least one heater for thermally cycling the nucleic acid sequences and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA011.14 Preferably, the CMOS circuitry activates the PCR outlet valve after a predetermined number of thermal cycles.
• GVA011.15 Preferably, the PCR section has a plurality of elongate PCR chambers having a longitudinal extent much greater than its lateral dimensions, and a plurality of heaters, each of the heaters being elongate and parallel with the longitudinal extent of the PCR chambers.
• GVA011.16 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GVA011.17 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GVA011.18 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GVA011.19 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GVA011.20 Preferably, the plurality of elongate heaters are independently operable.
• The mass-producible and inexpensive genetic analysis LOC device accepts a biological sample for analysis of its nucleic acid content via hybridization with an array of nucleic acid probes, with the requisite valve functionality being provided via reliable, easily manufacturable thermal bend actuated surface tension valves. The thermal bend actuated surface tension valves exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA012.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis comprising:
• a supporting substrate;
• an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids;
• a channel for directing a flow of liquid containing the target nucleic acid sequences, the channel having an inlet, an outlet and a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• an actuator valve with a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA012.2 Preferably, the meniscus anchor is an aperture and the valve heater extends about the periphery of the aperture.
• GVA012.3 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA012.4 Preferably, the LOC device also has CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve.
• GVA012.5 Preferably, the LOC device also has at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve.
• GVA012.6 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA012.7 Preferably, the LOC device also has a polymerase chain reaction (PCR) section for thermally cycling nucleic acid sequences and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature wherein the actuator valve is a PCR section outlet valve for retaining the nucleic acid sequences and a PCR mix of reagents in the PCR section during the thermal cycling, and the PCR outlet valve being configured to open in response to an activation signal such that amplicon can flow to the array of probes.
• GVA012.8 Preferably, the PCR section is configured to produce sufficient amplicon for more than 1000 of the probes in less than 10 minutes after the sample enters the sample inlet.
• GVA012.9 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA012.10 Preferably, the LOC device also has a temperature sensor, and wherein the PCR section has at least one heater for thermally cycling the nucleic acid sequences and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA012.11 Preferably, the CMOS circuitry activates the PCR outlet valve after a predetermined number of thermal cycles.
• GVA012.12 Preferably, the PCR section has a plurality of elongate PCR chambers having a longitudinal extent much greater than their lateral dimensions, and a plurality of heaters, each of the heaters being elongate and parallel with the longitudinal extent of the PCR chambers.
• GVA012.13 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GVA012.14 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GVA012.15 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GVA012.16 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GVA012.17 Preferably, the plurality of elongate heaters are independently operable.
• GVA012.18 Preferably, the LOC device also has an array of photodiodes for detecting hybridization of probes within the array of probes.
• The mass-producible and inexpensive genetic analysis LOC device accepts a biological sample for analysis of its nucleic acid content via hybridization with an array of nucleic acid probes, with the requisite valve functionality being provided via reliable, easily manufacturable boiling-initiation valves. The boiling-initiation valves exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA013.1 This aspect of the invention provides a test module for genetic analysis comprising:
• a casing for hand held portability;
• an inlet for receiving a liquid containing nucleic acid sequences;
• an outlet downstream of the inlet; and,
• a fault-tolerant multiple valve assembly having a plurality of flow-paths extending from the inlet to the outlet and a plurality of valves positioned along each of the flow-paths respectively.
• GVA013.2 Preferably, each of the flow-paths is configured for capillary driven flow of the liquid towards the outlet, and each of the valves are configured to arrest flow towards the outlet until opened.
• GVA013.3 Preferably, the test module also has a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA013.4 Preferably, the valves are bend actuated valves, each having a movable member configured for movement between a quiescent position and an actuated position displaced from the quiescent position and, an aperture at least partially defined by the movable member, the aperture configured to arrest the capillary driven flow by pinning a meniscus at the aperture wherein during use, displacement of the movable member to the actuated position unpins the meniscus from the aperture such that the capillary driven flow towards the outlet resumes.
• GVA013.5 Preferably, the bend actuated valve has a resistive element for causing differential thermal expansion to move the movable member.
• GVA013.6 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA013.7 Preferably, the valves are thermally actuated valves, each having an aperture for anchoring a meniscus to arrest the capillary driven flow, and a valve heater for heating the meniscus such that the meniscus unpins from the aperture such that the capillary driven flow towards the outlet resumes.
• GVA013.8 Preferably, the valve heater extends about the periphery of the aperture.
• GVA013.9 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA013.10 Preferably, the test module also has two of the flow-paths.
• GVA013.11 Preferably, the test module also has CMOS circuitry connected to the sensors for operative control of the valves and detecting faults including failure of either of the upstream valves to arrest the liquid flow.
• GVA013.12 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences by thermally cycling the nucleic acid sequences and a PCR mix of reagents through a denaturation temperature, an annealing temperature and a primer extension temperature; wherein,
• the fault-tolerant multiple valve assembly retains the nucleic acid sequences and a PCR mix of reagents in the PCR section during the thermal cycling.
• GVA013.13 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids;
• the array of probes is downstream of the fault-tolerant multiple valve assembly such that opening the fault-tolerant multiple valve assembly allows amplicon from the PCR section to contact the probes.
• GVA013.14 Preferably, the PCR section is configured to produce sufficient amplicon for more than 1000 of the probes in less than 10 minutes of thermal cycling.
• GVA013.15 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GVA013.16 Preferably, the test module also has a temperature sensor, and wherein the PCR section has at least one heater for thermally cycling the nucleic acid sequences and the PCR mix, the temperature sensor and the at least one heater being connected to the CMOS circuitry for feedback control of the at least one heater.
• GVA013.17 Preferably, the CMOS circuitry opens the fault-tolerant valve assembly after a predetermined number of thermal cycles.
• GVA013.18 Preferably, the PCR section has a plurality of elongate PCR chambers, each having a longitudinal extent much greater than its lateral dimensions, and a plurality of heaters, each of the heaters being elongate and parallel with the longitudinal extent of the PCR chambers.
• GVA013.19 Preferably, the plurality of elongate heaters are independently operable.
• GVA013.20 Preferably, the test module also has an array of photodiodes for detecting hybridization of probes within the array of probes.
• The mass-producible, inexpensive, portable genetic test module accepts a biological sample for analysis of its nucleic acid content, with the requisite valve functionality being provided via reliable, easily manufacturable fault-tolerant multiple-valve assemblies. Fault-tolerant multiple-valve assemblies provide the requisite level of reliability via individual valve fault tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA014.1 This aspect of the invention provides a microfluidic thermal bend actuated pressure pulse valve comprising:
• an inlet for receiving a flow of liquid;
• an outlet for outputting the flow of liquid;
• a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus;
• a movable member for contacting the liquid; and,
• a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus such that the liquid flow towards the outlet resumes.
• GVA014.2 Preferably, during use, the liquid flow from the inlet to the meniscus anchor is via capillary action and the liquid flow immediately downstream of the meniscus anchor to the outlet is via capillary action.
• GVA014.3 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA014.4 Preferably, the movable member at least partially defines the aperture.
• GVA014.5 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA014.6 Preferably, the microfluidic valve also has a single flow-path from the inlet to the outlet wherein the actuator reciprocates the movable member between the quiescent and displaced positions until the flow-path immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GVA014.7 Preferably, the microfluidic valve also has;
• a channel formed in a supporting substrate for at least partially defining the flow-path;
• CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve; and,
• at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator valve; wherein,
• the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA014.8 Preferably, the aperture is a nozzle in the movable member.
• GVA014.9 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA014.10 Preferably, the movable member is formed in one wall of the channel such that in the quiescent position, an interior surface of the movable member is coplanar with an interior surface of the wall.
• GVA014.11 Preferably, the channel is overlaid with a cap defining a cap channel, the cap channel defining the flow-path immediately downstream of the aperture in the movable member such that reciprocation of the movable member fills the cap channel until capillary driven flow to the outlet resumes.
• GVA014.12 Preferably, the channel has a cross sectional area transverse to the flow direction less than 400 square microns.
• GVA014.13 Preferably, the thermal expansion actuator comprises at least two dissimilar materials.
• GVA014.14 Preferably, one of the at least two dissimilar materials is an electrical insulator.
• GVA014.15 Preferably, the resistive element is a layer of titanium nitride.
• GVA014.16 Preferably, the thermal actuator has a layer of silicon dioxide adjacent the titanium nitride layer.
• The thermal bend actuated pressure pulse valves reliably and manufacturably provide microfluidic valving action, exploiting the intrinsic positive qualities of microfluidic device technologies and avoiding problematic aspects of such technologies.
• GVA015.1 This aspect of the invention provides a microfluidic thermal bend actuated surface tension valve comprising:
• an inlet for receiving a flow of liquid;
• an outlet for outputting the flow of liquid;
• a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus;
• a movable member for contacting the liquid; and,
• an actuator for displacing the movable member from a quiescent position to an actuated position where the meniscus is extended into contact with a surface downstream of the meniscus anchor such that the flow towards the outlet resumes.
• GVA015.2 Preferably, the meniscus anchor is an aperture at least partially defined by the movable member.
• GVA015.3 Preferably, the actuator is a resistive element for causing differential thermal expansion to move the movable member.
• GVA015.4 Preferably, the microfluidic valve also has a single flow-path from the inlet to the outlet.
• GVA015.5 Preferably, the microfluidic valve also has:
• a channel formed in a supporting substrate for at least partially defining the flow-path;
• CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve; and,
• at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the actuator; wherein,
• the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA015.6 Preferably, the surface downstream of the meniscus anchor, that the extended meniscus contacts, comprises inwardly converging side walls of the channel forming a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA015.7 Preferably, the aperture is partially defined by the movable member.
• GVA015.8 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA015.9 Preferably, the movable member is formed in one wall of the channel such that in the quiescent position, an interior surface of the movable member is coplanar with an interior surface of the wall.
• GVA015.10 Preferably, the channel is overlaid with a cap defining a cap channel containing the liquid, the cap channel defining the flow-path immediately upstream of the aperture until movement of the movable member allows capillary driven flow to the outlet to resume.
• GVA015.11 Preferably, the channel has a cross sectional area transverse to the flow direction less than 100,000 square microns.
• GVA015.12 Preferably, the channel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GVA015.13 Preferably, the channel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GVA015.14 Preferably, the channel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GVA015.15 Preferably, the actuator comprises at least two dissimilar materials.
• GVA015.16 Preferably, one of the at least two dissimilar materials is an electrical insulator.
• GVA015.17 Preferably, the resistive element is a layer of titanium nitride.
• GVA015.18 Preferably, actuator has a layer of silicon dioxide adjacent the titanium nitride layer.
• The thermal bend actuated surface tension valves reliably and manufacturably provide microfluidic valving action, exploiting the intrinsic positive qualities of microfluidic device technologies and avoiding problematic aspects of such technologies.
• GVA016.1 This aspect of the invention provides a microfluidic valve comprising:
• an inlet for receiving a flow of liquid;
• an outlet for outputting the flow of liquid;
• a meniscus anchor between the inlet and the outlet such that the flow from the inlet towards the outlet stops at the meniscus anchor where the liquid forms a meniscus; and,
• a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor such that the flow towards the outlet resumes.
• GVA016.2 Preferably, the meniscus anchor is an aperture and the valve heater extends about the periphery of the aperture.
• GVA016.3 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA016.4 Preferably, the microfluidic valve also has a single flow-path from the inlet to the outlet.
• GVA016.5 Preferably, the microfluidic valve also has:
• a channel formed in a supporting substrate for at least partially defining the flow-path;
• CMOS circuitry positioned between the channel and the supporting substrate for operatively controlling the actuator valve; and,
• at least one sensor responsive to the liquid flow wherein the at least one sensor provides feedback to the CMOS circuitry for use in the operative control of the heater.
• GVA016.6 Preferably, the microfluidic valve also has a roof layer enclosing the channel, wherein the aperture is formed in the roof layer and the heater is supported on the roof layer, outside the channel.
• GVA016.7 Preferably, the at least one sensor is a liquid sensor for sensing the presence or absence of liquid at a position in the channel.
• GVA016.8 Preferably, the roof layer is less than 5 microns thick.
• GVA016.9 Preferably, the heater is less than 4 microns thick.
• GVA016.10 Preferably, the channel is overlaid with a cap defining a cap channel, the cap channel defining the flow-path immediately downstream of the aperture such that unpinning the meniscus fills the cap channel until capillary driven flow to the outlet resumes to the outlet.
• GVA016.11 Preferably, the channel is overlaid with a cap defining a cap channel, the cap channel defining the flow-path immediately upstream of the aperture such that unpinning the meniscus initiates flow from the channel into the cap channel leading to the outlet.
• GVA016.12 Preferably, the channel has a cross sectional area transverse to the flow direction less than 100,000 square microns.
• GVA016.13 Preferably, the channel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GVA016.14 Preferably, the channel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GVA016.15 Preferably, the channel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GVA016.16 Preferably, the heater material is titanium nitride.
• The boiling-initiation valves reliably and manufacturably provide microfluidic valving action, exploiting the intrinsic positive qualities of microfluidic device technologies and avoiding problematic aspects of such technologies.
• GVA017.1 This aspect of the invention provides a fault-tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet;
• a plurality of flow-paths extending from the inlet to the outlet; and,
• a plurality of valves positioned along each of the flow-paths respectively.
• GVA017.2 Preferably, each of the flow-paths is configured for capillary driven flow of the liquid towards the outlet, and each of the valves has a meniscus anchor configured such that the liquid forms a meniscus at the meniscus anchor which stops the liquid flow towards the outlet.
• GVA017.3 Preferably, each of the valves has an actuator for dislodging the meniscus from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA017.4 Preferably, the multiple valve assembly of claim 3 further comprising a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA017.5 Preferably, the sensor in each of the flow-paths is positioned between two of the plurality of valves in each of the flow-paths, and an indication of liquid contact from any of the sensors is used to indicate that the meniscus anchor has failed in all the valves upstream of the sensor.
• GVA017.6 Preferably, the flow-paths are channels through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action.
• GVA017.7 Preferably, each of the valves has a movable member for contacting the liquid, and the actuator is a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA017.8 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA017.9 Preferably, the movable member partially defines the aperture.
• GVA017.10 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA017.11 Preferably, the aperture is a nozzle, and the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA017.12 Preferably, each of the valves has a movable member for contacting the liquid, and the actuator is a thermal expansion actuator for displacing the movable member from a quiescent position to an actuated position such that the meniscus is extended into contact with a surface downstream of the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA017.13 Preferably, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA017.14 Preferably, the movable member at least partially defines the aperture.
• GVA017.15 Preferably, the thermal expansion actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA017.16 Preferably, the aperture is a nozzle, and the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA017.17 Preferably, each of the valves has a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA017.18 Preferably, the meniscus anchor is an aperture and the valve heater extends about the periphery of the aperture.
• GVA017.19 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA017.20 Preferably, the microfluidic device has CMOS circuitry for operative control of each of the valves and detecting faults including failure of the upstream valves to arrest the liquid flow, wherein the valves are supported on the CMOS circuitry and each have electrical contacts extending directly to metal layers within the CMOS circuitry.
• Fault-tolerant multiple-valve assemblies manufacturably provide microfluidic valving action with the requisite level of reliability, through individual valve fault-tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA018.1 This aspect of the invention provides a fault-tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet;
• a plurality of flow-paths extending from the inlet to the outlet;
• a plurality of valves positioned along each of the flow-paths respectively; and,
• a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA018.2 Preferably, each of the flow-paths is configured for capillary driven flow of the liquid towards the outlet, and each of the valves has a meniscus anchor configured such that the liquid forms a meniscus at the meniscus anchor which stops the liquid flow towards the outlet.
• GVA018.3 Preferably, each of the valves has an actuator dislodging the meniscus from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA018.4 Preferably, the sensor in each of the flow-paths is positioned between two of the plurality of valves in each of the flow-paths, and an indication of liquid contact from any of the sensors is used to indicate that the meniscus anchor has failed in all the valves upstream of the sensor.
• GVA018.5 Preferably, the valves have a movable member for contacting the liquid, and a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus such that the liquid flow towards the outlet resumes.
• GVA018.6 Preferably, the channel is configured to draw liquid from the inlet to the outlet by capillary action.
• GVA018.7 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA018.8 Preferably, the movable member at least partially defines the aperture.
• GVA018.9 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA018.10 Preferably, the actuator valve reciprocates the movable member between the quiescent and displaced positions until the channel immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GVA018.11 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA018.12 Preferably, the valves each have a movable member for contacting the liquid, and an actuator for displacing the movable member from a quiescent position to an actuated position where the meniscus is extended into contact with a surface downstream of the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA018.13 Preferably, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA018.14 Preferably, the meniscus anchor is an aperture and the valve heater extends about the periphery of the aperture.
• GVA018.15 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA018.16 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA018.17 Preferably, the multiple valve assembly also has two of the flow-paths.
• GVA018.18 Preferably, the microfluidic device has CMOS circuitry for operative control of the valves and detecting faults including failure of the upstream valves to arrest the liquid flow, wherein the valves are supported on the CMOS circuitry and each have electrical contacts extending directly to metal layers within the CMOS circuitry.
• Fault-tolerant multiple-valve assemblies manufacturably provide microfluidic valving action with the requisite level of reliability, through individual valve fault-tolerance, optimally controlled via liquid detector sensor feedback, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA019.1 This aspect of the invention provides a fault-tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet;
• a plurality of flow-paths extending from the inlet to the outlet; and,
• a plurality of valves positioned along each of the flow-paths respectively; wherein,
• each of the valves has a movable member for contacting the liquid, and a thermal expansion actuator for displacing the movable member to generate a pulse in the liquid to dislodge the meniscus such that the liquid flow towards the outlet resumes.
• GVA019.2 Preferably, each of the flow-paths is configured for capillary driven flow of the liquid towards the outlet, and each of the valves has a meniscus anchor configured such that the liquid forms a meniscus at the meniscus anchor which stops the liquid flow towards the outlet.
• GVA019.3 Preferably, the flow-paths are channels through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action.
• GVA019.4 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA019.5 Preferably, the movable member at least partially defines the aperture.
• GVA019.6 Preferably, the thermal actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA019.7 Preferably, the actuator valve reciprocates the movable member between the quiescent and displaced positions until the channel immediately downstream of the aperture is filled enough for capillary action to re-establish the liquid flow in the flow direction.
• GVA019.8 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA019.9 Preferably, the multiple valve assembly also has a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA019.10 Preferably, the sensor in each of the flow-paths is positioned between two of the plurality of valves in each of the flow-paths, and an indication of liquid contact from any of the sensors is used to indicate that the meniscus anchor has failed in all the valves upstream of the sensor.
• GVA019.11 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA019.12 Preferably, the multiple valve assembly also has two of the flow-paths.
• GVA019.13 Preferably, the microfluidic device has CMOS circuitry for operative control of the valves and detecting faults including failure of the upstream valves to arrest the liquid flow, wherein the valves are supported on the CMOS circuitry and each have electrical contacts extending directly to metal layers within the CMOS circuitry.
• Fault-tolerant multiple thermal bend actuated pressure pulse valve assemblies manufacturably provide microfluidic valving action with the requisite level of reliability, through individual valve fault-tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA020.1 This aspect of the invention provides a fault-tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet;
• a plurality of flow-paths extending from the inlet to the outlet; and,
• a plurality of valves positioned along each of the flow-paths respectively; wherein,
• each of the valves has a meniscus anchor and a meniscus relocation mechanism, the meniscus anchor being configured to form a meniscus that stops the liquid flow towards the outlet, and the meniscus relocation mechanism being configured to deform the meniscus at the meniscus anchor such that the meniscus contacts a surface downstream of the meniscus anchor and the liquid flow towards the outlet resumes.
• GVA020.2 Preferably, the surface downstream of the meniscus anchor is configured to arrest capillary driven flow to the outlet and the meniscus relocation mechanism has a movable member for extending the meniscus from the meniscus anchor to the surface.
• GVA020.3 Preferably, the meniscus relocation mechanism has a thermal expansion actuator for displacing the movable member.
• GVA020.4 Preferably, the flow-paths are channels through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action.
• GVA020.5 Preferably, the movable member is configured for movement between a quiescent position and an actuated position displaced from the quiescent position and the meniscus anchor is an aperture configured to arrest the liquid flow by pinning the meniscus at the aperture.
• GVA020.6 Preferably, the movable member at least partially defines the aperture.
• GVA020.7 Preferably, the thermal expansion actuator has a resistive element for causing differential thermal expansion to move the movable member.
• GVA020.8 Preferably, the movable member is a cantilevered structure with the nozzle at the free end and the resistive element between the nozzle and the fixed end.
• GVA020.9 Preferably, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position.
• GVA020.10 Preferably, the multiple valve assembly also has a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA020.11 Preferably, the sensor in each of the flow-paths is positioned between two of the plurality of valves in each of the flow-paths, and an indication of liquid contact from any of the sensors is used to indicate that the meniscus anchor has failed in all the valves upstream of the sensor.
• GVA020.12 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA020.13 Preferably, the multiple valve assembly also has two of the flow-paths.
• GVA020.14 Preferably, the microfluidic device has CMOS circuitry for operative control of the valves and detecting faults including failure of the upstream valves to arrest the liquid flow, wherein the valves are supported on the CMOS circuitry and each have electrical contacts extending directly to metal layers within the CMOS circuitry.
• Fault-tolerant multiple thermal bend actuated surface tension valve assemblies manufacturably provide microfluidic valving action with the requisite level of reliability, through individual valve fault-tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA021.1 This aspect of the invention provides a fault-tolerant multiple valve assembly for a microfluidic device, the multiple valve assembly comprising:
• an inlet for receiving a liquid flowing through the microfluidic device;
• an outlet downstream of the inlet;
• a plurality of flow-paths extending from the inlet to the outlet; and,
• a plurality of valves positioned along each of the flow-paths respectively; wherein each valve has,
• a meniscus anchor for pinning a meniscus to arrest the flow of the liquid, and a valve heater for heating the meniscus such that the meniscus unpins from the meniscus anchor such that the liquid flow towards the outlet resumes.
• GVA021.2 Preferably, the meniscus anchor is an aperture and the valve heater extends about a periphery of the aperture.
• GVA021.3 Preferably, the valve heater is configured to boil liquid at the meniscus anchor to unpin the meniscus from the meniscus anchor.
• GVA021.4 Preferably, the flow-paths are channels through the microfluidic device, each channel being configured to draw liquid from the inlet to the outlet by capillary action.
• GVA021.5 Preferably, the multiple valve assembly also has a sensor in each of the flow-paths respectively, the sensor being responsive to contact with the liquid.
• GVA021.6 Preferably, the sensor in each of the flow-paths is positioned between two of the plurality of valves in each of the flow-paths, and an indication of liquid contact from any of the sensors is used to indicate that the meniscus anchor has failed in all the valves upstream of the sensor.
• GVA021.7 Preferably, each of the flow-paths has an upstream valve and a downstream valve, the sensor being positioned between the upstream valve and the downstream valves.
• GVA021.8 Preferably, the multiple valve assembly also has two of the flow-paths.
• GVA021.9 Preferably, the microfluidic device has CMOS circuitry for operative control of the valves and detecting faults including failure of the upstream valves to arrest the liquid flow, wherein the valves are supported on the CMOS circuitry and each have electrical contacts extending directly to metal layers within the CMOS circuitry.
• Fault-tolerant multiple boiling-initiation valve assemblies manufacturably provide microfluidic valving action with the requisite level of reliability, through individual valve fault-tolerance, and their design, fabrication, and operation exploit the intrinsic positive qualities of microfluidic device technologies and avoid problematic aspects of such technologies.
• GVA022.1 This aspect of the invention provides a microfluidic device comprising:
• a channel having an inlet, an outlet and an aperture between the inlet and the outlet; wherein,
• the aperture has a geometry that does not allow a meniscus of the fluid flow to anchor to the aperture, such that fluid flow from the inlet towards the outlet is uninterrupted.
• GVA022.2 Preferably, the channel is configured to draw fluid from the inlet to the outlet by capillary action.
• GVA022.3 Preferably, the aperture has a capillary initiation feature for shaping the meniscus at the aperture such that surface tension draws at least part of the meniscus past the aperture and towards the outlet.
• GVA022.4 Preferably, the microfluidic device also has:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate; and,
• a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer; wherein,
• at least one of the fluidic connections between the cap and the MST layer is the aperture.
• GVA022.5 Preferably, the channel extends through the MST layer and the cap connecting at least some of the fluidic connections.
• GVA022.6 Preferably, the MST layer has a roof layer and the aperture is partially formed in the roof layer.
• GVA022.7 Preferably, the roof layer is less than 5 microns thick.
• GVA022.8 Preferably, the channel section within the MST layer has a cross sectional area transverse to the flow direction less than 400 square microns.
• GVA022.9 Preferably, the fluid contains a biological sample including cells of different sizes, and at least one of the fluidic connections is an array of holes sized to prevent passage of cells larger than a predetermined threshold.
• GVA022.10 Preferably, the array of holes is part of a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GVA022.11 Preferably, the holes are configured such that the cells smaller than the predetermined threshold include pathogens.
• GVA022.12 Preferably, the microfluidic device also has a lysis section, the lysis section being configured to lyse pathogens and release genetic material within.
• GVA022.13 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the fluid.
• GVA022.14 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer for operative control of the PCR section.
• GVA022.15 Preferably, the microfluidic device also has an incubation section for incubating the fluid prior to amplifying the nucleic acid sequences in the fluid.
• GVA022.16 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences amplified by the PCR section.
• GVA022.17 Preferably, the microfluidic device also has an array of photodiodes for detecting hybridization of probes within the array of probes.
• GVA022.18 Preferably, the microfluidic device also has bond-pads and is configured for transmission of the hybridization data to an external device.
• GVA022.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GVA022.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The microfluidic aperture provides for reliable unpinned flow-through of fluid between two microfluidic levels, using an easily manufacturable and precise lithographic step.
• GHU001.1 This aspect of the invention provides a microfluidic device for analyzing a fluid sample containing target molecules, the microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate for processing the fluid sample, the MST layer having an array of probes configured to combine with the target molecules to form probe-target complexes; and,
• a humidifier configured to heat water for localized humidification of an area encompassing the MST layer.
• GHU001.2 Preferably, the humidifier has a water reservoir and an evaporator for exposing water supplied by the water reservoir to the area encompassing the MST layer and increasing the vapor pressure of the water in the area.
• GHU001.3 Preferably, the evaporator has an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU001.4 Preferably, the heater is annular and positioned about the aperture.
• GHU001.5 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU001.6 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU001.7 Preferably, the microfluidic device also has a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer.
• GHU001.8 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the cap such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU001.9 Preferably, the cap has a plurality of reagent reservoirs for different reagents.
• GHU001.10 Preferably, the microfluidic device also has a cap channel for a fluid flow, at least some of the reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel reaches the surface tension valve.
• GHU001.11 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU001.12 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of the MST channels and a corresponding plurality the fluidic connections to the cap.
• GHU001.13 Preferably, the cap channel and the reservoirs are formed in a unitary layer of material.
• GHU001.14 Preferably, the cap channel is formed in one surface of the unitary layer such that an outer surface of the MST layer encloses the cap channel.
• GHU001.15 Preferably, the cap has an exterior surface opposite said one surface, the exterior surface having an inlet for receiving fluid and feeding the fluid to the cap channel.
• GHU001.16 Preferably, the fluid sample is a biological sample and at least one of the fluid connections is an array of holes sized and configured to filter out cells larger than a threshold.
• GHU001.17 Preferably, the cap has a waste reservoir for collecting the cells greater than the threshold.
• GHU001.18 Preferably, the microfluidic device also has a dialysis section wherein the biological sample is blood and the array of holes is part of the dialysis section which, during use, removes erythrocytes from the fluid sample.
• GHU001.19 Preferably, the microfluidic device also has a nucleic acid amplification section wherein the target molecules are target nucleic acid sequences, and the nucleic acid amplification section is configured for amplifying the target nucleic acid sequences in the fluid sample.
• GHU001.20 Preferably, the nucleic acid amplification section is in the MST layer and one of the reagent reservoirs is a dNTP, primer and buffer reservoir and another of the reagent reservoirs is a polymerase reservoir, the dNTP, primer and buffer reservoir and the polymerase reservoir being directly connected to the nucleic acid amplification section by at least one of the fluidic connections configured for pinning a meniscus to retain the dNTPs, primers and buffer, and polymerase respectively.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, using an integral humidifier to prevent any dehumidification of the fluids that can interfere with any aspects of the process or analysis.
• GHU002.1 This aspect of the invention provides a microfluidic test module for analyzing genetic material, the test module comprising:
• a casing configured for hand-held portability;
• an array of probes housed in the casing, the probes being configured to hybridize with target nucleic acid sequences to form probe-target hybrids; wherein,
• the casing has a membrane seal for reducing dehumidification within the casing and providing pressure relief from small air pressure fluctuations.
• GHU002.2 Preferably, the microfluidic test module also has a humidifier for increasing the humidity within the casing wherein the array of probes is in a microfluidic device housed in the casing, the microfluidic device having a flow-path leading to the probes, and reagents for processing the genetic material, the flow-path being configured for fluidically connecting the genetic material with the reagents and the probes.
• GHU002.3 Preferably, the microfluidic device has a humidity sensor for sensing the humidity within the casing and the humidity sensor generates an output used for control of the humidifier.
• GHU002.4 Preferably, the microfluidic device has a supporting substrate, a microsystems technology (MST) layer configured for processing the genetic material, the MST layer containing the flow-path and the array of probes, and CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry being connected to the humidity sensor and configured for operative control of the humidifier.
• GHU002.5 Preferably, the microfluidic device has a nucleic acid amplification section for amplifying nucleic acid sequences within the genetic material, and the reagents include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GHU002.6 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU002.7 Preferably, the heater is annular and positioned about the aperture.
• GHU002.8 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU002.9 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU002.10 Preferably, the microfluidic test module also has a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer.
• GHU002.11 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the MST layer such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU002.12 Preferably, the cap has a plurality of reagent reservoirs for the reagents used to process the genetic material.
• GHU002.13 Preferably, the microfluidic test module also has a cap channel for a fluid flow, at least some of the reagent reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel reaches the surface tension valve.
• GHU002.14 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU002.15 Preferably, the surface tension valve for one of the reagent reservoirs has a different number of the MST channels and fluidic connections to the cap than the surface tension valve for another of the reagent reservoirs.
• GHU002.16 Preferably, the microfluidic device has an array of probes for hybridization with target nucleic acid sequences amplified by the nucleic acid amplification section.
• GHU002.17 Preferably, the microfluidic test module also has an array of photodiodes for sensing hybridization of any of the probes in the array.
• GHU002.18 Preferably, the microfluidic test module also has an excitation light source for illuminating the array of probes.
• GHU002.19 Preferably, the CMOS circuitry incorporates the array of photodiodes and is configured to generate a signal indicative of the probes that have hybridized.
• GHU002.20 Preferably, the microfluidic test module also has an electrical connection for transmitting the signal from the CMOS circuitry to an external device.
• The easily usable, mass-producible, inexpensive, and portable microfluidic test module processes and/or analyses fluids, using a membrane seal to prevent any dehumidification of the fluids that can interfere with any aspects of the process or analysis.
• GHU003.1 This aspect of the invention provides a test module for analyzing genetic material, the test module comprising:
• a casing configured for hand-held portability;
• an array of probes housed in the casing, the probes being configured to hybridize with target nucleic acid sequences to form probe-target hybrids; and,
• a humidifier for increasing the humidity within the casing.
• GHU003.2 Preferably, the array of probes is in a microfluidic device housed in the casing, the microfluidic device having a flow-path leading to the probes, and reagents for processing the genetic material, the flow-path being configured for fluidically connecting the genetic material with the reagents and the probes.
• GHU003.3 Preferably, the microfluidic device has a humidity sensor for sensing the humidity within the casing and the humidity sensor generates an output used for control of the humidifier.
• GHU003.4 Preferably, the microfluidic device has a supporting substrate, a microsystems technology (MST) layer configured for processing the genetic material, the MST layer containing the flow-path and the array of probes, and CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry being connected to the humidity sensor and configured for operative control of the humidifier.
• GHU003.5 Preferably, the microfluidic device has a nucleic acid amplification section for amplifying nucleic acid sequences within the genetic material and the reagents include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GHU003.6 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU003.7 Preferably, the heater is annular and positioned about the aperture.
• GHU003.8 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU003.9 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU003.10 Preferably, the test module also has a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer.
• GHU003.11 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the MST layer such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU003.12 Preferably, the cap has a plurality of reagent reservoirs for the reagents used to process the genetic material.
• GHU003.13 Preferably, the test module also has a cap channel for a fluid flow, at least some of the reagent reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel reaches the surface tension valve.
• GHU003.14 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU003.15 Preferably, the surface tension valve for one of the reagent reservoirs has a different number of the MST channels and fluidic connections to the cap than the surface tension valve for another of the reagent reservoirs.
• GHU003.16 Preferably, the microfluidic device has an array of probes for hybridization with target nucleic acid sequences amplified by the nucleic acid amplification section.
• GHU003.17 Preferably, the test module also has an array of photodiodes for sensing hybridization of any of the probes in the array.
• GHU003.18 Preferably, the test module also has an excitation light source for illuminating the array of probes.
• GHU003.19 Preferably, the CMOS circuitry incorporates the array of photodiodes and is configured to generate a signal indicative of the probes that have hybridized.
• GHU003.20 Preferably, the test module also has an electrical connection for transmitting the signal from the CMOS circuitry to an external device.
• The mass-producible, inexpensive, portable genetic test module accepts a biological sample for analysis of its nucleic acid content, using an integral humidifier to prevent any dehumidification of the fluids that can interfere with any aspects of the genetic analysis.
• GHU004.1 This aspect of the invention provides a test module for processing genetic material in a biological sample, the test module comprising:
• a casing;
• a microfluidic device housed in the casing, the microfluidic device having reagents for processing the genetic material;
• a humidifier for increasing the humidity within the casing;
• a humidity sensor for sensing the humidity within the casing; wherein,
• the humidity sensor generates an output used for control of the humidifier.
• GHU004.2 Preferably, the humidity sensor and the humidifier are formed in the microfluidic device.
• GHU004.3 Preferably, the microfluidic device has a supporting substrate, a microsystems technology (MST) layer configured for processing the genetic material, and CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry being connected to the humidity sensor and configured for operative control of the humidifier.
• GHU004.4 Preferably, the microfluidic device has a nucleic acid amplification section for amplifying nucleic acid sequences within the genetic material and the reagents include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GHU004.5 Preferably, the humidifier has a water reservoir and an evaporator.
• GHU004.6 Preferably, the evaporator has an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU004.7 Preferably, the heater is annular and positioned about the aperture.
• GHU004.8 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU004.9 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU004.10 Preferably, the microfluidic device has a cap overlying the MST layer, the cap having a plurality of fluidic connections for fluid communication between the cap and the MST layer.
• GHU004.11 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the cap such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU004.12 Preferably, the cap has a plurality of reagent reservoirs for the reagents used to process the genetic material.
• GHU004.13 Preferably, the cap has a cap channel for a fluid flow, at least some of the reagent reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel reaches the surface tension valve and removes the meniscus.
• GHU004.14 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU004.15 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of the MST channels and a corresponding plurality of the fluidic connections to the cap.
• GHU004.16 Preferably, the microfluidic device has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the nucleic acid amplification section, such that hybridization of the probes with the target nucleic acid sequences forms probe-target hybrids.
• GHU004.17 Preferably, the probe-target hybrids emit a fluorescence signal in response to an excitation light and the microfluidic device has an array of photodiodes for sensing the fluorescence signal from the probe-target hybrids.
• GHU004.18 Preferably, the test module also has an excitation light source for illuminating the array of probes.
• GHU004.19 Preferably, the CMOS circuitry incorporates the array of photodiodes to generate a signal indicative of the probes that have hybridized.
• GHU004.20 Preferably, the test module also has an electrical connection for transmitting the signal from the CMOS circuitry to an external device.
• The mass-producible, inexpensive, portable genetic test module accepts a biological sample for analysis of its nucleic acid content, using an integral humidifier to prevent any dehumidification of the fluids that can interfere with any aspects of the genetic analysis, with the humidifier being optimally controlled via a feedback control system.
• GHU006.1 This aspect of the invention provides a humidity sensor comprising:
• a pair of electrodes spaced adjacent each other and defining an air gap therebetween such that the electrodes provide a capacitor with air as dielectric material; and,
• circuitry for sensing capacitance connected to the electrodes such that changes in permittivity of the air in the air gap caused by changes in humidity, cause changes in capacitance; wherein,
• the circuitry generates a signal indicative of humidity in the air in response to sensed capacitance of the electrodes.
• GHU006.2 Preferably, the electrodes each have a comb-like structure with their respective teeth extending towards each other, and interleaved with each other such that the air gap has a serpentine shape.
• GHU006.3 Preferably, the electrodes are defined in a layer of conductive material supported on a substrate and lithographically etched to provide the comb-like structures.
• GHU006.4 Preferably, the conductive material is titanium nitride and the substrate is a planar surface on a LOC (lab-on-a-chip) device.
• GHU006.5 Preferably, the circuitry is CMOS circuitry on the LOC device.
• The easily manufacturable humidity sensor is used in a closed-loop humidity control system to prevent any dehumidification of the fluids that can interfere with any aspects of the fluidic processing or analysis.
• GHU007.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate for processing a fluid sample; and,
• a humidity sensor for sensing the humidity of an area encompassing the MST layer; wherein,
• the humidity sensor generates an output for humidity control of the area encompassing the MST layer.
• GHU007.2 Preferably, the microfluidic device also has a humidifier configured to heat water for localized humidification of the area encompassing the MST layer.
• GHU007.3 Preferably, the microfluidic device also has reagents for processing target nucleic acid sequences in the fluid sample wherein the MST layer has an array of probes configured to hybridize with the target nucleic acid sequences to form probe-target hybrids.
• GHU007.4 Preferably, the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to receive the output signal from the humidity sensor for operative control of the humidifier.
• GHU007.5 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU007.6 Preferably, the heater is annular and positioned about the aperture.
• GHU007.7 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU007.8 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU007.9 Preferably, the microfluidic device also has a cap overlying the MST layer, the cap having a plurality of fluidic connections for fluid communication between the cap and the MST layer.
• GHU007.10 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the cap such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU007.11 Preferably, the cap has a plurality of reagent reservoirs for the reagents used to process the target nucleic acid sequences.
• GHU007.12 Preferably, the microfluidic device also has a cap channel for a fluid flow, at least some of the reagent reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel removes the meniscus.
• GHU007.13 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU007.14 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of the MST channels and fluidic connections to the cap.
• GHU007.15 Preferably, the reagents include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GHU007.16 Preferably, the microfluidic device also has an array of photodiodes positioned in registration with each of the probes respectively wherein during use the probes emit a fluorescence signal in response to an excitation light to indicate hybridization with the target nucleic acid sequences, the photodiodes being configured for sensing hybridization of any of the probes in the array.
• GHU007.17 Preferably, the probes are fluorescence resonance energy transfer probes, each having a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GHU007.18 Preferably, the photodiodes are less than 249 microns from the array of probes.
• GHU007.19 Preferably, the CMOS circuitry incorporates the array of photodiodes and is configured to generate a signal indicative of the probes that have hybridized.
• GHU007.20 Preferably, the microfluidic device also has bond-pads for communication with an external device.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, using an integral humidifier to prevent any dehumidification of the fluids that can interfere with any aspects of the process or analysis, with the humidifier being optimally controlled via a feedback control system utilizing the humidity sensor.
• GHU008.1 This aspect of the invention provides a test module for analyzing a sample containing nucleic acid sequences, the test module comprising:
• a casing configured for hand-held portability;
• an array of probes housed in the casing, the probes being configured to hybridize with target nucleic acid sequences in the sample to form probe-target hybrids;
• a humidity sensor for sensing the humidity within the casing; wherein,
• the humidity sensor generates an output for humidity control within the casing.
• GHU008.2 Preferably, the test module also has a humidifier configured to heat water for humidifying the casing interior.
• GHU008.3 Preferably, the test module also has a microfluidic device mounted in the casing wherein the microfluidic device incorporates the array of probes, reagents for processing the target nucleic acid sequences, and a flow-path for fluidically connecting the sample with the reagents and the probes.
• GHU008.4 Preferably, the test module also has a microsystems technology (MST) layer configured for processing the target nucleic acid sequences, and a cap overlying the MST layer, the cap having a plurality of fluidic connections for fluid communications between the cap and the MST layer.
• GHU008.5 Preferably, the cap has a plurality of reagent reservoirs for the reagents used to process the target nucleic acid sequences.
• GHU008.6 Preferably, the microfluidic device has a supporting substrate, and CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry being connected to the humidity sensor and configured for operative control of the humidifier.
• 6. The test module test module according to claim 5 wherein the microfluidic device has a nucleic acid amplification section for amplifying target nucleic acid sequences within the sample, and the reagents include one or more of:
• polymerase;
• restriction enzymes;
• dNTPs and primers in buffer;
• lysis reagent; and,
• anticoagulant.
• GHU008.7 Preferably, the humidifier has a water reservoir and an evaporator, the evaporator having an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GHU008.8 Preferably, the heater is annular and positioned about the aperture.
• GHU008.9 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GHU008.10 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GHU008.11 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the cap such that the supply channel connects to the aperture via another of the fluidic connections.
• GHU008.12 Preferably, the test module also has a cap channel for a fluid flow, at least some of the reagent reservoirs being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel removes the meniscus.
• GHU008.13 Preferably, each of the surface tension valves has an MST channel in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GHU008.14 Preferably, the surface tension valve for one of the reagent reservoirs has a different number of the MST channels and fluidic connections to the cap than the surface tension valve for another of the reagent reservoirs.
• GHU008.15 Preferably, the probes emit a fluorescence signal in response to an excitation light to indicate hybridization with the target nucleic acid sequences and the CMOS circuitry has an array of photodiodes for sensing hybridization of any of the probes in the array.
• GHU008.16 Preferably, the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
• GHU008.17 Preferably, the test module also has an excitation light source for illuminating the array of probes.
• GHU008.18 Preferably, the probes are fluorescence resonance energy transfer probes, each having a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GHU008.19 Preferably, the CMOS circuitry incorporates the array of photodiodes and is configured to generate a signal indicative of the probes that have hybridized.
• GHU008.20 Preferably, the test module also has an electrical connection for transmitting the signal from the CMOS circuitry to an external device.
• The mass-producible, inexpensive, portable genetic test module accepts a biological sample for analysis of its nucleic acid content, using an integral humidifier to prevent any dehumidification of the fluids that can interfere with any aspects of the genetic analysis, with the humidifier being optimally controlled via a feedback control system utilizing the humidity sensor.
• GWM001.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate for analyzing a fluid; and,
• a waste reservoir for storage of waste material separated from the fluid.
• GWM001.2 Preferably, the waste reservoir is connected to a waste channel configured to feed waste material into the waste reservoir by capillary action.
• GWM001.3 Preferably, the microfluidic device also has a dialysis section wherein the fluid contains a biological sample that has constituents of different sizes, and the dialysis section separates the constituents of a predetermined size range into the waste material.
• GWM001.4 Preferably, the dialysis section has an array of holes sized to prevent passage of constituents greater than a threshold size.
• GWM001.5 Preferably, the waste reservoir is open to an exterior surface of the microfluidic device such that any excess waste material is removed by an external means.
• GWM001.6 Preferably, the microfluidic device also has a cap overlying the MST layer, the cap having a plurality of fluidic connections for fluid communication between the cap and the MST layer, wherein the waste reservoir and the waste channel are formed in the cap.
• GWM001.7 Preferably, the exterior surface is one side of the cap, the exterior surface having an inlet for receiving the fluid and feeding the fluid to the dialysis section via an input channel.
• GWM001.8 Preferably, the MST layer has a plurality of MST channels and the input channel is in fluid communication with the waste channel via a series of the MST channels in the dialysis section.
• GWM001.9 Preferably, one of the fluidic connections between the input channel and each of the series of MST channels in the dialysis section is the array of holes sized to prevent passage of constituents greater than a threshold size.
• GWM001.10 Preferably, the biological sample is blood and the dialysis section is configured to remove erythrocytes from the fluid.
• GWM001.11 Preferably, the cap has a cap channel leading from the dialysis section, and a plurality of reagent reservoirs, at least one of the reagent reservoirs being in fluid communication with the cap channel via a corresponding surface tension valve configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow removes the meniscus.
• GWM001.12 Preferably, each of the surface tension valves has one of the MST channels in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GWM001.13 Preferably, the surface tension valve for one of the reagent reservoirs has a plurality of the MST channels and corresponding plurality of the fluidic connections to the cap channel.
• GWM001.14 Preferably, the cap channel, the input channel and the reagent reservoirs are formed in a unitary layer of material.
• GWM001.15 Preferably, the cap channel is formed in one surface of the unitary layer such that an outer surface of the MST layer encloses the cap channel and the input channel.
• GWM001.16 Preferably, one of the reagent reservoirs is an anticoagulant reservoir in fluid communication with the input channel via the surface tension valve corresponding to the anticoagulant reservoir such that anticoagulant is mixed with the blood prior to entering the dialysis section.
• GWM001.17 Preferably, the MST layer has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the fluid.
• GWM001.18 Preferably, the MST layer an incubation section for incubating the fluid prior to amplifying the nucleic acid sequences in the fluid.
• GWM001.19 Preferably, the MST layer a hybridization section with an array of probes for hybridization with target nucleic acid sequences in the amplified nucleic acid sequences from the PCR section.
• GWM001.20 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating an array of photodiodes for detecting the fluorescent response from the FRET probes that hybridize.
• The mass-producible and inexpensive microfluidic device analyses fluids, with the waste generated by the device's functioning stored in a self-contained waste storage, obviating any need for expensive subsystems for off-board waste transport and storage, in turn providing for the low system component-count, low system complexity, and simple manufacturing procedures, further reducing the system cost.
• GWM002.1 This aspect of the invention provides a test module for analyzing fluid containing target nucleic acid sequences, the test module comprising:
• a casing for hand-held portability, the casing having a receptacle for receiving the fluid;
• an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids; and,
• a waste accumulator for storage of waste material separated from the fluid; wherein,
• the waste accumulator has a waste reservoir and a porous element to draw the waste material into the waste reservoir by capillary action.
• GWM002.2 Preferably, the test module also has a dialysis section for separating the waste material from the fluid, the fluid having constituents of different sizes, wherein the dialysis section separates the constituents of a predetermined size range into the waste material.
• GWM002.3 Preferably, the test module also has a waste channel from the dialysis section to the waste reservoir wherein the waste reservoir is open to the porous element.
• GWM002.4 Preferably, the dialysis section has an array of holes sized to prevent passage of cells greater than a threshold size.
• GWM002.5 Preferably, the dialysis section, the waste channel and the waste reservoir are formed in a LOC (lab-on-a-chip) device, the waste reservoir being open to an exterior surface of the LOC device and the porous element is a foam pad on the exterior surface to draw the waste material out of the waste reservoir.
• GWM002.6 Preferably, the LOC device has a supporting substrate and an MST (microsystems technology) layer incorporating the array of probes and a cap overlying the MST layer, the cap having a plurality of fluidic connections between the cap and the MST layer for fluid flow from the MST layer to the cap and fluid flow from the cap to the MST layer wherein the waste reservoir and the waste channel are formed in the cap.
• GWM002.7 Preferably, the exterior surface is one side of the cap, the exterior surface having an inlet for receiving the fluid and feeding the fluid to the dialysis section via an input channel.
• GWM002.8 Preferably, the test module also has a plurality of MST channels formed in the MST layer wherein the input channel is in fluid communication with the waste channel via a series of the MST channels in the dialysis section.
• GWM002.9 Preferably, one of the fluidic connections between the input channel and each of the series of MST channels in the dialysis section is the array of holes sized to prevent passage of cells greater than a threshold size.
• GWM002.10 Preferably, the fluid is blood and the dialysis section is configured to remove erythrocytes from the fluid.
• GWM002.11 Preferably, the test module also has a cap channel formed in the cap and a plurality of reagent reservoirs formed in the cap, the reagent reservoirs containing different reagents and each reservoir being in fluid communication with the cap channel via corresponding surface tension valves configured to retain the reagent by surface tension in a meniscus of the reagent until the fluid flow in the cap channel reaches the surface tension valve.
• GWM002.12 Preferably, each of the surface tension valves has one of the MST channels in the MST layer, a fluidic connection to the reagent reservoir and another fluidic connection to the cap channel such that the meniscus pins at the fluidic connection with the cap channel.
• GWM002.13 Preferably, the surface tension valve for one of the reagent reservoirs has a different number of the MST channels and the fluidic connections to the cap than the surface tension valve for another of the reagent reservoirs.
• GWM002.14 Preferably, the cap channel, the input channel and the reagent reservoirs are formed in a unitary layer of material.
• GWM002.15 Preferably, the cap channel and the input channel are formed in one surface of the unitary layer such that an outer surface of the MST layer encloses the cap channel and the input channel.
• GWM002.16 Preferably, one of the reagent reservoirs is an anticoagulant reservoir in fluid communication with the input channel via the surface tension valve corresponding to the anticoagulant reservoir such that anticoagulant is mixed with the blood prior to entering the dialysis section.
• GWM002.17 Preferably, the test module also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the fluid.
• GWM002.18 Preferably, the test module also has an incubation section for incubating the fluid prior to amplifying the nucleic acid sequences in the fluid.
• GWM002.19 Preferably, the test module also has a LED excitation light wherein the probes are FRET (fluorescence resonance energy transfer) probes that hybridize with target nucleic acid sequences and emit a fluorescence signal in response to excitation light from the excitation LED.
• GWM002.20 Preferably, the microfluidic device also has CMOS circuitry positioned between the MST layer and the supporting substrate, the CMOS circuitry incorporating an array of photodiodes for detecting the fluorescent response from the FRET probes that hybridize.
• The mass-producible, inexpensive, portable genetic test module accepts a biological sample for analysis of its nucleic acid content, with the waste generated by the module's functioning stored in a waste storage juxtaposed to a porous element. This waste management system provides for large-volume waste storage and capillary effect propulsion, while maintaining the low system component-count, low system complexity, and simple manufacturing procedures, further reducing the system cost.
• GDI001.1 This aspect of the invention provides a microfluidic device for dialysis of biological material, the microfluidic device comprising:
• a sample inlet for receiving a sample of biological material that has constituents of different sizes;
• a dialysis section for separating smaller constituents from larger constituents, the smaller constituents being smaller than a predetermined size threshold, and the larger constituents being larger than the predetermined size threshold; and,
• a microsystems technology (MST) layer for separately processing the smaller constituents and/or the larger constituents.
• GDI001.2 Preferably, the microfluidic device also has a first channel and a second channel and a plurality of apertures sized to correspond to the predetermined size threshold, wherein the first and second channels are in fluid communication via the plurality of apertures and the inlet is in fluid communication with the first channel such that only the smaller constituents flow to the second channel.
• GDI001.3 Preferably, the first and second channels are configured to fill with the sample received at the inlet by capillary action.
• GDI001.4 Preferably, the smaller constituents include targets such that processing of the smaller constituents by the MST layer includes detection of the targets.
• GDI001.5 Preferably, the targets are target cells and the MST layer has a lysis section connected to the target channel for lysing the target cells to release target nucleic acid sequences therein.
• GDI001.6 Preferably, the targets are target nucleic acid sequences and the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI001.7 Preferably, the microfluidic device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI001.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI001.9 Preferably, the microfluidic device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI001.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI001.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI001.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI001.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI001.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI001.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI001.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI001.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI001.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI001.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI001.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI002.1 This aspect of the invention provides a microfluidic device for dialysis of a fluid sample, the microfluidic device comprising:
• a first layer of material defining a first channel and a second channel, the first channel configured for receiving the sample which contains constituents of different sizes;
• a second layer having a plurality of apertures open to the first channel and at least one fluid connection leading from the apertures to the second channel for establishing fluid communication between the first channel and the second channel; wherein,
• the apertures are sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI002.2 Preferably, the second layer is a laminate with a roof layer, and the at least one fluid connection is a series of adjacent channels enclosed by the roof layer, the apertures being formed in the roof layer between the first channel and the adjacent channels.
• GDI002.3 Preferably, the first and second channels, and the series of adjacent channels are configured to fill with the sample by capillary action.
• GDI002.4 Preferably, the microfluidic device also has a MST layer for processing the sample wherein the smaller constituents include targets such that processing of the smaller constituents by the MST layer includes detection of the targets.
• GDI002.5 Preferably, the targets are target cells and the MST layer has a lysis section connected to the target channel for lysing the target cells to release target nucleic acid sequences therein.
• GDI002.6 Preferably, the targets are target nucleic acid sequences and the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI002.7 Preferably, the microfluidic device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI002.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI002.9 Preferably, the microfluidic device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI002.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI002.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI002.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI002.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI002.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI002.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI002.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI002.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI002.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI002.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI002.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI003.1 This aspect of the invention provides a microfluidic device for processing a fluid sample, the microfluidic device comprising:
• a first channel configured to fill with the sample by capillary action;
• a second channel configured to fill with the sample by capillary action;
• a plurality of fluid connections between the first channel and the second channel, each of the fluid connections being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel;
• a bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the plurality of fluid connections and is configured for uninterrupted capillary driven flow from the first channel to the second channel; wherein during use,
• flow from the bypass channel reaches the meniscus pinned at each of the fluid connections after the meniscus has formed such that the flow sequentially removes each of the menisci and sample flow from the first channel to the second channel is via the plurality of fluid connections as well as the bypass channel.
• GDI003.2 Preferably, the sample is a biological sample containing constituents of different sizes and the plurality of fluid connections and the bypass channel have apertures sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI003.3 Preferably, the first channel, the second channel, the plurality of fluid connections and the bypass channel are parts of a dialysis section within the microfluidic device for separately processing the smaller constituents and or the larger constituents.
• GDI003.4 Preferably, the biological sample is blood and the larger constituents include leukocytes and the smaller constituents include erythrocytes.
• GDI003.5 Preferably, the microfluidic device also has an MST layer for analysis of genetic material in the leukocytes wherein the MST layer has a lysis section for lysing the leukocytes to release target nucleic acid sequences therein.
• GDI003.6 Preferably, the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid.
• GDI003.7 Preferably, the microfluidic device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI003.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI003.9 Preferably, the microfluidic device also has CMOS circuitry for operative control of the PCR section, the CMOS circuitry having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI003.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI003.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI003.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the sample, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI003.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI003.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI003.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI003.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI003.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI003.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI003.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI003.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system. The special flow-channel structure provides for capillary-driven priming of the dialysis section without trapped air bubbles.
• GDI004.1 This aspect of the invention provides a lab-on-a-chip (LOC) device to for analyzing leukocytes in a blood sample, the LOC device comprising:
• a supporting substrate;
• an inlet for receiving a sample of blood including leukocytes and erythrocytes;
• a dialysis section for separating at least some of the erythrocytes from the leukocytes; and,
• a microsystems technology (MST) layer for separately analyzing the leukocytes; wherein,
• the inlet, the dialysis section and the MST layer are all supported on the supporting substrate.
• GDI004.2 Preferably, the dialysis section has a first channel and a second channel and a plurality of apertures sized to correspond to a predetermined size threshold, wherein the first and second channels are in fluid communication via the plurality of apertures and the inlet is in fluid communication with the first channel such that the erythrocytes flow to the second channel while the leukocytes remain in the first channel.
• GDI004.3 Preferably, the first and second channels are configured to fill with the sample received at the inlet by capillary action.
• GDI004.4 Preferably, the LOC device also has a first layer in which the first channel and the second channel are defined, and a second layer in which the apertures are defined, the second layer having at least one fluid connection from the apertures to the second channel for establishing fluid communication between the first channel and the second channel.
• GDI004.5 Preferably, the leukocytes contain target nucleic acid sequences and the MST layer has a lysis section for lysing the leukocytes to release the target nucleic acid sequences therein.
• GDI004.6 Preferably, the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI004.7 Preferably, the LOC device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI004.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI004.9 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI004.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI004.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI004.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI004.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI004.14 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI004.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI004.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI004.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI004.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI004.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI004.20 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive LOC device accepts a blood sample, uses a dialysis section for separating the leukocytes, and separately processes the nucleic acid content of the leukocytes separated from the blood sample.
• The dialysis section functionality extracts additional information from the blood sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI005.1 This aspect of the invention provides a lab-on-a-chip (LOC) device to separate pathogens from a biological sample for analysis, the LOC device comprising:
• a supporting substrate;
• an inlet for receiving the sample including pathogens and other constituents larger than the pathogens;
• a dialysis section for separating the pathogens into a portion of the sample; and,
• a microsystems technology (MST) layer for separately analyzing the pathogens; wherein,
• the inlet, the dialysis section and the MST layer are all supported on the supporting substrate.
• GDI005.2 Preferably, the dialysis section has a first channel and a second channel and a plurality of apertures sized to correspond to a predetermined size threshold, wherein the first and second channels are in fluid communication via the plurality of apertures and the inlet is in fluid communication with the first channel such that the pathogens flow to the second channel while the constituents larger than the predetermined size threshold remain in the first channel.
• GDI005.3 Preferably, the first and second channels are configured to fill with the sample received at the inlet by capillary action.
• GDI005.4 Preferably, the LOC device also has a first layer in which the first channel and the second channel are defined, and a second layer in which the apertures are defined, the second layer having at least one fluid connection from the apertures to the second channel for establishing fluid communication between the first channel and the second channel.
• GDI005.5 Preferably, the pathogens contain target nucleic acid sequences and the MST layer has a lysis section for lysing the pathogens to release the target nucleic acid sequences therein.
• GDI005.6 Preferably, the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI005.7 Preferably, the LOC device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI005.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI005.9 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI005.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI005.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI005.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI005.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI005.14 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI005.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI005.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI005.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI005.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI005.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI005.20 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample, uses a dialysis section for separating the pathogens in the sample, and separately processes the nucleic acid content of the pathogens separated from the sample.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI006.1 This aspect of the invention provides a lab-on-a-chip (LOC) device to separate pathogens and leukocytes from a blood sample for analysis, the LOC device comprising:
• a sample inlet for receiving the blood including pathogens, leukocytes and other constituents;
• a dialysis section for separating the pathogens into a first portion of the sample and separating the leukocytes into a second portion of the sample such that the remainder of the sample has constituents smaller than the leukocytes and larger than the pathogens; and,
• a microsystems technology (MST) layer for analyzing the pathogens and the leukocytes.
• GDI006.2 Preferably, the dialysis section has a leukocyte channel, a pathogen channel and a third channel for the remainder of the sample, the leukocyte channel being connected to the third channel by a first set of apertures and the pathogen channel being connected to the third channel by a second set of apertures, the first set of apertures being larger than the pathogens but smaller than the leukocytes, and the second set of apertures being larger than the pathogens but smaller than the constituents in the remainder of the sample.
• GDI006.3 Preferably, the leukocyte channel, the pathogen channel, the third channel and the plurality of apertures are configured to fill with the sample by capillary action.
• GDI006.4 Preferably, the leukocyte channel is connected to the sample inlet at an upstream end and connected to the MST layer downstream of the dialysis section, the pathogen channel is separately connected to the MST layer downstream of the dialysis section and the third channel is connected to a waste reservoir downstream of the dialysis section.
• GDI006.5 Preferably, the MST layer has a pathogen nucleic acid amplification section for amplifying pathogen targets which are target nucleic acid sequences in the pathogens, and a leukocyte nucleic acid amplification section for amplifying leukocyte targets which are target nucleic acid sequences in the leukocytes.
• GDI006.6 Preferably, the LOC device also has a pathogen lysis section upstream of the pathogen nucleic acid amplification section for lysing the pathogens to release the genetic material within, and a leukocyte lysis section upstream of the leukocyte nucleic acid amplification section for lysing the leukocytes to release the genetic material within.
• GDI006.7 Preferably, the LOC device also has a hybridization section having an array of probes including pathogen probes and leukocyte probes, wherein the pathogen probes are for hybridization with the pathogen targets and the leukocyte probes are for hybridization with the leukocyte targets.
• GDI006.8 Preferably, the probes are configured to form probe-target hybrids with the pathogen and leukocyte targets, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI006.9 Preferably, the LOC device also has CMOS circuitry for operative control of the pathogen and the leukocyte PCR sections, the CMOS circuitry having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI006.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI006.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI006.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI006.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI006.14 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI006.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI006.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI006.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI006.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI006.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI006.20 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive LOC device accepts a blood sample, uses a dialysis section for separating the leukocytes and pathogens from the sample, and separately processes the nucleic acid content of the leukocytes and pathogens separated from the blood sample.
• The dialysis section functionality extracts additional information from the blood sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI007.1 This aspect of the invention provides a lab-on-a-chip (LOC) device to separate constituents of intermediate size from larger and smaller constituents in a biological sample, the LOC device comprising:
• an inlet for receiving the biological sample including large constituents sized above an upper threshold, small constituents sized smaller than a lower threshold and intermediate constituents sized between the upper and the lower thresholds;
• a first channel in fluid communication with the inlet;
• first apertures sized to correspond to the upper threshold;
• a second channel in fluid communication with the first channel via the first apertures;
• second apertures sized to correspond to the lower threshold;
• a third channel in fluid communication with the second channel via the second apertures; and,
• a microsystems technology (MST) layer for analyzing the large, intermediate and or small constituents separately; wherein during use,
• the intermediate and the small constituents flow into the second channel via the first apertures while the large constituents are retained in the first channel, and the small constituents flow into the third channel while the intermediate constituents are retained in the second channel.
• GDI007.2 Preferably, the first channel, the second channel and the third channel are configured to fill with the sample received in the inlet by capillary action.
• GDI007.3 Preferably, the biological material is blood and the large constituents include leukocytes, and the MST layer is configured for detecting leukocyte targets, the leukocyte targets being target nucleic acid sequences within the leukocytes.
• GDI007.4 Preferably, the MST layer has a first lysis section for lysing the leukocytes to release the leukocyte targets therein.
• GDI007.5 Preferably, the small constituents include pathogens and the MST layer is configured for detecting pathogen targets, the pathogen targets being target nucleic acid sequences within the pathogens.
• GDI007.6 Preferably, the MST layer has a second lysis section for lysing the pathogens to release the pathogen targets therein.
• GDI007.7 Preferably, the intermediate constituents include erythrocytes and the MST layer is configured for detecting target proteins within the erythrocytes.
• GDI007.8 Preferably, the MST layer has an array probes for hybridization with the leukocyte targets and the pathogen targets to form probe-target hybrids, and hybridization or conjugation with the protein targets to form probe-target complexes.
• GDI007.9 Preferably, the LOC device also has a first nucleic acid amplification section for amplifying the leukocyte targets and a second nucleic acid amplification section for amplifying the pathogen targets.
• GDI007.10 Preferably, the LOC device also has CMOS circuitry for individual control of the first nucleic acid amplification section and the second nucleic acid amplification section.
• GDI007.11 Preferably, the probe-target hybrids are configured to fluoresce in response to an excitation light and the CMOS circuitry has a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI007.12 Preferably, the MST layer has a hybridization chamber array for containing the array of probes, and the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
• GDI007.13 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI007.14 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI007.15 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI007.16 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI007.17 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI007.18 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI007.19 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI007.20 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample, uses a dialysis section for separating cells of an intermediate size from larger and smaller cells in the sample, and separately processes the nucleic acid content of the separated cells.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GDI009.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for concentrating nucleated cells in a biological sample, the LOC device comprising:
• an inlet for receiving a sample of biological material including nucleated cells and other constituents, the nucleated cells all being larger than a predetermined threshold;
• a dialysis section for concentrating the nucleated cells into a portion of the sample such that the remainder of the sample contains the other constituents smaller than the predetermined threshold; and,
• a microsystems technology (MST) layer for processing the nucleated cells.
• GDI009.2 Preferably, the dialysis section has a target channel, a waste channel and a plurality of apertures fluidically connecting the target channel and the waste channel, the plurality of apertures being sized to correspond to the predetermined threshold.
• GDI009.3 Preferably, the target channel, the waste channel and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GDI009.4 Preferably, the target channel has an upstream end connected to the inlet and a downstream end connected to the MST layer.
• GDI009.5 Preferably, the waste channel has a downstream end connected to a waste reservoir.
• GDI009.6 Preferably, the MST layer has a nucleic acid amplification section for amplifying target nucleic acid sequences in the nucleated cells.
• GDI009.7 Preferably, the LOC device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI009.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI009.9 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI009.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI009.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI009.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI009.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI009.14 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI009.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI009.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI009.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI009.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI009.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI009.20 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• This LOC device design has the advantage of directly selecting the component of the sample which contains the target. This LOC device design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the LOC device. This LOC device design has the advantage of removing components of the sample which can inhibit later analytical steps. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This LOC device design has the advantage of removing components of the mixture which might clog the chambers or connections within the LOC device and degrade operation.
• GDI010.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for removing cell debris from a biological sample, the LOC device comprising:
• a dialysis section with a large constituent channel, a small constituent channel and a plurality of apertures for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel having an upstream end for receiving the biological sample, the biological sample being a liquid carrying a mixture of cell debris and target molecules, the small constituent channel having a downstream end for connection to a hybridization section with an array of probes for reaction with the target molecules to form probe-target complexes; wherein,
• the apertures are sized to allow the target molecules to flow into the small constituent channel but retain the cell debris larger than a threshold size in the large constituent channel.
• GDI010.2 Preferably, the large constituent channel and the small constituent channel have a common sidewall, and the plurality of apertures is a series of stoma extending through the common sidewall, each stoma having a small opening to the large constituent channel and inverse tapering to a large opening to the small constituent channel.
• GDI010.3 Preferably, the LOC device also has a plurality of the small constituent channels each sharing a common sidewall with the large constituent channel and fluidically connecting via a series of stoma.
• GDI010.4 Preferably, the small opening of each of the stoma has a height and a width dimension between 1 micron and 8 microns.
• GDI010.5 Preferably, the LOC device also has a waste reservoir wherein the large constituent channel has a downstream end connected to the waste reservoir.
• GDI010.6 Preferably, the LOC device also has a lysis section upstream of the dialysis section wherein the target molecules are target nucleic acid sequences and the lysis section is configured to lyse cells in the biological sample and release the target nucleic acid sequences therein.
• GDI010.7 Preferably, the LOC device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI010.8 Preferably, the probes are configured to hybridize with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI010.9 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI010.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI010.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI010.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI010.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI010.14 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI010.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI010.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI010.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI010.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the pre-programmed delay.
• GDI010.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI010.20 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• This LOC device design has the advantage of directly selecting the component of the sample which contains the target. This LOC device design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the LOC device. This LOC device design has the advantage of removing components of the sample which can inhibit later analytical steps. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This LOC device design has the advantage of removing components of the mixture which might clog the chambers or connections within the LOC device and degrade operation.
• GDI011.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for removing cell debris from a biological sample containing target nucleic acid sequences, the LOC device comprising:
• a dialysis section with a large constituent channel, a small constituent channel and a plurality of apertures for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel having an upstream end for receiving the biological sample, the biological sample being a liquid carrying a mixture of cell debris and target nucleic acid sequences, and the small constituent channel having a downstream end for connection to a hybridization section with an array of probes for hybridization with the target nucleic acid sequences to form probe-target complexes; wherein,
• the apertures are sized to allow the target nucleic acid sequences to flow into the small constituent channel but retain the cell debris larger than a threshold size in the large constituent channel.
• GDI011.2 Preferably, the large constituent channel and the small constituent channel have a common sidewall, and the plurality of apertures is a series of stoma extending through the common sidewall, each stoma having a small opening to the large constituent channel and tapering to a large opening to the small constituent channel.
• GDI011.3 Preferably, the LOC device also has a plurality of the small constituent channels each sharing a common sidewall with the large constituent channel and fluidically connecting via a series of stoma.
• GDI011.4 Preferably, the small opening of each of the stoma has a height and a width dimension between 1 micron and 8 microns.
• GDI011.5 Preferably, the LOC device also has a waste reservoir wherein the large constituent channel has a downstream end connected to the waste reservoir.
• GDI011.6 Preferably, the LOC device also has a lysis section upstream of the amplification section wherein the lysis section is configured to lyse cells in the biological sample and release the target nucleic acid sequences therein.
• GDI011.7 Preferably, the LOC device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI011.8 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI011.9 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI011.10 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI011.11 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI011.12 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI011.13 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI011.14 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI011.15 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI011.16 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI011.17 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI011.18 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI011.19 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• GDI011.20 Preferably, the reagent reservoirs each have a surface tension valve with an aperture configured to pin a meniscus that retains liquid reagents therein until contact with the sample removes the meniscus and the liquid reagents are added to the sample flow.
• This LOC device design has the advantage of removing components of the sample which can inhibit later analytical steps. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This LOC device design has the advantage of removing components of the mixture which might clog the chambers or connections within the LOC device and degrade operation.
• GDI013.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for removing cell debris from a biological sample containing target nucleic acid sequences, the LOC device comprising:
• an amplification section for amplifying the nucleic acid sequences;
• a dialysis section with a large constituent channel, a small constituent channel and a plurality of apertures for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel configured to receive the biological sample after nucleic acid amplification in the amplification section, the biological sample being a liquid mixture of the cell debris and the target nucleic acid sequences and the apertures are sized to allow the target nucleic acid sequences to flow into the small constituent channel while the cell debris larger than a threshold size is retained in the large constituent channel; and,
• an array of probes in fluid communication with the small constituent channel for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GDI013.2 Preferably, the large constituent channel and the small constituent channel have a common sidewall, and the plurality of apertures is a series of stoma extending through the common sidewall, each stoma having a small opening to the large constituent channel and tapering to a large opening to the small constituent channel.
• GDI013.3 Preferably, the dialysis section has a plurality of the small constituent channels each sharing a common sidewall with the large constituent channel and fluidically connecting via a series of stoma.
• GDI013.4 Preferably, the small opening of each of the stoma has a height and a width dimension between 1 micron and 8 microns.
• GDI013.5 Preferably, the LOC device also has a waste reservoir wherein the large constituent channel has a downstream end connected to the waste reservoir.
• GDI013.6 Preferably, the LOC device also has a lysis section upstream of the amplification section wherein the lysis section is configured to lyse cells in the biological sample and release the target nucleic acid sequences therein.
• GDI013.7 Preferably, the probe-target hybrids are fluorescent in response to an excitation light.
• GDI013.8 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI013.9 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI013.10 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI013.11 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI013.12 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI013.13 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI013.14 Preferably, each photodiode is less than 249 microns from the corresponding hybridization chamber.
• GDI013.15 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI013.16 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI013.17 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI013.18 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI013.19 Preferably, the LOC device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• GDI013.20 Preferably, the reagent reservoirs each have a corresponding outlet valve; wherein,
• the outlet valves are surface tension valves, each having an aperture configured to pin a meniscus that retains liquid reagents therein until contact with the sample removes the meniscus and the liquid reagents are added to the sample flow.
• This LOC device design has the advantage of directly selecting the component of the sample which contains the target. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This LOC device design has the advantage of removing components of the sample which can inhibit later analytical steps. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This LOC device design has the advantage of removing components of the mixture which might clog the chambers or connections within the LOC device and degrade operation.
• GDI014.1 This aspect of the invention provides a microfluidic device for processing a fluid sample, the microfluidic device comprising:
• a first channel configured to fill with the sample by capillary action;
• a second channel configured to fill with the sample by capillary action;
• a plurality of fluid connections between the first channel and the second channel, one of the fluid connections having an active valve to arrest the sample flow into the second channel and the remaining fluid connections each being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel, the fluid connection with the active valve being upstream of the remaining fluid connections; wherein during use,
• the sample flow into the second channel is initiated by activation of the active valve such that the sample flow in the second channel progressively removes the meniscus at each of the meniscus anchors.
• GDI014.2 Preferably, one of the remaining fluid connections has a liquid sensor for triggering the activation of the active valve.
• GDI014.3 Preferably, the sample is a biological sample containing constituents of different sizes and the plurality of fluid connections have apertures sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI014.4 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor to pin a meniscus of the fluid which arrests the flow, and a heater positioned adjacent the meniscus anchor such that activation of the heater boils the fluid at the meniscus anchor to unpin the meniscus such that the flow resumes.
• GDI014.5 Preferably, the active valve has a meniscus anchor to pin a meniscus of the fluid which arrests the flow, and a bend actuator configured to bend upon activation and unpin the meniscus from the meniscus anchor such that the flow resumes.
• GDI014.6 Preferably, the first channel, the second channel and the plurality of fluid connections are parts of a dialysis section within the microfluidic device for separately processing the smaller constituents and or the larger constituents.
• GDI014.7 Preferably, the biological sample is blood and the larger constituents include leukocytes and the smaller constituents include erythrocytes.
• GDI014.8 Preferably, the microfluidic device also has an MST layer for analysis of genetic material in the leukocytes wherein the MST layer has a lysis section for lysing the leukocytes to release target nucleic acid sequences therein.
• GDI014.9 Preferably, the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid.
• GDI014.10 Preferably, the microfluidic device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI014.11 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI014.12 Preferably, the microfluidic device also has CMOS circuitry for operative control of the PCR section, the CMOS circuitry having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI014.13 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI014.14 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI014.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI014.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI014.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI014.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI014.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI014.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system. The special flow-channel structure with an active valve provides for capillary-driven priming of the dialysis section without trapped air bubbles.
• GDI015.1 This aspect of the invention provides a microfluidic device for processing a sample fluid containing target molecules, the microfluidic device comprising:
• a dialysis device for receiving the sample and concentrating the target molecules in a portion of the sample;
• a lab-on-a-chip (LOC) for analyzing the target molecules; and,
• a cap overlaying the LOC and the dialysis device for establishing fluid communication between the LOC and the dialysis device.
• GDI015.2 Preferably, the fluid sample is a sample of biological material including cells of different sizes, and the dialysis device has at least two channels with a plurality of apertures fluidically connecting the channels, the plurality of apertures being sized to correspond to a predetermined threshold size of the cells in the fluid sample.
• GDI015.3 Preferably, the at least two channels and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GDI015.4 Preferably, the at least two channels includes a target channel and a waste channel, the target channel being connected to the cap for capillary driven flow to the LOC.
• GDI015.5 Preferably, the target molecules are target nucleic acid sequences within cells in the sample fluid, and the LOC has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI015.6 Preferably, the target nucleic acid sequences are within the cells smaller than the predetermined threshold.
• GDI015.7 Preferably, the LOC has a hybridization section with an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GDI015.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being configured to emit photons of light in response to an excitation electric current.
• GDI015.9 Preferably, the LOC has CMOS circuitry for operative control of the PCR section, the CMOS circuitry having a photosensor for sensing photons emitted by the probe-target hybrids.
• GDI015.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI015.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI015.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI015.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI015.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI015.15 Preferably, the photodiodes are less than 1600 microns from the corresponding hybridization chamber.
• GDI015.16 Preferably, the probes have an electrochemiluminescent (ECL) luminophore that emits a photon when in an excited state.
• GDI015.17 Preferably, the hybridization chambers have electrodes for exciting the ECL luminophores with electrical current.
• GDI015.18 Preferably, the ECL probes each have a luminophore and a quencher positioned proximate the luminophore for quenching photons emitted by the luminophore such that hybridization with one of the target nucleic acid sequences moves the quencher away from the luminophore such that the photons are not quenched.
• GDI015.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI015.20 Preferably, the cap has at least one channel for fluid communication between the dialysis device and the LOC, and a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic multichip assembly accepts a biochemical sample, uses a dialysis chip for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The microfluidic multichip assembly provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI016.1 This aspect of the invention provides a dialysis device for dialysis of a fluid containing constituents of different sizes, the dialysis device comprising:
• a first layer of material defining a first channel and a second channel, the first channel configured for receiving the fluid which contains constituents of different sizes;
• a second layer having a plurality of apertures open to the first channel and at least one fluid connection leading from the apertures to the second channel for establishing fluid communication between the first channel and the second channel; wherein,
• the apertures are sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI016.2 Preferably, the second layer is a laminate with a roof layer, and the at least one fluid connection is a series of adjacent channels enclosed by the roof layer, the apertures being formed in the roof layer between the first channel and the adjacent channels.
• GDI016.3 Preferably, the first and second channels, and the series of adjacent channels are configured to fill with the sample by capillary action.
• GDI016.4 Preferably, the dialysis device also has a bypass channel between the first channel and the second channel, wherein each of the adjacent channels are configured to pin a meniscus of the fluid that arrests capillary flow between the first channel and the second channel, and the bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the series of adjacent channels and is configured for uninterrupted capillary driven flow from the first channel to the second channel, such that flow from the bypass channel reaches the meniscus pinned at each of the adjacent channels after the meniscus has formed to sequentially remove each of the menisci and flow from the first channel to the second channel is via the adjacent channels as well as the bypass channel.
• GDI016.5 Preferably, the sample is a biological sample containing constituents of different sizes and the adjacent channels and the bypass channel have apertures sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI016.6 Preferably, the biological sample is blood and the larger constituents include leukocytes and the smaller constituents include erythrocytes.
• GDI016.7 Preferably, the adjacent channels are a series of parallel, adjacent channels extending normal to the first channel and the second channel.
• GDI016.8 Preferably, the dialysis device also has a waste reservoir for receiving constituents not required for downstream processing or analysis.
• GDI016.9 Preferably, the apertures are holes with diameters less than 8 microns.
• GDI016.10 Preferably, the smaller constituents include targets such that processing of the smaller constituents includes detection of the targets.
• GDI016.11 Preferably, the dialysis device also has a lysis section connected to the target channel for lysing the target cells to release target nucleic acid sequences therein.
• GDI016.12 Preferably, the dialysis device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI016.13 Preferably, the dialysis device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI016.14 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being electrochemiluminescent in response to an electrical pulse.
• GDI016.15 Preferably, the dialysis device also has CMOS circuitry for operative control of the nucleic acid amplification section and the hybridization section, the CMOS circuitry also having a photosensor for sensing electrochemiluminescence emission from the probe-target hybrids.
• GDI016.16 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI016.17 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI016.18 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI016.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI016.20 Preferably, the dialysis device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic multichip assembly accepts a biochemical sample, uses a dialysis chip for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The microfluidic multichip assembly provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI017.1 This aspect of the invention provides a dialysis device with capillary-driven, flow-channel structure for dialysis of a fluid containing constituents of different sizes, the dialysis device comprising:
• a first channel configured to fill with the fluid by capillary action;
• a second channel configured to fill with the fluid by capillary action;
• a plurality of fluid connections between the first channel and the second channel, each of the fluid connections being configured to pin a meniscus of the fluid that arrests capillary flow between the first channel and the second channel;
• a bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the plurality of fluid connections and is configured for uninterrupted capillary driven flow from the first channel to the second channel; wherein during use,
• flow from the bypass channel reaches the meniscus pinned at each of the fluid connections after the meniscus has formed such that the flow sequentially removes each of the menisci and sample flow from the first channel to the second channel is via the plurality of fluid connections as well as the bypass channel.
• GDI017.2 Preferably, the fluid is a biological sample containing constituents of different sizes and the plurality of fluid connections and the bypass channel have apertures sized in accordance with a predetermined size threshold such that the constituents flowing to the second channel are smaller constituents that are smaller than the predetermined size threshold, and the constituents retained in the first channel include larger constituents which are larger than the predetermined size threshold.
• GDI017.3 Preferably, the biological sample is blood and the larger constituents include leukocytes and the smaller constituents include erythrocytes.
• GDI017.4 Preferably, the dialysis device also has:
• a first layer defining the first channel and the second channel; and,
• a second layer defining the plurality of fluid connections and the bypass channel.
• GDI017.5 Preferably, the second layer is a laminate with a roof layer, and the plurality of fluid connections is a series of adjacent channels enclosed by the roof layer, the apertures being formed in the roof layer between the first channel and the adjacent channels.
• GDI017.6 Preferably, the adjacent channels are a series of parallel, adjacent channels extending normal to the first channel and the second channel.
• GDI017.7 Preferably, the dialysis device also has a waste reservoir for receiving constituents not required for downstream processing or analysis.
• GDI017.8 Preferably, the apertures are holes with diameters less than 8 microns.
• GDI017.9 Preferably, the smaller constituents include targets such that processing of the smaller constituents includes detection of the targets.
• GDI017.10 Preferably, the dialysis device also has a lysis section connected to the target channel for lysing the target cells to release target nucleic acid sequences therein.
• GDI017.11 Preferably, the dialysis device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI017.12 Preferably, the dialysis device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI017.13 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being electrochemiluminescent in response to an electrical pulse.
• GDI017.14 Preferably, the dialysis device also has CMOS circuitry for operative control of the nucleic acid amplification section and the hybridization section, the CMOS circuitry also having a photosensor for sensing electrochemiluminescence emission from the probe-target hybrids.
• GDI017.15 Preferably, the CMOS circuitry is configured to provide the electrical pulse to the electrodes.
• GDI017.16 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI017.17 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI017.18 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI017.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI017.20 Preferably, the dialysis device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic multichip assembly accepts a biochemical sample, uses a dialysis chip for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The microfluidic multichip assembly provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• The special flow-channel structure provides for capillary-driven priming of the dialysis chip without trapped air bubbles.
• GDI019.1 This aspect of the invention provides a dialysis device to separate pathogens from a biological sample, the dialysis device comprising:
• a first channel for receiving the biological sample;
• a second channel; and,
• a plurality of apertures; wherein,
• the second channel is fluidically connected to the first channel via the apertures such that the pathogens flow from the first channel to the second channel and larger constituents in the biological sample remain in the first channel.
• GDI019.2 Preferably, the dialysis device also has a series of adjacent channels extending between the first channel and the second channel wherein the apertures are at an upstream end of the adjacent channels, each of the adjacent channels being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel; and,
• a bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the adjacent channels and is configured for uninterrupted capillary driven flow from the first channel to the second channel; wherein during use,
• flow from the bypass channel reaches the meniscus pinned at each of the adjacent channels after the meniscus has formed such that the flow sequentially removes each of the menisci and sample flow from the first channel to the second channel is via the adjacent channels as well as the bypass channel.
• GDI019.3 Preferably, the dialysis device also has:
• a cap layer in which the first channel and the second channel are formed; and,
• an supporting layer in which the adjacent channels are formed, the cap layer overlaying the supporting layer.
• GDI019.4 Preferably, the supporting layer is a laminate that includes a roof layer, the roof layer defining the apertures.
• GDI019.5 Preferably, the adjacent channels are parallel and extend normal to the first channel and the second channel.
• GDI019.6 Preferably, the biological sample is blood and the cap has a reagent reservoir containing anticoagulant for addition to the blood.
• GDI019.7 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent in the reservoir, such that the sample flow removes the meniscus from the meniscus anchor to entrain the anticoagulant into the sample flow.
• The easily usable, mass-producible, and inexpensive multichip lab accepts a biological sample, uses a dialysis chip for separating the pathogens in the sample, and separately processes the nucleic acid content of the pathogens separated from the sample. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The multichip lab provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI023.1 This aspect of the invention provides a dialysis device to separate nucleated cells in a biological sample from smaller constituents, the dialysis device comprising:
• a first channel for receiving the biological sample;
• a second channel; and,
• a plurality of apertures; wherein,
• the second channel is fluidically connected to the first channel via the apertures, the apertures being smaller than the nucleated cells such that the smaller constituents flow from the first channel to the second channel and the nucleated cells in the biological sample remain in the first channel.
• GDI023.2 Preferably, the dialysis device also has a series of adjacent channels extending between the first channel and the second channel wherein the apertures are at an upstream end of the adjacent channels, each of the adjacent channels being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel; and,
• a bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the adjacent channels and is configured for uninterrupted capillary driven flow from the first channel to the second channel; wherein during use,
• flow from the bypass channel reaches the meniscus pinned at each of the adjacent channels after the meniscus has formed such that the flow sequentially removes each of the menisci and sample flow from the first channel to the second channel is via the adjacent channels as well as the bypass channel.
• GDI023.3 Preferably, the dialysis device also has:
• a cap layer in which the first channel and the second channel are formed; and,
• an supporting layer in which the adjacent channels are formed, the cap layer overlaying the supporting layer.
• GDI023.4 Preferably, the supporting layer is a laminate that includes a roof layer, the roof layer defining the apertures.
• GDI023.5 Preferably, the adjacent channels are parallel and extend normal to the first channel and the second channel.
• GDI023.6 Preferably, the biological sample is blood and the cap has a reagent reservoir containing anticoagulant for addition to the blood.
• GDI023.7 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent in the reservoir, such that the sample flow removes the meniscus from the meniscus anchor to entrain the anticoagulant into the sample flow.
• GDI023.8 Preferably, the nucleated cells are leukocytes.
• This multichip lab design has the advantage of directly selecting the component of the sample which contains the target. This multichip lab design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the multichip lab. This multichip lab design has the advantage of removing components of the sample which can inhibit later analytical steps. This multichip lab design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target. This multichip lab design has the advantage of removing components of the mixture which might clog the chambers or connections within the multichip lab and degrade operation.
• The multichip lab provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI028.1 This aspect of the invention provides a test module for analyzing a sample fluid containing target molecules, the test module comprising:
• an outer casing with a receptacle for receiving the sample fluid; and,
• a microfluidic device having:
• a dialysis device in fluid communication with the receptacle and configured to separate the target molecules from other constituents of the sample;
• a lab-on-a-chip (LOC) device for analyzing the target molecules; and,
• a cap overlaying the LOC device and the dialysis device for establishing fluid communication between the LOC device and the dialysis device.
• GDI028.2 Preferably, the fluid sample is a sample of biological material including cells of different sizes, and the dialysis device has at least two channels with a plurality of apertures fluidically connecting the channels, the plurality of apertures being sized to correspond to a predetermined threshold size of the cells in the fluid sample.
• GDI028.3 Preferably, the at least two channels and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GDI028.4 Preferably, the at least two channels includes a target channel and a waste channel, the target channel being connected to the cap for capillary driven flow to the LOC device.
• GDI028.5 Preferably, the target molecules are target nucleic acid sequences within cells in the sample fluid, and the LOC device has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI028.6 Preferably, the target nucleic acid sequences are within the cells smaller than the predetermined threshold.
• GDI028.7 Preferably, the LOC device has a hybridization section with an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GDI028.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being configured to emit photons of light in response to an excitation electric current.
• GDI028.9 Preferably, the LOC device has CMOS circuitry for operative control of the PCR section, the CMOS circuitry having a photosensor for sensing photons emitted by the probe-target hybrids.
• GDI028.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI028.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI028.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI028.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI028.14 Preferably, the LOC device has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI028.15 Preferably, the photodiodes are less than 1600 microns from the corresponding hybridization chamber.
• GDI028.16 Preferably, the probes have an electrochemiluminescent (ECL) luminophore that emits a photon when in an excited state.
• GDI028.17 Preferably, the hybridization chambers have electrodes for exciting the ECL luminophores with electrical current.
• GDI028.18 Preferably, the ECL probes each have a luminophore and a quencher positioned proximate the luminophore for quenching photons emitted by the luminophore such that hybridization with one of the target nucleic acid sequences moves the quencher away from the luminophore such that the photons are not quenched.
• GDI028.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI028.20 Preferably, the cap has at least one channel for fluid communication between the dialysis device and the LOC device, and a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biochemical sample, uses a dialysis chip for separating the cells of different dimensions, and separately processes the nucleic acid content of the cells separated based on their dimensions. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The microfluidic multichip assembly provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI030.1 This aspect of the invention provides a test module for concentrating pathogens in a biological sample, the test module comprising:
• an outer casing with a receptacle for receiving the sample;
• a dialysis device in fluid communication with the receptacle and configured to separate the pathogens from other constituents in the sample; and,
• a lab-on-a-chip (LOC) device being in fluid communication with the dialysis device and configured to analyze the pathogens.
• GDI030.2 Preferably, the dialysis device has a first channel for receiving the biological sample, a second channel, and a plurality of apertures, the second channel being fluidically connected to the first channel via the apertures such that the pathogens flow from the first channel to the second channel and larger constituents in the biological sample remain in the first channel.
• GDI030.3 Preferably, the dialysis device also has a series of adjacent channels extending between the first channel and the second channel wherein the apertures are at an upstream end of the adjacent channels, each of the adjacent channels being configured to pin a meniscus of the sample that arrests capillary flow between the first channel and the second channel and, a bypass channel between the first channel and the second channel, the bypass channel joining the second channel upstream of the adjacent channels and is configured for uninterrupted capillary driven flow from the first channel to the second channel, wherein during use, flow from the bypass channel reaches the meniscus pinned at each of the adjacent channels after the meniscus has formed such that the flow sequentially removes each of the menisci and sample flow from the first channel to the second channel is via the adjacent channels as well as the bypass channel.
• GDI030.4 Preferably, the second channel supplies a target channel and the first channel supplies a waste channel, the target channel being configured for capillary driven flow to the LOC device.
• GDI030.5 Preferably, the pathogens contain target nucleic acid sequences, and the LOC device has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI030.6 Preferably, the LOC device has a lysis section for lysing the pathogens to release the target nucleic acid sequences therein.
• GDI030.7 Preferably, the LOC device has a hybridization section with an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GDI030.8 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being configured to emit photons of light in response to an excitation electric current.
• GDI030.9 Preferably, the LOC device has CMOS circuitry for operative control of the PCR section, the CMOS circuitry having a photosensor for sensing photons emitted by the probe-target hybrids.
• GDI030.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI030.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI030.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI030.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI030.14 Preferably, the LOC device has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI030.15 Preferably, the photodiodes are less than 1600 microns from the corresponding hybridization chamber.
• GDI030.16 Preferably, the probes have an electrochemiluminescent (ECL) luminophore that emits a photon when in an excited state.
• GDI030.17 Preferably, the hybridization chambers have electrodes for exciting the ECL luminophores with electrical current.
• GDI030.18 Preferably, the ECL probes each have a luminophore and a quencher positioned proximate the luminophore for quenching photons emitted by the luminophore such that hybridization with one of the target nucleic acid sequences moves the quencher away from the luminophore such that the photons are not quenched.
• GDI030.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI030.20 Preferably, the LOC device and the dialysis device are fluidically connected via a cap, the cap has at least one channel for fluid communication between the dialysis device and the LOC device, and a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biological sample, uses a dialysis chip for separating the pathogens in the sample, and separately processes the nucleic acid content of the pathogens separated from the sample. The dialysis chip functionality extract additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The microfluidic multichip assembly provides for higher modularity. Surface-micromachined chips in the assembly would each be much smaller and disproportionately cheaper that a monolithic chip capable of providing the total functionality of the assembly. Alternatively a large yet cost-effective dialysis chip can provide a high level of dialysis capacity providing for further increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• Functionally optimal, less expensive fabrication process is utilized to fabricate each surface-micromachined chip constituent of the microfluidic multichip assembly, ie, the bioMST-only dialysis chip skips all of the CMOS process steps and uses an inexpensive glass wafer.
• GDI039.1 This aspect of the invention provides a microfluidic device for removing cell debris from a biological sample, the microfluidic device comprising:
• a dialysis section with a large constituent channel, a small constituent channel and a plurality of apertures for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel having an upstream end for receiving the biological sample, the biological sample being a liquid carrying a mixture of cell debris and target molecules, the small constituent channel having a downstream end for connection to a hybridization section with an array of probes for reaction with the target molecules to form probe-target complexes; wherein,
• the apertures are spaced along the large constituent channel at a spacing between 1 micron and 10 microns and each of the apertures is sized to allow the target molecules to flow into the small constituent channel but retain the cell debris larger than a threshold size in the large constituent channel.
• GDI039.2 Preferably, the large constituent channel and the small constituent channel have a common sidewall, and the plurality of apertures is a series of stoma extending through the common sidewall, each stoma having a small opening to the large constituent channel and inverse tapering to a large opening to the small constituent channel.
• GDI039.3 Preferably, the microfluidic device also has a plurality of the small constituent channels each sharing a common sidewall with the large constituent channel and fluidically connecting via a series of the stoma.
• GDI039.4 Preferably, the small opening of each of the stoma has a height and a width dimension between 1 micron and 8 microns.
• GDI039.5 Preferably, the microfluidic device also has a waste reservoir wherein the large constituent channel has a downstream end connected to the waste reservoir.
• GDI039.6 Preferably, the microfluidic device also has a lysis section upstream of the dialysis section wherein the target molecules are target nucleic acid sequences and the lysis section is configured to lyse cells in the biological sample and release the target nucleic acid sequences therein.
• GDI039.7 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI039.8 Preferably, the probes are configured to hybridize with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI039.9 Preferably, the microfluidic device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI039.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI039.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI039.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI039.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI039.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI039.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI039.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI039.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI039.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI039.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI039.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the sample constituents of different dimensions, and separately processes the sample constituents separated based on their dimensions.
• The dialysis section directly selects the component of the sample which contains the target, removing unwanted components of the processed mixture which may interfere with later detection of the target, inhibit later analytical steps, or might clog the chambers or connections within the microfluidic device and degrade operation. The dialysis section functionality also extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system. Also the dialysis section being fabricated via only surface-micromachining, provides for the simple and inexpensive manufacturing procedures, leading into the further reduction of assay system costs.
• GDI040.1 This aspect of the invention provides a microfluidic device for removing cell debris from a biological sample, the microfluidic device comprising:
• a dialysis section with a large constituent channel and a small constituent channel, and a series of stoma for fluid communication between the large constituent channel and the small constituent channel, the large constituent channel having an upstream end for receiving the biological sample, the biological sample being a liquid carrying a mixture of cell debris and target molecules, the small constituent channel having a downstream end for connection to a hybridization section with an array of probes for reaction with the target molecules to form probe-target complexes; wherein,
• each of the stoma is tapered in a counter-flow direction such that each have a small opening to the large constituent channel and a large opening to the small constituent channel, the small openings being sized to allow the target molecules to flow into the small constituent channel but retain the cell debris larger than a threshold size in the large constituent channel.
• GDI040.2 Preferably, the stoma are spaced between 1 micron and 10 microns along the large constituent channel.
• GDI040.3 Preferably, the microfluidic device also has a plurality of the small constituent channels each sharing a common sidewall with the large constituent channel and fluidically connecting via a series of the stoma.
• GDI040.4 Preferably, the small opening of each of the stoma has a height and a width dimension between 1 micron and 8 microns.
• GDI040.5 Preferably, the microfluidic device also has a waste reservoir wherein the large constituent channel has a downstream end connected to the waste reservoir.
• GDI040.6 Preferably, the microfluidic device also has a lysis section upstream of the dialysis section wherein the target molecules are target nucleic acid sequences and the lysis section is configured to lyse cells in the biological sample and release the target nucleic acid sequences therein.
• GDI040.7 Preferably, the microfluidic device also has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI040.8 Preferably, the probes are configured to hybridize with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI040.9 Preferably, the microfluidic device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI040.10 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences.
• GDI040.11 Preferably, the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI040.12 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI040.13 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI040.14 Preferably, the microfluidic device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI040.15 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI040.16 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GDI040.17 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI040.18 Preferably, the FRET probes each have a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to the excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI040.19 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device, and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDI040.20 Preferably, the microfluidic device also has a plurality of reservoirs for holding liquid reagents for addition to the sample.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a dialysis section for separating the sample constituents of different dimensions, and separately processes the sample constituents separated based on their dimensions.
• The dialysis section directly selects the component of the sample which contains the target, removing unwanted components of the processed mixture which may interfere with later detection of the target, inhibit later analytical steps, or might clog the chambers or connections within the microfluidic device and degrade operation. The dialysis section functionality also extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section incorporates stomata based on trapezoidal planform pillars; this aspect of the design reduces, for a given lithographic space width, the stomata's dimensions as required for many applications.
• The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system. Also the dialysis section being fabricated via only surface-micromachining, provides for the simple and inexpensive manufacturing procedures, leading into the further reduction of assay system costs.
• GDI041.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing leukocytes in a blood sample, the LOC device comprising:
• a supporting substrate;
• an inlet for receiving a sample of blood;
• a dialysis section for separating erythrocytes from leukocytes in the blood; and,
• a microsystems technology (MST) layer for analyzing the leukocytes; wherein,
• the inlet, the dialysis section and the MST layer are all supported on the supporting substrate.
• GDI041.2 Preferably, the LOC device also has a reagent reservoir containing anticoagulant wherein the reagent reservoir has an outlet with a surface tension valve for retaining the anticoagulant with a meniscus until contact with the sample removes the meniscus.
• GDI041.3 Preferably, the LOC device also has a cap overlying the MST layer, the reagent reservoir being formed in the cap wherein the cap has a flow-path configured to draw the sample from the inlet to the surface tension valve by capillary action.
• GDI041.4 Preferably, the reagent reservoir contains anticoagulant and erythrocyte lysis buffer to lyse the erythrocytes.
• GDI041.5 Preferably, the dialysis section has a first channel and a second channel and a plurality of apertures sized to correspond to a predetermined size threshold, the first and second channels being in fluid communication via the plurality of apertures and the inlet being in fluid communication with the first channel such that the erythrocytes flow to the second channel while the leukocytes remain in the first channel.
• GDI041.6 Preferably, the first and second channels are configured to fill with the sample received at the inlet by capillary action.
• GDI041.7 Preferably, the LOC device also has a first layer in which the first channel and the second channel are defined, and a second layer in which the apertures are defined, the second layer having at least one fluid connection from the apertures to the second channel for establishing fluid communication between the first channel and the second channel.
• GDI041.8 Preferably, the LOC device also has a lysis section wherein the leukocytes contain target nucleic acid sequences and the lysis section is configured to lyse the leukocytes to release the target nucleic acid sequences therein.
• GDI041.9 Preferably, the MST layer has a nucleic acid amplification section for amplifying the target nucleic acid sequences.
• GDI041.10 Preferably, the LOC device also has a hybridization section having an array of probes for hybridization with the target nucleic acid sequences amplified by the nucleic acid amplification section.
• GDI041.11 Preferably, the probes are configured to form probe-target hybrids with the target nucleic acid sequences, the probe-target hybrids being fluorescent in response to an excitation light.
• GDI041.12 Preferably, the LOC device also has CMOS circuitry for operative control of the nucleic acid amplification section, the CMOS circuitry also having a photosensor for sensing fluorescence emission from the probe-target hybrids.
• GDI041.13 Preferably, the hybridization section has an array of hybridization chambers containing the probes for hybridization with the target nucleic acid sequences, and the photosensor is an array of photodiodes positioned adjacent each of the hybridization chambers respectively.
• GDI041.14 Preferably, the CMOS circuitry has a digital memory for storing data relating to the processing of the fluid, the data including the probe details and location of each of the probes in the array of hybridization chambers.
• GDI041.15 Preferably, the CMOS circuitry has at least one temperature sensor for sensing the temperature at the array of hybridization chambers.
• GDI041.16 Preferably, the LOC device also has a heater controlled by the CMOS circuitry using feedback from the temperature sensor for maintaining the probes and the target nucleic acid sequences at a hybridization temperature.
• GDI041.17 Preferably, the photodiodes are less than 249 microns from the corresponding hybridization chamber.
• GDI041.18 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes, and each probe has a fluorophore and a quencher, the fluorophore being configured to emit a fluorescence signal to the photodiode in response to an excitation light when the FRET probe has formed a probe-target hybrid, the CMOS circuitry being configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished, the digital memory including the predetermined delay.
• GDI041.19 Preferably, the hybridization chambers have an optical window positioned to expose the FRET probes to the excitation light.
• GDI041.20 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device and is configured to convert output from the photodiodes into a signal indicative of the FRET probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• The easily usable, mass-producible, and inexpensive LOC device accepts a blood sample, adds an erythrocyte lysis buffer from a reagent reservoir to lyse erythrocytes, uses a dialysis section for separating the leukocytes from the lysed erythrocytes, and separately processes the nucleic acid content of the leukocytes separated from the blood sample.
• The dialysis section functionality extracts additional information from the blood sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis system being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC001.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one elongate PCR chamber having a longitudinal extent much greater than its lateral dimensions; and,
• at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR chamber; wherein,
• the elongate heater element extends parallel with the longitudinal extent of the PCR chamber.
• GPC001.2 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chamber is a section of the microchannel.
• GPC001.3 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC001.4 Preferably, the PCR section has a plurality of the elongate PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC001.5 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC001.6 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC001.7 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC001.8 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heaters.
• GPC001.9 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequences.
• GPC001.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC001.11 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC001.12 Preferably, the elongate heaters are placed above the roof defining the top of the microchannel.
• GPC001.13 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GPC001.14 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC001.15 Preferably, at least one of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heaters.
• GPC001.16 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC001.17 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GPC001.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• an array of sensors for detecting hybridization of probes within the array of probes.
• GPC001.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GPC001.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR chamber is of an elongated geometry which provides for rapid temperature cycling of the mixture and capillary action propulsion of the mixture. The rapid temperature cycling capability increases the assay speed. The capillary action propulsion simplifies the design of the assay system, further increasing the reliability and reducing the cost of the assay system.
• GPC002.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having a PCR chamber; and,
• a heater element for heating the nucleic acid sequences within the PCR chamber; wherein,
• the PCR chamber is between the heater element and the supporting substrate.
• GPC002.2 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the PCR chamber is a section of the microchannel.
• GPC002.3 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC002.4 Preferably, the PCR section has a plurality of the PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the PCR chambers.
• GPC002.5 Preferably, each of the channel sections has a plurality of the heater elements.
• GPC002.6 Preferably, the plurality of heater elements are elongate, aligned with the longitudinal extent of the channel section and positioned end to end along the channel section.
• GPC002.7 Preferably, each of the plurality of elongate heater elements is independently operable.
• GPC002.8 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater elements.
• GPC002.9 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequences.
• GPC002.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC002.11 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC002.12 Preferably, the elongate heaters are supported on a roof layer that encloses the microchannel.
• GPC002.13 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC002.14 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC002.15 Preferably, at least one of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heater elements.
• GPC002.16 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC002.17 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GPC002.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• an array of sensors for detecting hybridization of probes within the array of probes.
• GPC002.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GPC002.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample. The PCR chamber's heater is above the chamber, maximizing heat transfer into the PCR mixture and minimizing heat transfer into the substrate.
• GPC003.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having a microchannel configured to have a plurality of mutually parallel, adjacent channel sections, and a plurality of elongate heaters positioned end to end along each of the channel sections; wherein,
• each of the heaters are independently operable for two-dimensional control of heat flux density to the PCR section.
• GPC003.2 Preferably, the microchannel is configured in a serpentine configuration with a series of wide meanders to form the plurality of mutually parallel, adjacent channel sections.
• GPC003.3 Preferably, the microfluidic device also has a supporting substrate and CMOS circuitry wherein the CMOS circuitry is between the supporting substrate and the PCR section for controlling of the heaters.
• GPC003.4 Preferably, the microfluidic device also has at least one sensor wherein the CMOS circuitry uses the sensor for feedback control of the heaters.
• GPC003.5 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GPC003.6 Preferably, the microchannel is configured to draw a liquid containing the nucleic acid sequences through all the channel sections by capillary action.
• GPC003.7 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequences.
• GPC003.8 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC003.9 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC003.10 Preferably, the elongate heaters are placed above the roof defining the top of the microchannel.
• GPC003.11 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing cells larger than the predetermined threshold.
• GPC003.12 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC003.13 Preferably, each of the channel sections have a cross-sectional area between 1 square micron and 400 square microns.
• GPC003.14 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC003.15 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GPC003.16 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, and a photosensor for detecting probe-target hybrids within the array of probes.
• GPC003.17 Preferably, the CMOS circuitry incorporates the photosensor.
• GPC003.18 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC003.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GPC003.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The heat flux density into the PCR chamber is controlled two-dimensionally, with the resulting two-dimensional temperature distribution being highly uniform, providing for uniform amplification characteristics and improved amplification specificity.
• GPC004.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one heater; and,
• at least one sensor; wherein,
• the at least one sensor is configured to provide output for feedback control of the at least one heater.
• GPC004.2 Preferably, the microfluidic device also has CMOS circuitry connected to the at least one heater and the at least one sensor such that the CMOS circuitry uses the output for feedback control of the at least one heater.
• GPC004.3 Preferably, the microfluidic device also has a plurality of the temperature sensors and a plurality of liquid sensors, wherein the PCR section has a plurality of heaters and a plurality of the liquid sensors such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the temperature sensors.
• GPC004.4 Preferably, the PCR section has a plurality of elongate PCR chambers, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GPC004.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC004.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC004.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC004.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GPC004.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC004.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC004.11 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, nucleic acid polymerase and buffer to amplify the nucleic acid sequences.
• GPC004.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC004.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC004.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GPC004.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC004.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC004.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR, and a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC004.18 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, and a photosensor for detecting probe-target hybrids within the array of probes.
• GPC004.19 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• GPC004.20 Preferably, the PCR section has a thermal cycle time less than 11 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR section is controlled by a feedback control system, resulting into the faster and more precise temperature cycling. The faster temperature cycling capability increases the assay speed. The more precise temperature cycling capability provides for the improved amplification specificity.
• GPC005.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one heater; and,
• at least one temperature sensor; wherein,
• the at least one temperature sensor is configured to provide output for feedback control of the at least one heater.
• GPC005.2 Preferably, the microfluidic device also has CMOS circuitry connected to the at least one heater and the at least one temperature sensor such that the CMOS circuitry uses the output for feedback control of the at least one heater.
• GPC005.3 Preferably, the microfluidic device also has a plurality of the temperature sensors and a plurality of liquid sensors wherein the PCR section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the temperature sensors.
• GPC005.4 Preferably, the PCR section has a plurality of elongate PCR chambers each having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GPC005.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC005.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC005.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC005.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GPC005.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC005.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC005.11 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, nucleic acid polymerase and buffer to amplify the nucleic acid sequences.
• GPC005.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC005.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC005.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GPC005.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC005.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC005.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC005.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GPC005.19 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• GPC005.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR section is controlled by a temperature feedback control system, resulting into the faster and more precise temperature cycling. The faster temperature cycling capability increases the assay speed. The more precise temperature cycling capability provides for the improved amplification specificity.
• GPC006.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one heater for thermally cycling the nucleic acid sequences through a denaturation phase, an annealing phase and a primer extension phase; wherein during use,
• the PCR section has a thermal cycle time of less than 30 seconds.
• GPC006.2 Preferably, the PCR section has a thermal cycle time of less than 11 seconds.
• GPC006.3 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GPC006.4 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GPC006.5 Preferably, the microfluidic device also has at least one sensor and CMOS circuitry connected to the at least one heater and the at least one sensor such that the CMOS circuitry uses output from the at least one sensor for feedback control of the at least one heater.
• GPC006.6 Preferably, the microfluidic device also has a plurality of liquid sensors wherein the PCR section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the at least one temperature sensor.
• GPC006.7 Preferably, the PCR section has a plurality of elongate PCR chambers, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GPC006.8 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC006.9 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC006.10 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC006.11 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GPC006.12 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC006.13 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC006.14 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequences.
• GPC006.15 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC006.16 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC006.17 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GPC006.18 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences amplified by the PCR section to form probe-target hybrids.
• GPC006.19 Preferably, the array of probes has more than 1000 probes and the PCR section is configured to generate enough amplicon to hybridize with all the probes in less than 10 minutes.
• GPC006.20 Preferably, the microfluidic device also has an array of photodiodes for detecting probe-target hybrids within the array of probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample. The PCR section has a short thermal cycle time, increasing the assay speed.
• GPC007.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a microsystems technology (MST) layer having a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one heater for thermally cycling the nucleic acid sequences through a denaturation phase, an annealing phase and a primer extension phase; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry being connected to the at least one heater for operative control of the at least one heater during the thermal cycling.
• GPC007.2 Preferably, the microfluidic device also has at least one sensor such that the CMOS circuitry uses output from the at least one sensor for feedback control of the at least one heater.
• GPC007.3 Preferably, the microfluidic device also has a plurality of the temperature sensors and a plurality of liquid sensors wherein the PCR section has a plurality of heaters and a plurality of the liquid sensors such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the temperature sensors.
• GPC007.4 Preferably, the PCR section has a plurality of elongate PCR chambers, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GPC007.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC007.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC007.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC007.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GPC007.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC007.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC007.11 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, nucleic acid polymerase and buffer to amplify the nucleic acid sequences.
• GPC007.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC007.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC007.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GPC007.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold from cells smaller than the predetermined threshold.
• GPC007.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC007.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR and a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC007.18 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC007.19 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• GPC007.20 Preferably, the PCR section has a thermal cycle time less than 11 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR section is controlled by an on-chip semiconductor control system, resulting into the faster and more precise temperature cycling. The faster temperature cycling capability increases the assay speed. The more precise temperature cycling capability provides for the improved amplification specificity.
• GPC008.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a microsystems technology (MST) layer having a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having at least one heater for thermally cycling the nucleic acid sequences through a denaturing phase, an annealing phase and a primer extension phase; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry configured to energize the at least one heater with a pulse width modulated (PWM) signal during the thermal cycling.
• GPC008.2 Preferably, the microfluidic device also has at least one sensor such that the CMOS circuitry uses output from the at least one sensor for feedback control of the at least one heater.
• GPC008.3 Preferably, the PCR section has a plurality of heaters and at least one temperature sensor and at least one liquid sensor such that the CMOS circuitry controls initial activation of the heaters in response to the at least one liquid sensor and heater power in response to the at least one temperature sensor.
• GPC008.4 Preferably, the PCR section has a plurality of elongate PCR chambers each having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GPC008.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC008.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC008.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC008.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GPC008.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC008.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC008.11 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase and buffer to amplify the nucleic acid sequences.
• GPC008.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC008.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC008.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GPC008.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC008.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC008.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC008.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GPC008.19 Preferably, during use, the PCR section has a thermal cycle time determined by the CMOS circuitry.
• GPC008.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR section is controlled by a PWM control system, resulting into the faster and more precise temperature cycling. The faster temperature cycling capability increases the assay speed. The more precise temperature cycling capability provides for the improved amplification specificity.
• GPC009.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section with a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining a flow-path for the sample such that flow of the sample along the PCR microchannel is driven by capillary action; and,
• the PCR microchannel has a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC009.2 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC009.3 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC009.4 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GPC009.5 Preferably, the microfluidic device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel; and,
• the elongate heater element extends parallel to the PCR microchannel.
• GPC009.6 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GPC009.7 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC009.8 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GPC009.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC009.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC009.11 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GPC009.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC009.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC009.14 Preferably, the elongate heaters are supported on a wall defining one side of the microchannel.
• GPC009.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC009.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC009.17 Preferably, at least one of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heaters.
• GPC009.18 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC009.19 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GPC009.20 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR chamber is of a small cross-section which provides for rapid temperature cycling of the mixture and capillary action propulsion of the mixture. The rapid temperature cycling capability increases the assay speed. The capillary action propulsion simplifies the design of the assay system, further increasing the reliability and reducing the cost of the assay system.
• GPC010.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having a PCR chamber; and,
• a heater element for heating the nucleic acid sequences within the PCR chamber; wherein during use,
• the heater element heats the nucleic acid sequences at a rate more than 80 K per second.
• GPC010.2 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 100 K per second.
• GPC010.3 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 1000 K per second.
• GPC010.4 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 10,000 K per second.
• GPC010.5 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 100,000 K per second.
• GPC010.6 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 1000,000 K per second.
• GPC010.7 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 10,000,000 K per second.
• GPC010.8 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 20,000,000 K per second.
• GPC010.9 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 40,000,000 K per second.
• GPC010.10 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 80,000,000 K per second.
• GPC010.11 Preferably, during use, the heater element heats the nucleic acid sequences at a rate more than 160,000,000 K per second.
• GPC010.12 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the PCR chamber is a section of the microchannel.
• GPC010.13 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC010.14 Preferably, the PCR section has a plurality of the PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the PCR chambers.
• GPC010.15 Preferably, each of the channel sections has a plurality of the heater elements.
• GPC010.16 Preferably, the plurality of heater elements are elongate, aligned with the longitudinal extent of the channel section and positioned end to end along the channel section.
• GPC010.17 Preferably, each of the plurality of elongate heater elements is independently operable.
• GPC010.18 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater elements.
• GPC010.19 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GPC010.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample. The PCR section has a fast temperature rate of change, providing for the short thermal cycle time, increasing the overall assay speed.
• GPC011.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, the PCR section having a PCR chamber; and,
• a heater element for heating the nucleic acid sequences within the PCR chamber; wherein during use,
• the PCR section generates sufficient amplicon for hybridization with an array of probes in less than 10 minutes.
• GPC011.2 Preferably, during use the PCR section generates sufficient amplicon for hybridization with an array of probes in less than 220 seconds.
• GPC011.3 Preferably, during use the PCR section generates sufficient amplicon for hybridization with an array of probes in less than 80 seconds.
• GPC011.4 Preferably, during use the PCR section generates sufficient amplicon for hybridization with an array of probes in less than 30 seconds.
• GPC011.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the PCR chamber is a section of the microchannel.
• GPC011.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GPC011.7 Preferably, the PCR section has a plurality of the PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the PCR chambers.
• GPC011.8 Preferably, each of the channel sections has a plurality of the heater elements.
• GPC011.9 Preferably, the plurality of heater elements are elongate, aligned with the longitudinal extent of the channel section and positioned end to end along the channel section.
• GPC011.10 Preferably, each of the plurality of elongate heater elements is independently operable.
• GPC011.11 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater elements.
• GPC011.12 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GPC011.13 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC011.14 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC011.15 Preferably, the elongate heaters are embedded a roof layer that encloses the microchannel.
• GPC011.16 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC011.17 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC011.18 Preferably, each of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heater elements.
• GPC011.19 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC011.20 Preferably, the microfluidic device also has a hybridization section that has the array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample. The PCR section is capable of rapid nucleic acid amplification, increasing the overall assay speed.
• GPC012.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section with a PCR microchannel for thermally cycling a PCR mixture of the sample, polymerase, dNTP's and buffer solution to amplify the nucleic acid sequences, the PCR microchannel defining a flow-path for the sample such that flow of the sample along the PCR microchannel is driven by capillary action; wherein,
• the PCR mixture has a volume less than 400 nanoliters.
• GPC012.2 Preferably, the PCR mixture has a volume less than 170 nanoliters.
• GPC012.3 Preferably, the PCR mixture has a volume less than 70 nanoliters.
• GPC012.4 Preferably, the PCR mixture has a volume less than 30 nanoliters.
• GPC012.5 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC012.6 Preferably, the microfluidic device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel; and,
• the elongate heater element extends parallel to the PCR microchannel.
• GPC012.7 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GPC012.8 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC012.9 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GPC012.10 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC012.11 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC012.12 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heaters.
• GPC012.13 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the PCR mixture.
• GPC012.14 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC012.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC012.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC012.17 Preferably, at least one of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heaters.
• GPC012.18 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC012.19 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GPC012.20 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample. The PCR section provides for capillary action propulsion of the PCR mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system.
• The microfluidic device uses a low PCR mixture volume, providing for rapid nucleic acid amplification, increasing the overall assay speed, and also providing for low reagent expenditure. The low reagent expenditure, in turn, allows total on-chip reagent storage which reduces the component-count and assembly complexity of the assay system, further reducing the assay system cost.
• GPC014.1 This aspect of the invention provides a test module for amplifying nucleic acid sequences in a sample fluid, the test module comprising:
• an outer casing with receptacle for receiving the sample fluid;
• a polymerase chain reaction (PCR) section with a PCR microchannel for thermally cycling a PCR mixture of the sample fluid, polymerase, dNTP's, primers, and buffer solution to amplify the nucleic acid sequences; wherein,
• the PCR mixture has a volume less than 400 nanoliters.
• GPC014.2 Preferably, the PCR mixture has a volume less than 170 nanoliters.
• GPC014.3 Preferably, the PCR mixture has a volume less than 70 nanoliters.
• GPC014.4 Preferably, the PCR mixture has a volume less than 30 nanoliters.
• GPC014.5 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC014.6 Preferably, the test module also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel; and,
• the elongate heater element extends parallel to the PCR microchannel.
• GPC014.7 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GPC014.8 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC014.9 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GPC014.10 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC014.11 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC014.12 Preferably, the test module also has at least one temperature sensor for feedback control of the elongate heaters.
• GPC014.13 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the PCR mixture.
• GPC014.14 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC014.15 Preferably, the test module also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC014.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC014.17 Preferably, at least one of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heaters.
• GPC014.18 Preferably, the test module also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC014.19 Preferably, the test module also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GPC014.20 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a sample containing nucleic acids and then using the module's PCR section amplifies the nucleic acid targets in the sample. The PCR section provides for capillary action propulsion of the PCR mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system.
• The module uses a low PCR mixture volume, providing for rapid nucleic acid amplification, increasing the overall assay speed, and also providing for low reagent expenditure. The low reagent expenditure, in turn, allows total on-chip reagent storage which reduces the component-count and assembly complexity of the assay system, further reducing the assay system cost.
• GPC017.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing buffer, dNTPs, primers, and polymerase for addition to the sample; and,
• a polymerase chain reaction (PCR) section for thermal cycling the sample, buffer, dNTPs, primers, and polymerase to amplify the target nucleic acid sequences.
• GPC017.2 Preferably, the PCR section has a plurality of elongate PCR chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the PCR chambers.
• GPC017.3 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC017.4 Preferably, the PCR section has a plurality of the elongate PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC017.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the thermal cycling.
• GPC017.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC017.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC017.8 Preferably, the elongate heaters are independently operable.
• GPC017.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC017.10 Preferably, the PCR section has an active valve for retaining liquid in the PCR section while the elongate heaters thermally cycle the PCR mixture to amplify the nucleic acid sequences.
• GPC017.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC017.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC017.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC017.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC017.15 Preferably, the LOC device also has a photosensor for detecting hybridization of the probe-target hybrids.
• GPC017.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC017.17 Preferably, the liquid in the PCR section has a volume less than 400 nanoliters.
• GPC017.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC017.19 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• GPC017.20 Preferably, the liquid in the PCR section has a volume less than 30 nanoliters.
• This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device has the advantage of fewer chemical steps during operation, which will result in simpler, more reliable operation. This LOC device design will increase the speed of sample analysis. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GPC018.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing buffer, dNTPs, primers, and polymerase for addition to the sample to form an amplification mix; and,
• a nucleic acid amplification section for controlling the temperature of the amplification mix during isothermal amplification of the target nucleic acid sequences.
• GPC018.2 Preferably, the nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC018.3 Preferably, the nucleic acid amplification section has a microchannel with an amplification inlet and a amplification outlet, and the elongate amplification chambers are sections of the microchannel.
• GPC018.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC018.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the thermal cycling.
• GPC018.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC018.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC018.8 Preferably, the elongate heaters are independently operable.
• GPC018.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC018.10 Preferably, the nucleic acid amplification section has an active valve for retaining liquid in the nucleic acid amplification section while the elongate heaters control the temperature of the amplification mixture to amplify the nucleic acid sequences.
• GPC018.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC018.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC018.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC018.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC018.15 Preferably, the LOC device also has a photosensor for detecting hybridization of the probe-target hybrids.
• GPC018.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC018.17 Preferably, the liquid in the nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC018.18 Preferably, the amplification microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC018.19 Preferably, the liquid in the nucleic acid amplification section has a volume less than 30 nanoliters.
• GPC018.20 Preferably, the CMOS circuitry has bond-pads for communication with an external device.
• This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device has the advantage of fewer chemical steps during operation, which will result in simpler, more reliable operation. This
• LOC device design will increase the speed of sample analysis. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage that thermal cycling is not required, which simplifies thermal control electronics, allows more uniform temperature control, and reduces degradation of materials in the LOC device. This LOC device design will reduce the degree of evaporation during operation, allowing improved control over the physical and chemical conditions within the LOC device.
• GPC019.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a whole blood sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the whole blood sample;
• a plurality of reagent reservoirs containing buffer, dNTPs, primers, and polymerase for addition to the whole blood sample; and,
• a polymerase chain reaction (PCR) section for thermal cycling the whole blood sample, buffer, dNTPs, primers, and polymerase to amplify the target nucleic acid sequences.
• GPC019.2 Preferably, the PCR section has a plurality of elongate PCR chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the PCR chambers.
• GPC019.3 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC019.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC019.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the thermal cycling.
• GPC019.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC019.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC019.8 Preferably, the elongate heaters are independently operable.
• GPC019.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC019.10 Preferably, the PCR section has an active valve for retaining liquid in the PCR section while the elongate heaters thermally cycle the PCR mixture to amplify the nucleic acid sequences.
• GPC019.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC019.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC019.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC019.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC019.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC019.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC019.17 Preferably, the liquid in the PCR section has a volume less than 400 nanoliters.
• GPC019.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC019.19 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• GPC019.20 Preferably, the liquid in the PCR section has a volume less than 30 nanoliters.
• This LOC device design has the advantage of being optimised to handle the constituents of blood samples, including leukocytes, erythrocytes, pathogens, and soluble materials. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device has the advantage of fewer chemical steps during operation, which will result in simpler, more reliable operation. This LOC device design will increase the speed of sample analysis. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GPC023.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing buffer, dNTPs, primers, and polymerase derived from a thermophilic bacterium for addition to the sample to form an amplification mixture; and,
• a nucleic acid amplification section for controlling the temperature of the amplification mixture during amplification of the target nucleic acid sequences.
• GPC023.2 Preferably, the polymerase is derived from Thermus aquaticus DNA polymerase.
• GPC023.3 Preferably, the polymerase is derived from Thermococcus litoralis DNA polymerase.
• GPC023.4 Preferably, the polymerase is derived from Pyrococcus furiosus DNA polymerase.
• GPC023.5 Preferably, the polymerase is derived from Pyrococcus abyssi DNA polymerase.
• GPC023.6 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the amplification mixture is thermally cycled in the PCR section to amplify the nucleic acid sequences.
• GPC023.7 Preferably, the PCR section has a plurality of elongate PCR chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the PCR chambers.
• GPC023.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir and mix with the sample.
• GPC023.9 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GPC023.10 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC023.11 Preferably, the microfluidic device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the thermal cycling.
• GPC023.12 Preferably, a plurality of elongate heaters are positioned end to end along the channel section, each of the heaters being independently operable.
• GPC023.13 Preferably, the LOC device also has a supporting substrate, wherein the CMOS circuitry is between the microchannel and the supporting substrate, the CMOS circuitry incorporating at least one temperature sensor for feedback control of the elongate heaters.
• GPC023.14 Preferably, the PCR section has an active valve for retaining liquid in the PCR section while the elongate heaters control the temperature of the amplification mixture to amplify the nucleic acid sequences.
• GPC023.15 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC023.16 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC023.17 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC023.18 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC023.19 Preferably, the amplification microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC023.20 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device has the advantage of fewer chemical steps during operation, which will result in simpler, more reliable operation. This LOC device design will increase the speed of sample analysis. This enzyme amplifies the target DNA more rapidly (higher processivity) than other polymerases.
• GPC027.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing buffer solution, dNTPs, primers, and polymerase for addition to the sample to form an amplification mixture; and,
• a nucleic acid amplification section for maintaining the amplification mixture at a predetermined temperature during isothermal amplification of the target nucleic acid sequences.
• GPC027.2 Preferably, the nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC027.3 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC027.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC027.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the isothermal amplification.
• GPC027.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC027.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC027.8 Preferably, the elongate heaters are independently operable.
• GPC027.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC027.10 Preferably, the nucleic acid amplification section has an active valve for retaining liquid in the nucleic acid amplification section during isothermal amplification.
• GPC027.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC027.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC027.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC027.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC027.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC027.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC027.17 Preferably, the liquid in the nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC027.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC027.19 Preferably, the LOC device also has a dialysis section upstream of the nucleic acid amplification section, wherein the sample includes constituents of different sizes, the dialysis section being configured for separating constituents smaller than a predetermined threshold from constituents larger than the predetermined threshold.
• GPC027.20 Preferably, the nucleic acid sequences are contained within cells and organisms smaller than the predetermined threshold size.
• This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantage that thermal cycling is not required, which simplifies thermal control electronics, allows more uniform temperature control, and reduces degradation of materials in the LOC device. This LOC device design will reduce the degree of evaporation during operation, allowing improved control over the physical and chemical conditions within the LOC device.
• GPC028.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing dNTPs, primers, recombinase, DNA polymerase and buffer solution for addition to the sample to form an amplification mixture; and,
• a nucleic acid amplification section for maintaining the amplification mixture at a predetermined temperature during recombinase polymerase amplification (RPA) of the target nucleic acid sequences.
• GPC028.2 Preferably, the nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC028.3 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC028.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC028.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the RPA.
• GPC028.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC028.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC028.8 Preferably, the elongate heaters are independently operable.
• GPC028.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC028.10 Preferably, the nucleic acid amplification section has an active valve for retaining liquid in the nucleic acid amplification section during isothermal amplification.
• GPC028.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC028.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC028.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC028.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC028.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC028.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC028.17 Preferably, the liquid in the nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC028.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC028.19 Preferably, the LOC device also has a dialysis section upstream of the nucleic acid amplification section, wherein the sample includes constituents of different sizes, the dialysis section being configured for separating constituents smaller than a predetermined threshold from constituents larger than the predetermined threshold.
• GPC028.20 Preferably, the nucleic acid sequences are contained within cells and organisms smaller than the predetermined threshold size.
• This LOC device has the advantage that thermal cycling is not required, which simplifies thermal control electronics, allows more uniform temperature control, and reduces degradation of materials in the LOC device. This LOC device design will reduce the degree of evaporation during operation, allowing improved control over the physical and chemical conditions within the LOC device. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. Recombinase polymerase amplification has the additional advantage that it performs amplification to detectable levels within 15 minutes from single copies of target nucleic acid. This LOC device has the advantage of less complex design and fabrication requirements, which will result in simpler, more reliable fabrication. This LOC device has the advantage of fewer chemical steps during operation, which will result in simpler, more reliable operation.
• GPC029.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing dNTPs, primers, nicking enzymes, buffer solution and DNA polymerase for addition to the sample to form an amplification mixture; and,
• a nucleic acid amplification section for maintaining the amplification mixture at a predetermined temperature during isothermal amplification of the target nucleic acid sequences.
• GPC029.2 Preferably, the nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC029.3 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC029.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC029.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the isothermal amplification.
• GPC029.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC029.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC029.8 Preferably, the elongate heaters are independently operable.
• GPC029.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC029.10 Preferably, the nucleic acid amplification section has an active valve for retaining liquid in the nucleic acid amplification section during isothermal amplification.
• GPC029.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC029.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC029.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC029.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC029.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC029.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC029.17 Preferably, the liquid in the nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC029.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC029.19 Preferably, the LOC device also has a dialysis section upstream of the nucleic acid amplification section, wherein the sample includes constituents of different sizes, the dialysis section being configured for separating constituents smaller than a predetermined threshold from constituents larger than the predetermined threshold.
• GPC029.20 Preferably, the nucleic acid sequences are contained within cells and organisms smaller than the predetermined threshold size.
• This LOC device has the advantage that thermal cycling is not required, which simplifies thermal control electronics, allows more uniform temperature control, and reduces degradation of materials in the LOC device. This LOC device design will reduce the degree of evaporation during operation, allowing improved control over the physical and chemical conditions within the LOC device. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GPC030.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing dNTPs, primers, reverse transcriptase, RNA polymerase, and buffer solution for addition to the sample to form an amplification mixture; and,
• a nucleic acid amplification section for maintaining the amplification mixture at a predetermined temperature during isothermal amplification of the target nucleic acid sequences.
• GPC030.2 Preferably, the nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC030.3 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC030.4 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC030.5 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for operative control of the at least one heater during the isothermal amplification.
• GPC030.6 Preferably, each of the channel sections along each of the wide meanders has a plurality of the elongate heaters.
• GPC030.7 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC030.8 Preferably, the elongate heaters are independently operable.
• GPC030.9 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the elongate heaters.
• GPC030.10 Preferably, the nucleic acid amplification section has an active valve for retaining liquid in the nucleic acid amplification section during isothermal amplification.
• GPC030.11 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the nucleic acid amplification section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the nucleic acid amplification section resumes.
• GPC030.12 Preferably, the LOC device also has a liquid sensor downstream of the active valve, the liquid sensor being configured to detect the liquid at the liquid sensor location for feedback control of the valve heater.
• GPC030.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GPC030.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC030.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC030.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC030.17 Preferably, the liquid in the nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC030.18 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC030.19 Preferably, the LOC device also has a dialysis section upstream of the nucleic acid amplification section, wherein the sample includes constituents of different sizes, the dialysis section being configured for separating constituents smaller than a predetermined threshold from constituents larger than the predetermined threshold.
• GPC030.20 Preferably, the nucleic acid sequences are contained within cells and organisms smaller than the predetermined threshold size.
• This LOC device has the advantage that thermal cycling is not required, which simplifies thermal control electronics, allows more uniform temperature control, and reduces degradation of materials in the LOC device. This LOC device design will reduce the degree of evaporation during operation, allowing improved control over the physical and chemical conditions within the LOC device. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target RNA sequence.
• GPC031.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of nucleic acid sequences in a sample, the LOC device comprising:
• a sample inlet for receiving the sample;
• a first polymerase chain reaction (PCR) section for thermal cycling a first PCR mixture of dNTP's, primers, and buffer solution together with the sample and polymerase, to amplify the nucleic acid sequences; and,
• a second PCR section downstream of the first PCR section for thermally cycling a second PCR mixture of dNTPs, primers, and buffer solution together with polymerase, and at least some of the amplicon from the first PCR section.
• GPC031.2 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section while the nucleic acid sequences in the sample are amplified.
• GPC031.3 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the first PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the first PCR section resumes.
• GPC031.4 Preferably, the LOC device also has a second PCR mix reservoir with an outlet valve and a second polymerase reservoir with an outlet valve for adding the PCR mix and the polymerase to the amplicon from the first PCR section prior to amplification in the second PCR section.
• GPC031.5 Preferably, the outlet valves are surface tension valves each having an aperture configured to pin a meniscus that retains liquid reagents therein until contact with the amplicon removes the meniscus and the PCR mix and the polymerase are added to the amplicon flow into the second PCR section.
• GPC031.6 Preferably, the second PCR section has an active valve for retaining liquid in the second PCR section while the nucleic acid sequences in the amplicon from the first PCR section are amplified.
• GPC031.7 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the second PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the second PCR section resumes.
• GPC031.8 Preferably, the LOC device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the second PCR section amplicon.
• GPC031.9 Preferably, the hybridization section has a photosensor for detecting hybridization of probes within the array of probes.
• GPC031.10 Preferably, the LOC device also has a dialysis section downstream of the second PCR section and upstream of the hybridization section, wherein the second PCR section amplicon contains nucleic acid sequences and cell debris, such that the dialysis section is configured to remove the cell debris larger than the nucleic acid sequences.
• GPC031.11 Preferably, the first PCR section has a PCR microchannel for holding the sample during amplification and at least one elongate heater for thermal cycling of the nucleic acid sequences within the sample, the elongate heater being aligned with the PCR microchannel.
• GPC031.12 Preferably, the PCR mixture has a volume less than 400 nanoliters.
• GPC031.13 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC031.14 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GPC031.15 Preferably, the first PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC031.16 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GPC031.17 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GPC031.18 Preferably, each of the plurality of elongate heaters is independently operable.
• GPC031.19 Preferably, the LOC device also has at least one temperature sensor for feedback control of the elongate heaters.
• GPC031.20 Preferably, the first PCR section has a thermal cycle time of less than 30 seconds.
• This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. Two phases of amplification allows protocols to be further refined for highly sensitive detection which is resistant to the presence of similar genetic material, and chemical contaminants which can interfere with a single-stage amplification scheme. The product from the first amplification stage can also be subdivided to enable multiple parallel reactions in the second amplification phase, which increases the multiplexing capability of the LOC device. LOC device designs with parallel reaction sites enable parallel diagnostic tests to be performed on a very small sample volume, which increases the range and quality of diagnostic data obtained.
• GPC033.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing dNTP's, primers, polymerase and buffer solution for addition to the sample; and,
• a nucleic acid amplification section for thermal control of the sample to perform a first stage amplification of the target nucleic acid sequences and subsequent thermal control of amplicon from the first stage amplification, to perform a second stage amplification for further amplifying the target nucleic acid sequences.
• GPC033.2 Preferably, the nucleic acid amplification section is configured for whole genome amplification during the first stage amplification, and amplification of a predetermined nucleic acid sequence during the second stage amplification.
• GPC033.3 Preferably, the nucleic acid amplification section is configured for polymerase chain reaction (PCR) amplification, and the thermal control includes thermal cycling of the sample.
• GPC033.4 Preferably, the nucleic acid amplification section is configured for isothermal whole genome amplification, and the thermal control includes maintaining the sample at a predetermined temperature.
• GPC033.5 Preferably, the nucleic acid amplification section has a first nucleic acid amplification section for performing the first stage amplification, and a second nucleic acid amplification section, downstream of the first nucleic acid amplification section, for performing the first stage amplification.
• GPC033.6 Preferably, the first nucleic acid amplification section is configured to amplify a first predetermined nucleic acid sequence and the second nucleic acid amplification section is configured to amplify a second predetermined nucleic acid sequence, the first predetermined nucleic acid sequence being a subsection of the second predetermined nucleic acid sequence.
• GPC033.7 Preferably, the reagent reservoirs include first reagent reservoirs containing first dNTP's, primers, polymerase and buffer solution for addition to the sample prior to amplification in the first nucleic acid amplification section, and second reagent reservoirs containing second dNTP's, primers, polymerase and buffer solution for addition to the amplicon from the first nucleic acid amplification section prior to amplification in the second nucleic acid amplification section.
• GPC033.8 Preferably, the reagent reservoirs each have a surface tension valve with an aperture configured to pin a meniscus that retains liquid reagents therein until contact with the sample removes the meniscus.
• GPC033.9 Preferably, the first nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC033.10 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC033.11 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC033.12 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for the thermal control of the sample during amplification.
• GPC033.13 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the at least one heater.
• GPC033.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC033.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC033.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC033.17 Preferably, the liquid in the first nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC033.18 Preferably, the microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC033.19 Preferably, each of the channel sections has a plurality of the heaters, each of the heaters being elongate and positioned end to end along the channel section and, the CMOS circuitry being configured for independent operation of each of the plurality of elongate heaters.
• GPC033.20 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the array of probes and the supporting substrate.
• This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. Two phases of amplification allows protocols to be further refined for highly sensitive detection which is resistant to the presence of similar genetic material, and chemical contaminants which can interfere with a single-stage amplification scheme. The product from the first amplification stage can also be subdivided to enable multiple parallel reactions in the second amplification phase, which increases the multiplexing capability of the LOC device. LOC device designs with parallel reaction sites enable parallel diagnostic tests to be performed on a very small sample volume, which increases the range and quality of diagnostic data obtained. Non-specific nucleic acid amplification before specific detection or additional amplification allows higher sensitivity to rare targets. Non-specific amplification prior to more specific steps also increases the versatility of the LOC device platform by decreasing the amount of additional development required to add new tests to the existing diagnostic panel.
• GPC034.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a sample containing target nucleic acid sequences, the LOC device comprising:
• a sample inlet for receiving the sample;
• a plurality of reagent reservoirs containing dNTP's, primers, polymerase and buffer solution for addition to the sample;
• a first nucleic acid amplification section for thermal control of the sample to amplify the target nucleic acid sequences; and,
• a second nucleic acid amplification section for thermal control of amplicon from the first nucleic acid amplification section to further amplify the target nucleic acid sequences.
• GPC034.2 Preferably, the first nucleic acid amplification section is configured for whole genome amplification, and the second nucleic acid amplification section is configured for amplification of a predetermined nucleic acid sequence.
• GPC034.3 Preferably, the first nucleic acid amplification section is configured for polymerase chain reaction (PCR) amplification, and the thermal control includes thermal cycling of the sample.
• GPC034.4 Preferably, the first nucleic acid amplification section is configured for isothermal whole genome amplification, and the thermal control includes maintaining the sample at a predetermined temperature.
• GPC034.5 Preferably, the second nucleic acid amplification section is a PCR amplification section configured to amplify a predetermined nucleic acid sequence.
• GPC034.6 Preferably, the first nucleic acid amplification section is configured to amplify a first predetermined nucleic acid sequence and the second nucleic acid amplification section is configured to amplify a second predetermined nucleic acid sequence, the first predetermined nucleic acid sequence being a subsection of the second predetermined nucleic acid sequence.
• GPC034.7 Preferably, the reagent reservoirs include first reagent reservoirs containing first dNTP's, primers, polymerase and buffer solution for addition to the sample prior to amplification in the first nucleic acid amplification section, and second reagent reservoirs containing second dNTP's, primers, polymerase and buffer solution for addition to the amplicon from the first nucleic acid amplification section prior to amplification in the second nucleic acid amplification section.
• GPC034.8 Preferably, the reagent reservoirs each have a surface tension valve with an aperture configured to pin a meniscus that retains liquid reagents therein until contact with the sample removes the meniscus.
• GPC034.9 Preferably, the first nucleic acid amplification section has a plurality of elongate amplification chambers, each chamber having at least one elongate heater extending parallel to the longitudinal extent of the amplification chambers.
• GPC034.10 Preferably, the nucleic acid amplification section has a microchannel, and the elongate amplification chambers are sections of the microchannel.
• GPC034.11 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate amplification chambers.
• GPC034.12 Preferably, the LOC device also has CMOS circuitry connected to the at least one heater for the thermal control of the sample during amplification.
• GPC034.13 Preferably, the LOC device also has at least one temperature sensor connected to the CMOS circuitry for feedback control of the at least one heater.
• GPC034.14 Preferably, the LOC device also has an array of probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GPC034.15 Preferably, the LOC device also has a photosensor for detecting the probe-target hybrids.
• GPC034.16 Preferably, the photosensor is an array of photodiodes in registration with the array of probes.
• GPC034.17 Preferably, the liquid in the first nucleic acid amplification section has a volume less than 400 nanoliters.
• GPC034.18 Preferably, the microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC034.19 Preferably, each of the channel sections has a plurality of the heaters, each of the heaters being elongate and positioned end to end along the channel section and, the CMOS circuitry being configured for independent operation of each of the plurality of elongate heaters.
• GPC034.20 Preferably, the LOC device also has a supporting substrate wherein the CMOS circuitry is positioned between the array of probes and the supporting substrate.
• This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. Two phases of amplification allows protocols to be further refined for highly sensitive detection which is resistant to the presence of similar genetic material, and chemical contaminants which can interfere with a single-stage amplification scheme. The product from the first amplification stage can also be subdivided to enable multiple parallel reactions in the second amplification phase, which increases the multiplexing capability of the LOC device. Performing the two amplification stages in separate LOC device modules has the advantage of facilitating the addition of extra reagents between amplification phases. This enhances chemical optimisation for sensitivity and specificity of the LOC device results. LOC device designs with parallel reaction sites enable parallel diagnostic tests to be performed on a very small sample volume, which increases the range and quality of diagnostic data obtained. Non-specific nucleic acid amplification before specific detection or additional amplification allows higher sensitivity to rare targets. Non-specific amplification prior to more specific steps also increases the versatility of the LOC device platform by decreasing the amount of additional development required to add new tests to the existing diagnostic panel.
• GPC035.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for amplifying nucleic acid sequences in a sample, the LOC device comprising:
• a supporting substrate;
• a plurality of functional sections for processing the sample, one of the functional sections being a nucleic acid amplification section for amplifying nucleic acid sequences in the sample; wherein,
• the nucleic acid amplification section is supported on the supporting substrate and the supporting substrate defines a trench for thermally insulating the nucleic acid amplification section from one or more of the other functional sections.
• GPC035.2 Preferably, one of the functional sections is a hybridization section with an array of probes for hybridization with target nucleic acid sequences and the trench extends between the nucleic acid amplification section and the hybridization section to thermally insulate the hybridization section from heat generated by the nucleic acid amplification section.
• GPC035.3 Preferably, the supporting substrate is a layer of material having opposing surfaces separated by a substrate thickness, the functional sections being formed on one surface of the supporting substrate and the trench being defined in the other surface.
• GPC035.4 Preferably, the LOC device also has CMOS circuitry supported on the supporting substrate wherein the trench extends through the substrate to the CMOS circuitry.
• GPC035.5 Preferably, during use the trench contains air.
• GPC035.6 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GPC035.7 Preferably, the LOC device also has a temperature sensor and a microsystems technology (MST) layer which incorporates the functional sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GPC035.8 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC035.9 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC035.10 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC035.11 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC035.12 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GPC035.13 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GPC035.14 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GPC035.15 Preferably, the LOC device also has a photosensor for detecting hybridization of any probes within the hybridization section.
• GPC035.16 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GPC035.17 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GPC035.18 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GPC035.19 Preferably, the isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC035.20 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• The easily usable, mass-producible, and inexpensive genetic analysis LOC device accepts a sample containing nucleic acids and then in the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The PCR chamber is thermally isolated with a thermal isolation trench, providing for rapid temperature cycling of the mixture, reducing the power requirements, and reducing the impact of the thermal cycling on the rest of the device, allowing for a wider assay chemistry. The rapid temperature cycling capability increases the assay speed.
• GPC036.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences; and,
• a nucleic acid amplification section for amplifying the nucleic acid sequences, the nucleic acid amplification section having an amplification chamber and a heater element; wherein,
• the heater element is bonded to an interior surface of the amplification chamber.
• GPC036.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the amplification chamber is a PCR chamber such that the heater element thermally cycles the sample together with dNTPs, primers, polymerase and a buffer solution to amplify the nucleic acid sequences.
• GPC036.3 Preferably, the microfluidic device also has a supporting substrate for supporting the PCR section wherein the PCR section has a microchannel with a PCR inlet and a PCR outlet, the PCR chamber being a section of the microchannel, and the interior surface to which the heater element is bonded is a base of the microchannel, closest to the supporting substrate.
• GPC036.4 Preferably, the PCR section has a plurality of the PCR chambers, and the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the PCR chambers.
• GPC036.5 Preferably, each of the channel sections has a plurality of the heater elements.
• GPC036.6 Preferably, the plurality of heater elements are elongate, aligned with the longitudinal extent of the channel section and positioned end to end along the channel section.
• GPC036.7 Preferably, each of the plurality of elongate heater elements is independently operable.
• GPC036.8 Preferably, the microfluidic device also has at least one temperature sensor for feedback control of the elongate heater elements.
• GPC036.9 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section for the duration of the thermal cycling.
• GPC036.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GPC036.11 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GPC036.12 Preferably, the PCR microchannel has a cross sectional area transverse to the flow of between 400 square microns and 1 square micron.
• GPC036.13 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GPC036.14 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GPC036.15 Preferably, each of the channel sections has a liquid sensor proximate one end, the liquid sensors being configured to detect the liquid at the liquid sensors location for feedback control of the heater elements.
• GPC036.16 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for nucleic acid amplification, and a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GPC036.17 Preferably, the reservoir has a vent for ingress of air as the reagent flows out of the reagent reservoir.
• GPC036.18 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC036.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GPC036.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's nucleic acid amplification chamber amplifies the nucleic acid targets in the sample.
• GPC037.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for amplifying nucleic acid sequences, the LOC device comprising:
• an inlet for receiving a sample containing genetic material;
• a supporting substrate;
• a plurality of reagent reservoirs; and,
• a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material; wherein,
• the nucleic acid amplification section is supported on the supporting substrate.
• GPC037.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GPC037.3 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GPC037.4 Preferably, the PCR section has a PCR microchannel in which the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC037.5 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC037.6 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC037.7 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC037.8 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC037.9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling.
• GPC037.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GPC037.11 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC037.12 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GPC037.13 Preferably, the LOC device also has a hybridization section downstream of the isothermal nucleic acid amplification section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC037.14 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GPC037.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chamber array.
• GPC037.16 Preferably, the isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the sample at a reaction temperature, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC037.17 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC037.18 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC037.19 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GPC037.20 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• The easily usable, mass-producible, and inexpensive LOC device accepts a sample containing nucleic acids and then utilizing the LOC device's nucleic acid amplification section amplifies the nucleic acid targets in the sample, utilizing reagents stored in the LOC device's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC038.1 This aspect of the invention provides a microfluidic device for amplifying nucleic acid sequences, the microfluidic device comprising:
• an inlet for receiving a sample containing genetic material;
• a plurality of reagent reservoirs containing reagents for addition to the sample; and,
• a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GPC038.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GPC038.3 Preferably, the microfluidic device also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GPC038.4 Preferably, the PCR section has a PCR microchannel in which the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC038.5 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC038.6 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC038.7 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC038.8 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC038.9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling.
• GPC038.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GPC038.11 Preferably, the microfluidic device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC038.12 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GPC038.13 Preferably, the microfluidic device also has a hybridization section downstream of the isothermal nucleic acid amplification section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC038.14 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GPC038.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chamber array.
• GPC038.16 Preferably, the isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the sample at a reaction temperature, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC038.17 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC038.18 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC038.19 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GPC038.20 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's nucleic acid amplification section amplifies the nucleic acid targets in the sample, utilizing reagents stored in the microfluidic device's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The reagent reservoirs, being integral to the microfluidic device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC039.1 This aspect of the invention provides a test module for amplifying nucleic acid sequences, the test module comprising:
• an outer casing with receptacle for receiving a sample containing genetic material;
• a plurality of reagent reservoirs containing reagents for addition to the sample; and,
• a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GPC039.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GPC039.3 Preferably, the test module also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GPC039.4 Preferably, the PCR section has a PCR microchannel in with the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC039.5 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC039.6 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC039.7 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC039.8 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC039.9 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling.
• GPC039.10 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GPC039.11 Preferably, the test module also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC039.12 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GPC039.13 Preferably, the test module also has a hybridization section downstream of the isothermal nucleic acid amplification section that has an array of probes for hybridization with target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC039.14 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GPC039.15 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chamber array.
• GPC039.16 Preferably, the isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the sample at a reaction temperature, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC039.17 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC039.18 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 2,500 square microns.
• GPC039.19 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GPC039.20 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• The easily usable, mass-producible, inexpensive, and portable test-module accepts a sample containing nucleic acids and then using the module's nucleic acid amplification section amplifies the nucleic acid targets in the sample, utilizing reagents stored in the test-module's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The reagent reservoirs, being integral to the test-module and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC040.1 This aspect of the invention provides a microfluidic device for amplifying nucleic acid sequences, the microfluidic device comprising:
• an inlet for receiving a sample containing genetic material;
• a plurality of reagent reservoirs containing reagents for addition to the sample;
• a first nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section.
• GPC040.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GPC040.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC040.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GPC040.5 Preferably, the microfluidic device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC040.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GPC040.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC040.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GPC040.9 Preferably, the microfluidic device also has a first hybridization section downstream of the first isothermal nucleic acid amplification section that has a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, a second hybridization section downstream of the second isothermal nucleic acid amplification section that has a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC040.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences.
• GPC040.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and section array of probes.
• GPC040.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the sample at a reaction temperature, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC040.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC040.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GPC040.15 Preferably, the microfluidic device also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GPC040.16 Preferably, the first PCR section has a PCR microchannel in which the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC040.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC040.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC040.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GPC040.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids and then utilizing the device's parallel nucleic acid amplification section amplifies the nucleic acid targets in the sample, utilizing reagents stored in the microfluidic device's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the microfluidic device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC041.1 This aspect of the invention provides a test module for amplifying nucleic acid sequences, the test module comprising:
• an outer casing with receptacle for receiving a sample containing genetic material;
• a plurality of reagent reservoirs containing reagents for addition to the sample;
• a first nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section.
• GPC041.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GPC041.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC041.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GPC041.5 Preferably, the test module also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC041.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GPC041.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC041.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GPC041.9 Preferably, the test module also has a first hybridization section downstream of the first isothermal nucleic acid amplification section that has a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, a second hybridization section downstream of the second isothermal nucleic acid amplification section that has a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GPC041.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences.
• GPC041.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chamber array.
• GPC041.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC041.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC041.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GPC041.15 Preferably, the test module also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GPC041.16 Preferably, the first PCR section has a PCR microchannel in which the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC041.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC041.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC041.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GPC041.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, and portable test-module accepts a sample containing nucleic acids and then using the module's parallel nucleic acid amplification section amplifies the nucleic acid targets in the sample, utilizing reagents stored in the test-module's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the test-module and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC042.1 This aspect of the invention provides a microfluidic device for amplifying DNA and RNA, the microfluidic device comprising:
• an inlet for receiving a sample containing genetic material including DNA and RNA;
• a plurality of reagent reservoirs containing reagents for addition to the sample;
• a first nucleic acid amplification section for amplifying at least some of the genetic material; and,
• a second nucleic acid amplification section for amplifying at least some of the genetic material in parallel with the first nucleic acid amplification section.
• GPC042.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second PCR section configured for amplifying the RNA in the genetic material.
• GPC042.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences in the RNA being different to the second set of complementary nucleic acid sequences.
• GPC042.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GPC042.5 Preferably, the microfluidic device also has a photosensor, a first hybridization section downstream of the first PCR section, a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GPC042.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section configured for amplifying the RNA in the genetic material.
• GPC042.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC042.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GPC042.9 Preferably, the microfluidic device also has further comprising a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section and a second hybridization section downstream of the second isothermal nucleic acid amplification section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GPC042.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences, and the second hybridization section has a second hybridization chamber array for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GPC042.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second arrays of hybridization chambers.
• GPC042.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC042.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC042.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GPC042.15 Preferably, the microfluidic device also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GPC042.16 Preferably, the first PCR section has a PCR microchannel where, during use, the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC042.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC042.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC042.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GPC042.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing DNA and RNA sequences and then utilizing the device's parallel nucleic acid amplification section amplifies the target DNA and RNA sequences in the sample, utilizing reagents stored in the microfluidic device's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of the target DNA and RNA sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the microfluidic device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GPC043.1 This aspect of the invention provides a test module for amplifying DNA and RNA, the test module comprising:
• an outer casing with receptacle for receiving a sample containing genetic material including DNA and RNA;
• a plurality of reagent reservoirs containing reagents for addition to the sample;
• a first nucleic acid amplification section for amplifying at least some of the genetic material; and,
• a second nucleic acid amplification section for amplifying at least some of the genetic material in parallel with the first nucleic acid amplification section.
• GPC043.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second PCR section configured for amplifying the RNA in the genetic material.
• GPC043.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences in the RNA being different to the second set of complementary nucleic acid sequences.
• GPC043.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GPC043.5 Preferably, the test module also has a photosensor, a first hybridization section downstream of the first PCR section, a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GPC043.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section configured for amplifying the RNA in the genetic material.
• GPC043.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GPC043.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GPC043.9 Preferably, the test module also has further comprising a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section and a second hybridization section downstream of the second isothermal nucleic acid amplification section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GPC043.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences, and the second hybridization section has a second hybridization chamber array for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GPC043.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second arrays of hybridization chambers.
• GPC043.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC043.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GPC043.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GPC043.15 Preferably, the test module also has CMOS circuitry and a temperature sensor, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GPC043.16 Preferably, the first PCR section has a PCR microchannel where, during use, the genetic material is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GPC043.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GPC043.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective channel sections of the PCR microchannel, the channel sections being parallel and adjacent to each other such that the PCR microchannel has a serpentine configuration.
• GPC043.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GPC043.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, and portable test-module accepts a sample containing DNA and RNA sequences and then using the module's parallel nucleic acid amplification section amplifies the target DNA and RNA sequences in the sample, utilizing reagents stored in the test-module's reagent reservoirs.
• The nucleic acid amplification section provides for capillary action propulsion of the amplification mixture, simplifying the design of the assay system, further increasing the reliability and reducing the cost of the assay system. The amplification of the target DNA and RNA sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the test-module and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GLY001.1 This aspect of the invention provides a microfluidic device for lysing cells in a fluid, the microfluidic device comprising:
• an inlet for receiving a fluid containing cells;
• a lysis reagent reservoir containing a lysis reagent and having an outlet valve; and,
• a lysis section in fluid communication with the inlet for holding the fluid during lysis of the cells; wherein,
• the outlet valve is upstream of the lysis section and configured to open as the fluid flows into the lysis section thereby adding the lysis reagent to the fluid.
• GLY001.2 Preferably, the outlet valve is a surface tension valve with an aperture configured to pin a meniscus that retains the lysis reagent therein until contact with the fluid removes the meniscus and the lysis reagent is added to the fluid flow into the lysis section.
• GLY001.3 Preferably, the lysis section is configured as a lysis channel to draw the fluid from the inlet by capillary action, the lysis section having an active valve at a downstream end of the lysis channel for retaining the fluid during lysis of the cells.
• GLY001.4 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the lysis section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GLY001.5 Preferably, the microchannel has a cross sectional area transverse to the flow of between 8 square microns and 20,000 square microns.
• GLY001.6 Preferably, the microfluidic device also has a supporting substrate wherein the inlet, the lysis section and the lysis reagent reservoir are supported on the supporting substrate and configured as a lab-on-a-chip (LOC) device.
• GLY001.7 Preferably, the microfluidic device also has CMOS circuitry between the supporting substrate and the lysis section, the CMOS circuitry being configured for operative control of the valve heater.
• GLY001.8 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section with at least one heater for thermally cycling the fluid to amplify nucleic acid sequences released from the cells.
• GLY001.9 Preferably, the microfluidic device also has at least one sensor wherein the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY001.10 Preferably, the microfluidic device also has a plurality of heaters and a plurality of temperature sensors and a plurality of liquid sensors such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the temperature sensors.
• GLY001.11 Preferably, each of the plurality of heaters is independently operable.
• GLY001.12 Preferably, the PCR section is configured as a PCR microchannel with the cross sectional area between 1 square micron and 400 square microns, the PCR section also having an active valve at a downstream end of the PCR microchannel for retaining the fluid during amplification of the nucleic acid sequences.
• GLY001.13 Preferably, the microfluidic device also has a PCR mix reservoir and a polymerase reservoir wherein the PCR mix reservoir contains dNTPs, primers and buffer solution and the polymerase reservoir contains polymerase enzymes.
• GLY001.14 Preferably, the microfluidic device also has a cap which defines the lysis reagent reservoir, the PCR mix reservoir, the polymerase reservoir and the lysis section, and the PCR section is between the cap and the supporting substrate.
• GLY001.15 Preferably, the microfluidic device also has a dialysis section wherein the cells in the fluid are of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY001.16 Preferably, the nucleic acid sequences are from the cells larger than the predetermined threshold.
• GLY001.17 Preferably, the microfluidic device also has an anticoagulant reservoir wherein the fluid is a whole blood sample such that anticoagulant is added to the whole blood upstream of the dialysis section and the dialysis section is configured to concentrate the leukocytes into a portion of the whole blood sample.
• GLY001.18 Preferably, the microfluidic device also has a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with predetermined target nucleic acid sequences in the amplicon from the PCR section; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GLY001.19 Preferably, the hybridization section has electrodes positioned for receiving an electrical pulse, and the probes are electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes.
• GLY001.20 Preferably, the photosensor is an array of photodiodes positioned in registration with the ECL probes respectively.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a chemical lysis subunit for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• GLY002.1 This aspect of the invention provides a microfluidic device for lysing cells in a fluid, the microfluidic device comprising:
• an inlet channel for receiving a fluid containing cells;
• a lysis section in fluid communication with the inlet channel for holding the fluid during lysis of the cells; and,
• a lysis heater for heating the fluid in the lysis section such that the cells lyse; wherein,
• the inlet channel is configured for filling the lysis section by capillary action.
• GLY002.2 Preferably, the lysis section is configured as a lysis microchannel to draw the fluid from the inlet by capillary action, the lysis section having an active valve at a downstream end of the lysis microchannel for retaining the fluid during lysis of the cells.
• GLY002.3 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the lysis section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GLY002.4 Preferably, the microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GLY002.5 Preferably, the microfluidic device also has a supporting substrate wherein the inlet, the lysis section is supported on the supporting substrate and configured as a lab-on-a-chip (LOC) device.
• GLY002.6 Preferably, the microfluidic device also has CMOS circuitry between the supporting substrate and the lysing chamber, the CMOS circuitry being configured for operative control of the lysis heater and the valve heater at the downstream end of the microchannel.
• GLY002.7 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section with at least one heater for thermally cycling the fluid to amplify nucleic acid sequences released from the cells.
• GLY002.8 Preferably, the microfluidic device also has at least one sensor wherein the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY002.9 Preferably, the microfluidic device also has a plurality of temperature sensors and a plurality of liquid sensors wherein the PCR section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensors and heater power in response to the temperature sensors.
• GLY002.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GLY002.11 Preferably, the PCR section is configured as a PCR microchannel with the same cross section as the lysis microchannel, the PCR section also having an active valve at a downstream end of the PCR microchannel for retaining the fluid during amplification of the nucleic acid sequences.
• GLY002.12 Preferably, the microfluidic device also has a PCR mix reservoir and a polymerase reservoir wherein the PCR mix reservoir contains dNTPs, primers and buffer solution and the polymerase reservoir contains polymerase enzymes.
• GLY002.13 Preferably, the microfluidic device also has a cap which defines the PCR mix reservoir and the polymerase reservoir, wherein the lysis section and the PCR section are between the cap and the supporting substrate.
• GLY002.14 Preferably, the microfluidic device also has a dialysis section wherein the cells in the fluid are of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY002.15 Preferably, the nucleic acid sequences are from the cells larger than the predetermined threshold.
• GLY002.16 Preferably, the microfluidic device also has an anticoagulant reservoir wherein the fluid is a whole blood sample such that anticoagulant is added to the whole blood upstream of the dialysis section and the dialysis section is configured to concentrate the leukocytes into a portion of the whole blood sample.
• GLY002.17 Preferably, the microfluidic device also has a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with predetermined target nucleic acid sequences in the amplicon from the PCR section; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GLY002.18 Preferably, the hybridization section has electrodes positioned for receiving an electrical pulse, and the probes are electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes.
• GLY002.19 Preferably, the photosensor is an array of photodiodes positioned in registration with the ECL probes respectively.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses a thermal lysis subunit for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• GLY003.1 This aspect of the invention provides a microfluidic device for lysing cells in a fluid, the microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving fluid containing cells;
• a lysis section in fluid communication with the inlet, the lysis section having at least one heater for heating the fluid;
• a reservoir containing a lysis reagent; and,
• CMOS circuitry between the supporting substrate and the lysis section, the CMOS circuitry being configured for selectively lysing the cells using thermal lysis in the lysis section, chemically lysing the cells using the lysis reagent, or both chemically and thermally lysing the cells using the lysis section and the lysis reagent.
• GLY003.2 Preferably, the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences released from the cells, the PCR section having at least one PCR heater and at least one sensor connected to the CMOS circuitry; wherein,
• the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY003.3 Preferably, the microfluidic device also has a temperature sensor and a liquid sensor wherein the PCR section has a plurality of heaters and the CMOS circuitry is configured to control initial activation of the heaters in response to the liquid sensor and heater power in response to the temperature sensor.
• GLY003.4 Preferably, the PCR section has a plurality of elongate PCR chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the PCR chambers.
• GLY003.5 Preferably, the PCR section has a microchannel with a PCR inlet and a PCR outlet, and the elongate PCR chambers are sections of the microchannel.
• GLY003.6 Preferably, the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
• GLY003.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GLY003.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GLY003.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GLY003.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GLY003.11 Preferably, the PCR section has a active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GLY003.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GLY003.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GLY003.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined number of thermal cycles.
• GLY003.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY003.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GLY003.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GLY003.18 Preferably, the microfluidic device also has a hybridization section with an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids; and,
• an array of photodiodes for detecting the probe-target hybrids within the array of probes.
• GLY003.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GLY003.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a biochemical sample, uses chemical and thermal lysis subunits for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The option of using chemical and thermal lysing processes simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• GLY004.1 This aspect of the invention provides a test module for lysing cells in a fluid, the test module comprising:
• an outer casing with receptacle for receiving a fluid containing cells;
• a lysis reagent reservoir containing a lysis reagent and having an outlet valve; and,
• a lysis section in fluid communication with the receptacle for holding the fluid during lysis of the cells; wherein,
• the outlet valve is upstream of the lysis section and configured to open as the fluid flows into the lysis section thereby adding the lysis reagent to the fluid.
• GLY004.2 Preferably, the outlet valve is a surface tension valve with an aperture configured to pin a meniscus that retains the lysis reagent therein until contact with the fluid removes the meniscus and the lysis reagent is added to the fluid flow into the lysis section.
• GLY004.3 Preferably, the lysis section is configured as a lysis microchannel to draw the fluid from the receptacle by capillary action, the lysis section having an active valve at a downstream end of the lysis microchannel for retaining the fluid during lysis of the cells.
• GLY004.4 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the lysis section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GLY004.5 Preferably, the microchannel has a cross sectional area transverse to the flow of between 8 square microns and 20,000 square microns.
• GLY004.6 Preferably, the test module also has a supporting substrate wherein the lysis section and the lysis reagent reservoir are supported on the supporting substrate and configured as a lab-on-a-chip (LOC) device.
• GLY004.7 Preferably, the test module also has CMOS circuitry between the supporting substrate and the lysis section, the CMOS circuitry being configured for operative control of the valve heater at the downstream end of the microchannel.
• GLY004.8 Preferably, the test module also has a polymerase chain reaction (PCR) section with at least one heater for thermally cycling the fluid to amplify nucleic acid sequences released from the cells.
• GLY004.9 Preferably, the test module also has at least one sensor wherein the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY004.10 Preferably, the test module also has a plurality of heaters and at least one temperature sensor and at least one liquid sensor such that the CMOS circuitry controls initial activation of the heaters in response to the at least one liquid sensor and heater power in response to the at least one temperature sensor.
• GLY004.11 Preferably, each of the plurality of heaters is independently operable.
• GLY004.12 Preferably, the PCR section is configured as a PCR microchannel with the same cross section as the lysis microchannel, the PCR section also having an active valve at a downstream end of the PCR microchannel for retaining the fluid during amplification of the nucleic acid sequences.
• GLY004.13 Preferably, the test module also has a PCR mix reservoir and a polymerase reservoir wherein the PCR mix reservoir contains dNTPs, primers and buffer solution and the polymerase reservoir contains polymerase enzymes.
• GLY004.14 Preferably, the test module also has a cap which defines the lysis reagent reservoir, the PCR mix reservoir and the polymerase reservoir, wherein the lysis section and the PCR section are between the cap and the supporting substrate.
• GLY004.15 Preferably, the test module also has a dialysis section wherein the cells in the fluid are of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY004.16 Preferably, the nucleic acid sequences are from the cells larger than the predetermined threshold.
• GLY004.17 Preferably, the test module also has an anticoagulant reservoir wherein the fluid is a whole blood sample such that anticoagulant is added to the whole blood upstream of the dialysis section and the dialysis section is configured to concentrate the leukocytes into a portion of the whole blood sample.
• GLY004.18 Preferably, the test module also has a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with predetermined target nucleic acid sequences in the amplicon from the PCR section; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GLY004.19 Preferably, the hybridization section has electrodes positioned for receiving an electrical pulse, and the probes are electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes.
• GLY004.20 Preferably, the photosensor is an array of photodiodes positioned in registration with the ECL probes respectively.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a biochemical sample, uses a chemical lysis subunit for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the module, provides for simple assay procedures, low assay system complexity, leading into an inexpensive assay system.
• GLY005.1 This aspect of the invention provides a test module for lysing cells in a fluid, the test module comprising:
• • an outer casing with receptacle for receiving a sample that is a fluid containing cells;
• a lysis section in fluid communication with the receptacle for holding the fluid during lysis of the cells; and,
• a lysis heater for heating the fluid in the lysis section to lyse cells; wherein,
• the test module is configured for drawing the fluid from the receptacle to the lysis section by capillary action.
• GLY005.2 Preferably, the lysis section is configured as a lysis microchannel to draw the fluid from the inlet by capillary action, the lysis section having an active valve at a downstream end of the lysis microchannel for retaining the fluid during lysis of the cells.
• GLY005.3 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the lysis section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GLY005.4 Preferably, the microchannel has a cross sectional area transverse to the flow of between 1 square micron and 400 square microns.
• GLY005.5 Preferably, the test module also has a supporting substrate wherein the lysis section is supported on the supporting substrate and configured as a lab-on-a-chip (LOC) device.
• GLY005.6 Preferably, the test module also has CMOS circuitry between the supporting substrate and the lysis section, the CMOS circuitry being configured for operative control of the lysis heater and the valve heater at the downstream end of the microchannel.
• GLY005.7 Preferably, the test module also has a polymerase chain reaction (PCR) section with at least one heater for thermally cycling the fluid to amplify nucleic acid sequences released from the cells.
• GLY005.8 Preferably, the test module also has at least one sensor wherein the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY005.9 Preferably, the test module also has at least one temperature sensor and at least one liquid sensor wherein the PCR section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the at least one liquid sensor and heater power in response to the at least one temperature sensor.
• GLY005.10 Preferably, each of the plurality of heaters is independently operable.
• GLY005.11 Preferably, the PCR section is configured as a PCR microchannel with the same cross section as the lysis microchannel, the PCR section also having an active valve at a downstream end of the PCR microchannel for retaining the fluid during amplification of the nucleic acid sequences.
• GLY005.12 Preferably, the test module also has a PCR mix reservoir and a polymerase reservoir wherein the PCR mix reservoir contains dNTPs, primers and buffer solution and the polymerase reservoir contains polymerase enzymes.
• GLY005.13 Preferably, the test module also has a cap which defines the PCR mix reservoir and the polymerase reservoir, wherein the lysis section and the PCR section are between the cap and the supporting substrate.
• GLY005.14 Preferably, the test module also has a dialysis section wherein the cells in the fluid are of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY005.15 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GLY005.16 Preferably, the test module also has an anticoagulant reservoir wherein the fluid is a whole blood sample such that anticoagulant is added to the whole blood upstream of the dialysis section.
• GLY005.17 Preferably, the test module also has a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with predetermined target nucleic acid sequences in the amplicon from the PCR section; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GLY005.18 Preferably, the hybridization section has electrodes positioned for receiving an electrical pulse, and the probes are electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being configured to emit a photon of light when excited by current between the electrodes.
• GLY005.19 Preferably, the photosensor is an array of photodiodes positioned in registration with the ECL probes respectively.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a biochemical sample, uses a thermal lysis subunit for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the module, provides for simple assay procedures, low assay system complexity, leading into an inexpensive assay system.
• The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• GLY006.1 This aspect of the invention provides a test module for lysing cells in a fluid, the test module comprising:
• an outer casing with receptacle for receiving a fluid containing cells;
• a lysis reagent reservoir containing a lysis reagent and having a valve; and,
• a lysis section in fluid communication with the receptacle for holding the fluid during lysis of the cells, the lysis section having a lysis heater for heating the fluid in the lysis section; wherein,
• the valve is upstream of the lysis section and configured to open as the fluid flows into the lysis section thereby adding the lysis reagent to the fluid.
• GLY006.2 Preferably, the valve is a surface tension valve with an aperture configured to pin a meniscus that retains the lysis reagent therein until contact with the fluid removes the meniscus and the lysis reagent is added to the fluid flow into the lysis section.
• GLY006.3 Preferably, the lysis section has a lysis microchannel configured to draw the fluid from the receptacle by capillary action, the lysis section having an active valve at a downstream end of the lysis microchannel for retaining the fluid during lysis of the cells.
• GLY006.4 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the lysis section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GLY006.5 Preferably, the lysis microchannel has a cross sectional area transverse to the flow of between 8 square microns and 20,000 square microns.
• GLY006.6 Preferably, the test module also has a supporting substrate wherein the lysis section and the lysis reagent reservoir are supported on the supporting substrate and configured as a lab-on-a-chip (LOC) device.
• GLY006.7 Preferably, the test module also has CMOS circuitry between the supporting substrate and the lysis section, the CMOS circuitry being configured for operative control of the valve heater at the downstream end of the microchannel and the lysis heater in the lysis section.
• GLY006.8 Preferably, the test module also has a polymerase chain reaction (PCR) section with at least one heater for thermally cycling the fluid to amplify nucleic acid sequences released from the cells.
• GLY006.9 Preferably, the test module also has at least one sensor wherein the CMOS circuitry is configured to use the sensor for feedback control of the at least one PCR heater.
• GLY006.10 Preferably, the test module also has a plurality of heaters and at least one temperature sensor and at least one liquid sensor such that the CMOS circuitry controls initial activation of the heaters in response to the at least one liquid sensor and heater power in response to the at least one temperature sensor.
• GLY006.11 Preferably, each of the plurality of heaters is independently operable.
• GLY006.12 Preferably, the PCR section is configured as a PCR microchannel with the same cross section as the lysis microchannel, the PCR section also having an active valve at a downstream end of the PCR microchannel for retaining the fluid during amplification of the nucleic acid sequences.
• GLY006.13 Preferably, the test module also has a PCR mix reservoir and a polymerase reservoir wherein the PCR mix reservoir contains dNTPs, primers and buffer solution and the polymerase reservoir contains polymerase enzymes.
• GLY006.14 Preferably, the test module also has a cap which defines the lysis reagent reservoir, the PCR mix reservoir and the polymerase reservoir, wherein the lysis section and the PCR section are between the reservoirs and the supporting substrate.
• GLY006.15 Preferably, the test module also has a dialysis section wherein the cells in the fluid are of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLY006.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GLY006.17 Preferably, the test module also has an anticoagulant reservoir wherein the fluid is a whole blood sample such that anticoagulant is added to the whole blood upstream of the dialysis section and the dialysis section is configured to concentrate the leukocytes into a portion of the whole blood sample.
• GLY006.18 Preferably, the test module also has a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with predetermined target nucleic acid sequences in the amplicon from the PCR section; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GLY006.19 Preferably, the array of probes are fluorescent probes for hybridization with the target nucleic acid sequences to form probe-target hybrids, the probe-target hybrids being configured to emit a photon of light when exposed to an excitation light.
• GLY006.20 Preferably, the photosensor is an array of photodiodes positioned in registration with the fluorescent probes respectively.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a biochemical sample, uses chemical and thermal lysis subunits for lysing cells or cell organelles, and processes the lysates.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the module, provides for simple assay procedures, low assay system complexity, leading into an inexpensive assay system.
• The option of using chemical and thermal lysing processes simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• GIN001.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• an inlet for receiving a fluid;
• a microsystems technology (MST) layer for processing the fluid, the MST layer incorporating an incubation section, the incubation section having at least one heater for maintaining the fluid at a predetermined incubation temperature; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry being connected to the at least one heater for operative control of the at least one heater.
• GIN001.2 Preferably, the microfluidic device also has at least one sensor wherein the CMOS circuitry uses the sensor for feedback control of the at least one heater.
• GIN001.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN001.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN001.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN001.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN001.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN001.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN001.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN001.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN001.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN001.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN001.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN001.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN001.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN001.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN001.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN001.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN001.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN001.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation section incubates the fluid at the requisite temperature for the requisite period of time for the required reaction. The microfluidic device is mass-produced inexpensively using microsystem technology (MST).
• The incubation section being integral to the device, provides for simple procedures and low system complexity, with the low system complexity in turn providing for an inexpensive system.
• GIN002.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a fluid sample;
• an incubation section for maintaining the fluid sample at an incubation temperature, the incubation section having at least one heater; and,
• at least one sensor; wherein,
• the at least one sensor is configured to provide output for feedback control of the at least one heater.
• GIN002.2 Preferably, the microfluidic device also has CMOS circuitry connected to the at least one heater and the at least one sensor such that the CMOS circuitry uses the sensor for feedback control of the at least one heater.
• GIN002.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN002.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN002.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN002.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN002.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN002.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN002.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN002.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN002.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN002.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN002.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN002.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN002.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN002.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN002.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN002.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN002.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN002.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation section incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The incubation section is controlled by a feedback control system, resulting into the faster and more precise temperature control. The faster temperature control capability increases the overall process speed. The more precise temperature cycling capability provides for the improved reaction conditions. The incubation section being integral to the device, provides for simple procedures and low system complexity, with the low system complexity in turn providing for an inexpensive system.
• GIN003.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a fluid sample;
• an incubation section for maintaining the fluid sample at an incubation temperature, the incubation section having at least one heater and at least one temperature sensor; wherein,
• the at least one temperature sensor is configured to provide output for feedback control of the at least one heater.
• GIN003.2 Preferably, the microfluidic device also has CMOS circuitry connected to the at least one heater and the at least one temperature sensor such that the CMOS circuitry uses the temperature sensor for feedback control of the at least one heater.
• GIN003.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN003.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN003.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN003.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN003.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN003.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN003.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN003.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN003.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN003.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN003.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN003.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN003.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN003.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN003.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN003.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN003.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN003.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation section incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The incubation section is controlled by a temperature feedback control system, resulting into the faster and more precise temperature control. The faster temperature control capability increases the overall process speed. The more precise temperature cycling capability provides for the improved reaction conditions. The incubation section being integral to the device, provides for simple procedures and low system complexity, with the low system complexity in turn providing for an inexpensive system.
• GIN004.1 This aspect of the invention provides a lab-on-a-chip (LOC) device comprising:
• a supporting substrate;
• a sample inlet for receiving a fluid sample;
• an incubation section in fluid communication with the sample inlet, the incubation section having at least one heater; and,
• CMOS circuitry between the supporting substrate and the incubation section; wherein,
• the CMOS circuitry is connected to the at least one heater for maintaining the fluid sample at an incubation temperature for an incubation period.
• GIN004.2 Preferably, the LOC device also has at least one temperature sensor wherein the CMOS circuitry uses the temperature sensor for feedback control of the at least one heater.
• GIN004.3 Preferably, the LOC device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN004.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN004.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN004.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN004.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN004.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN004.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN004.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN004.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN004.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN004.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN004.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined time interval.
• GIN004.15 Preferably, the LOC device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN004.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN004.17 Preferably, the LOC device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN004.18 Preferably, the LOC device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN004.19 Preferably, the LOC device also has a nucleic acid amplification section.
• GIN004.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation section incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The incubation section is controlled by an on-chip semiconductor control system, resulting into the faster and more precise temperature control. The faster temperature control capability increases the overall process speed. The more precise temperature cycling capability provides for the improved reaction conditions. The incubation section and its on-chip semiconductor control system being integral to the device, provides for simple procedures and low system complexity, with the low system complexity in turn providing for an inexpensive system.
• GIN005.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a fluid sample;
• an incubation section in fluid communication with the sample inlet, the incubation section having at least one heater for maintaining the fluid sample at an incubation temperature for an incubation period; and,
• CMOS circuitry to energize the at least one heater with a pulse width modulated (PWM) signal.
• GIN005.2 Preferably, the microfluidic device also has at least one temperature sensor wherein the CMOS circuitry uses the temperature sensor for feedback control of the at least one heater.
• GIN005.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN005.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN005.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN005.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN005.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN005.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN005.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN005.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN005.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN005.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN005.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN005.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN005.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN005.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN005.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN005.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN005.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN005.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation section incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The incubation section is controlled by a PWM control system, resulting into the faster and more precise temperature control. The faster temperature control capability increases the overall process speed. The more precise temperature cycling capability provides for the improved reaction conditions. The incubation section and its PWM control system being integral to the device, provides for simple procedures and low system complexity, with the low system complexity in turn providing for an inexpensive system.
• GIN006.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a fluid sample;
• an incubation section having an elongate incubation chamber with a longitudinal extent much greater than the lateral dimensions, and at least one heater for maintaining the fluid sample at an incubation temperature for an incubation period; wherein,
• the at least one heater is also elongated with a lateral extent parallel to that of the elongate incubation chamber.
• GIN006.2 Preferably, the microfluidic device also has CMOS circuitry, and at least one temperature sensor wherein the CMOS circuitry is connected to the at least one heater and the at least one temperature sensor such that the CMOS circuitry uses the temperature sensor for feedback control of the at least one heater.
• GIN006.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN006.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN006.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN006.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN006.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN006.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN006.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN006.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN006.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN006.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN006.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN006.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN006.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN006.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN006.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN006.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN006.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN006.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation chamber incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The incubation chamber is of an elongated geometry which provides for rapid temperature control of the mixture and capillary action propulsion of the mixture. The rapid temperature control capability increases the overall process speed. The capillary action propulsion simplifies the design of the system, further increasing the reliability and reducing the cost of the system.
• GIN007.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a sample inlet for receiving a fluid sample;
• an incubation section having an incubation chamber, and at least one heater for maintaining the fluid sample at an incubation temperature for a period; wherein,
• the incubation chamber is between the at least one heater element and the supporting substrate.
• GIN007.2 Preferably, the microfluidic device also has CMOS circuitry, and at least one temperature sensor wherein the CMOS circuitry is connected to the at least one heater and the at least one temperature sensor such that the CMOS circuitry uses the temperature sensor for feedback control of the at least one heater.
• GIN007.3 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the incubation section has a plurality of heaters such that the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN007.4 Preferably, the incubation section has a plurality of elongate chambers having a longitudinal extent much greater than its lateral dimensions, and each of the heaters are elongate and parallel with the longitudinal extent of the incubation chambers.
• GIN007.5 Preferably, the incubation section has a microchannel with an incubation inlet and an incubation outlet, and the elongate incubation chambers are sections of the microchannel.
• GIN007.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences from the incubation inlet to the incubation outlet by capillary action.
• GIN007.7 Preferably, the microchannel has a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate incubation chambers.
• GIN007.8 Preferably, the channel section forming each of the wide meanders has a plurality of the elongate heaters.
• GIN007.9 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GIN007.10 Preferably, each of the plurality of elongate heaters is independently operable.
• GIN007.11 Preferably, the incubation section has an active valve at the incubation outlet for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN007.12 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN007.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN007.14 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN007.15 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN007.16 Preferably, the dialysis section is upstream of the incubation section and the cells smaller than the predetermined threshold contain nucleic acid sequences to be digested by restriction enzymes in the incubation section.
• GIN007.17 Preferably, the microfluidic device also has a reagent reservoir for holding a reagent used in the incubator; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GIN007.18 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GIN007.19 Preferably, the microfluidic device also has a nucleic acid amplification section.
• GIN007.20 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation chamber incubates the fluid at the requisite temperature for the requisite period of time for the required reaction. The incubation chamber's heater is above the chamber, maximizing heat transfer into the incubation mixture and minimizing heat transfer into the substrate.
• GIN008.1 This aspect of the invention provides a microfluidic device comprising:
• an inlet for receiving a fluid;
• an incubation section having a microchannel configured to have a plurality of mutually parallel, adjacent channel sections, and a plurality of elongate heaters positioned end to end along each of the channel sections; wherein,
• each of the heaters are independently operable for two-dimensional control of heat flux density to the incubation section.
• GIN008.2 Preferably, the microchannel is configured in a serpentine configuration with a series of wide meanders to form the plurality of mutually parallel, adjacent channel sections.
• GIN008.3 Preferably, the microfluidic device also has a supporting substrate and
• CMOS circuitry wherein the CMOS circuitry is between the supporting substrate and the incubator section for control of the heaters.
• GIN008.4 Preferably, the microfluidic device also has at least one sensor wherein the CMOS circuitry uses the sensor for feedback control of the heaters.
• GIN008.5 Preferably, the microfluidic device also has at least one temperature sensor and at least one liquid sensor wherein the CMOS circuitry controls initial activation of the heaters in response to the liquid sensor and subsequent control of the heaters in response to the temperature sensor.
• GIN008.6 Preferably, the fluid sample is a biological material containing nucleic acid sequences and the microchannel is configured to draw a liquid containing the nucleic acid sequences through all the channel sections by capillary action.
• GIN008.7 Preferably, the incubation section has an active valve at a downstream end for retaining the liquid in the incubation section while the elongate heaters maintain a mixture of restriction enzymes and the nucleic acid sequences at a temperature suitable for strand restriction digestion.
• GIN008.8 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the fluid in the incubation section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the incubation section resumes.
• GIN008.9 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GIN008.10 Preferably, the CMOS circuitry activates the valve heater after a predetermined incubation time.
• GIN008.11 Preferably, the microfluidic device also has a dialysis section wherein the fluid sample is biological material including cells of different sizes, the dialysis section being configured for concentrating cells smaller than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells larger than the predetermined threshold.
• GIN008.12 Preferably, the dialysis section is upstream of the incubation section and the cells larger than the predetermined threshold contain nucleic acid sequences to be digested by the restriction enzymes in the incubation section.
• GIN008.13 Preferably, the microfluidic device also has a reagent reservoir for holding the restriction enzymes used in the incubator and a surface tension valve having an aperture configured to pin a meniscus of solution containing the restriction enzymes such that the meniscus retains the restriction enzymes in the reagent reservoir until contact with the fluid sample removes the meniscus and the restriction enzymes flow out of the reagent reservoir.
• GIN008.14 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, and a photosensor for detecting the probe-target hybrids.
• GIN008.15 Preferably, the microfluidic device also has a nucleic acid amplification section downstream of the incubation section.
• GIN008.16 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section having a thermal cycle time between 0.45 seconds and 1.5 seconds.
• GIN008.17 Preferably, the microfluidic device also has a lysis section to disrupt cellular and intracellular membranes in the cells contained in the sample, the lysis section being upstream of the incubation section and downstream of the dialysis section.
• GIN008.18 Preferably, the fluid sample is blood and the cells larger than the predetermined threshold include leukocytes.
• GIN008.19 Preferably, the microfluidic device also has an anticoagulant reservoir for adding anticoagulant to the sample upstream of the dialysis section.
• GIN008.20 Preferably, the photosensor is an array of photodiodes positioned in registration with the probes.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a fluid and then utilizing the device's incubation chamber incubates the fluid at the requisite temperature for the requisite period of time for the required reaction.
• The heat flux density into the incubation chamber is controlled two-dimensionally, with the resulting two-dimensional temperature distribution being highly uniform, providing for uniform incubation characteristics and improved reaction outcome.
• GMI001.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences;
• a reagent reservoir containing a reagent; and,
• a mixing section for mixing the nucleic acid sequences with the reagent; wherein during use,
• the sample flows from the sample inlet to the PCR section via the mixing section.
• GMI001.2 Preferably, the reagent reservoir has a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GMI001.3 Preferably, the microfluidic device also has an incubation section downstream of the mixing section, the incubation section being configured to maintain a mixture of the sample and the restriction enzymes at an incubation temperature for restriction digestion of the nucleic acid sequences.
• GMI001.4 Preferably, the mixing section is a microchannel defining a tortuous flow-path having a length sufficient for diffusive mixing of the restriction enzymes and the sample.
• GMI001.5 Preferably, the microchannel has a serpentine configuration.
• GMI001.6 Preferably, the cross sectional area transverse to the flow is between 20,000 square microns and 8 square microns.
• GMI001.7 Preferably, the microfluidic device also has a lysis section upstream of the mixing section, the lysis section being configured to lyse cells within the sample to release genetic material therein.
• GMI001.8 Preferably, the incubation section has an incubation heater configured for heating the nucleic acid sequences and the restriction enzymes to an incubation temperature.
• GMI001.9 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer in which the lysis section, the incubation section and the PCR section are formed.
• GMI001.10 Preferably, the microfluidic device also has CMOS circuitry and at least one temperature sensor, the CMOS circuitry being positioned between the supporting substrate and the MST layer, and the temperature sensor being configured for feedback control of the incubation heater.
• GMI001.11 Preferably, the lysis section has an active valve at a downstream end to retain liquid for a predetermined period of time.
• GMI001.12 Preferably, the outlet valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the lysis section, the boiling-initiated valve having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GMI001.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GMI001.14 Preferably, the microfluidic device also has a cap overlying the MST layer, wherein the cap has the restriction enzyme reservoir, the mixing section and a plurality of PCR reagent reservoirs formed therein.
• GMI001.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GMI001.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GMI001.17 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GMI001.18 Preferably, each of the channel sections has a plurality of heaters.
• GMI001.19 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GMI001.20 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing a fluid, then using a microfluidic mixer, adds and mixes the requisite reagents into the input fluid or into the mixtures derived from the input fluid.
• The microfluidic mixer is a reliable and easily manufacturable mixer and is integral to the device, providing for a reliable, easily assembled, and inexpensive microfluidic system with a low component-count.
• GMI002.1 This aspect of the invention provides a microfluidic device comprising:
• a sample inlet for receiving a sample of biological material having nucleic acid sequences;
• a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences;
• a reagent reservoir containing a reagent; and,
• a diffusion mixing section for mixing the nucleic acid sequences with the reagent, the diffusion mixing section having a microchannel defining a tortuous flow-path having a length sufficient for diffusive mixing of the reagent and the sample; wherein during use,
• the sample flows from the sample inlet to the PCR section via the diffusion mixing section.
• GMI002.2 Preferably, the reagent reservoir has a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GMI002.3 Preferably, the microfluidic device also has an incubation section downstream of the mixing section, the incubation section being configured to maintain a mixture of the sample and the restriction enzymes at an incubation temperature for restriction digestion of the nucleic acid sequences.
• GMI002.4 Preferably, the microchannel has a serpentine configuration.
• GMI002.5 Preferably, the cross sectional area transverse to the flow is between 8 square micron and 20,000 square microns.
• GMI002.6 Preferably, the microfluidic device also has a lysis section upstream of the mixing section, the lysis section being configured to lyse cells within the sample to release genetic material therein.
• GMI002.7 Preferably, the microfluidic device also has an anticoagulant reservoir upstream of the lysis section wherein the sample is whole blood and the anticoagulant reservoir has a surface tension valve with an aperture configured to pin a meniscus of the anticoagulant that retains the anticoagulant until contact with the blood removes the meniscus to add the anticoagulant to the blood.
• GMI002.8 Preferably, the incubation section has an incubation heater configured for heating the nucleic acid sequences and the restriction enzymes to an incubation temperature.
• GMI002.9 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer in which the lysis section, the incubation section and the PCR section are formed.
• GMI002.10 Preferably, the microfluidic device also has CMOS circuitry and at least one temperature sensor, the CMOS circuitry being positioned between the supporting substrate and the MST layer, and the temperature sensor being configured for feedback control of the incubation heater.
• GMI002.11 Preferably, the lysis section has an active valve at a downstream end to retain liquid for a predetermined period of time.
• GMI002.12 Preferably, the outlet valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the lysis section, the boiling-initiated valve having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GMI002.13 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GMI002.14 Preferably, the microfluidic device also has a cap overlying the MST layer, wherein the cap has the restriction enzyme reservoir, a plurality of PCR reagent reservoirs and the mixing section formed therein.
• GMI002.15 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GMI002.16 Preferably, the nucleic acid sequences are from the cells smaller than the predetermined threshold.
• GMI002.17 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GMI002.18 Preferably, each of the channel sections has a plurality of heaters.
• GMI002.19 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GMI002.20 Preferably, the PCR section has a thermal cycle time of less than 30 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing nucleic acids, then using a diffusion mixer, adds and mixes the requisite reagents into the sample or into the mixtures derived from the sample, and then utilizing the device's PCR chamber amplifies the nucleic acid targets in the sample.
• The diffusion mixer is a reliable and easily manufacturable mixer and is integral to the device, providing for a reliable, easily assembled, and inexpensive microfluidic system with a low component-count.
• The microfluidic PCR chamber is an easily manufacturable PCR cycler and is integral to the device, in turn providing for an easily assembled and inexpensive microfluidic system with a low component-count.
• GMI005.1 This aspect of the invention provides a microfluidic device comprising:
• a supporting substrate;
• a microsystems technologies (MST) layer on the supporting substrate, the MST layer being configured to combine at least two liquids to form a flow of combined liquids, the MST layer having a diffusion mixing section for diffusive mixing of the at least two liquids, the mixing section having a microchannel defining a flow-path for the combined liquids; wherein,
• the channel cross section transverse to the flow direction is less than 100,000 square microns.
• GMI005.2 Preferably, the microchannel cross section transverse to the flow direction is less than 16,000 square microns.
• GMI005.3 Preferably, the microchannel cross section transverse to the flow direction is less than 2500 square microns.
• GMI005.4 Preferably, the microchannel cross section transverse to the flow direction is between 1 square micron and 400 square microns.
• GMI005.5 Preferably, one of the at least two liquids contains genetic material from a biological sample, and another of the at least two liquids is a reagent used for analyzing the genetic material.
• GMI005.6 Preferably, the microfluidic device of claim 5 further comprising a sample inlet for receiving the biological sample having nucleic acid sequences, a polymerase chain reaction (PCR) section for amplifying the nucleic acid sequences, a reagent reservoir containing the reagent; wherein,
• the microchannel defines a flow-path having a length sufficient for diffusive mixing of the reagent and the sample; wherein during use,
• the sample flows from the sample inlet to the PCR section via the diffusion mixing section.
• GMI005.7 Preferably, the reagent reservoir has a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GMI005.8 Preferably, the microfluidic device also has an incubation section downstream of the diffusion mixing section, the incubation section configured to maintain a mixture of the sample and the restriction enzymes at an incubation temperature for restriction digestion of the nucleic acid sequences.
• GMI005.9 Preferably, the microfluidic device also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• a photosensor for detecting hybridization of probes within the array of probes.
• GMI005.10 Preferably, the microfluidic device also has a lysis section upstream of the diffusion mixing section, the lysis section being configured to lyse cells within the sample to release genetic material therein.
• GMI005.11 Preferably, the microfluidic device also has an anticoagulant reservoir upstream of the lysis section wherein the sample is whole blood and the anticoagulant reservoir has a surface tension valve with an aperture configured to pin a meniscus of the anticoagulant that retains the anticoagulant until contact with the blood removes the meniscus to add the anticoagulant to the blood.
• GMI005.12 Preferably, the incubation section has an incubation heater configured for heating the nucleic acid sequences and the restriction enzymes to an incubation temperature.
• GMI005.13 Preferably, the MST layer incorporates the lysis section, the mixing section, the incubation section and the PCR section.
• GMI005.14 Preferably, the microfluidic device also has CMOS circuitry and at least one temperature sensor, the CMOS circuitry being positioned between the supporting substrate and the MST layer, and the temperature is configured for feedback control of the incubation heater.
• GMI005.15 Preferably, the lysis section has an active valve at a downstream end to retain liquid for a predetermined period of time.
• GMI005.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the lysis section, the boiling-initiated valve having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GMI005.17 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GMI005.18 Preferably, the microfluidic device also has a cap overlying the MST layer, wherein the cap has the restriction enzyme reservoir and a plurality of PCR reagent reservoirs formed therein.
• GMI005.19 Preferably, the microfluidic device also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• The easily usable, mass-producible, and inexpensive microfluidic device accepts a sample containing a fluid, then using a diffusion mixer, adds and mixes the requisite reagents into the input fluid or into the mixtures derived from the input fluid.
• The diffusion mixer utilizes a small-cross-section microchannel, increasing the efficiency of mixing. The diffusion mixer is a reliable and easily manufacturable mixer and is integral to the device, providing for a reliable, easily assembled, and inexpensive microfluidic system with a low component-count.
• GMI008.1 This aspect of the invention provides a test module for analysis of genetic material in a biological sample, the test module comprising:
• an outer casing with an inlet for receiving the sample;
• a diffusion mixing section for diffusive mixing of the sample with at least one other liquid, the diffusion mixing section having a microchannel defining a flow-path for a combined flow of the sample and the at least one other liquid; wherein,
• the channel cross section transverse to the flow direction is less than 100,000 square microns.
• GMI008.2 Preferably, the microchannel cross section transverse to the flow direction is less than 16,000 square microns.
• GMI008.3 Preferably, the microchannel cross section transverse to the flow direction is less than 2500 square microns.
• GMI008.4 Preferably, the microchannel cross section transverse to the flow direction is between 1 square micron and 400 square microns.
• GMI008.5 Preferably, the at least one other liquid is a reagent used for analyzing the genetic material.
• GMI008.6 Preferably, the test module of claim 5 further comprising a microfluidic device mounted in the outer casing for fluid communication with the inlet, the microfluidic device having a supporting substrate and a microsystems technology (MST) layer formed on the supporting substrate, the MST layer incorporating the diffusion mixing section, a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences from the genetic material, and a reagent reservoir containing the reagent; wherein,
• the microchannel defines a flow-path having a length sufficient for diffusive mixing of the reagent and the sample; wherein during use,
• the sample flows to the PCR section via the diffusion mixing section.
• GMI008.7 Preferably, the reagent reservoir has a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GMI008.8 Preferably, the test module also has an incubation section downstream of the diffusion mixing section, the incubation section being configured to maintain a mixture of the sample and the restriction enzymes at an incubation temperature for restriction digestion of the nucleic acid sequences.
• GMI008.9 Preferably, the test module also has a hybridization section that has an array of probes for hybridization with target nucleic acid sequences in the sample; and,
• • a photosensor for detecting hybridization of probes within the array of probes.
• GMI008.10 Preferably, the test module also has a lysis section upstream of the diffusion mixing section, the lysis section being configured to lyse cells within the sample to release genetic material therein.
• GMI008.11 Preferably, the test module also has an anticoagulant reservoir upstream of the lysis section wherein the sample is whole blood and the anticoagulant reservoir has a surface tension valve with an aperture configured to pin a meniscus of the anticoagulant that retains the anticoagulant until contact with the blood removes the meniscus to add the anticoagulant to the blood.
• GMI008.12 Preferably, the incubation section has an incubation heater configured for heating the nucleic acid sequences and the restriction enzymes to an incubation temperature.
• GMI008.13 Preferably, the MST layer incorporates the lysis section, the mixing section, the incubation section and the PCR section.
• GMI008.14 Preferably, the test module also has CMOS circuitry and at least one temperature sensor, the CMOS circuitry being positioned between the supporting substrate and the MST layer, and the temperature is configured for feedback control of the incubation heater.
• GMI008.15 Preferably, the lysis section has an active valve at a downstream end to retain liquid for a predetermined period of time.
• GMI008.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the lysis section, the boiling-initiated valve having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the lysis section resumes.
• GMI008.17 Preferably, the meniscus anchor is an aperture and the valve heater is positioned adjacent a periphery of the aperture.
• GMI008.18 Preferably, the test module also has a cap overlying the MST layer, wherein the cap has the restriction enzyme reservoir and a plurality of PCR reagent reservoirs formed therein.
• GMI008.19 Preferably, the test module also has a dialysis section wherein the biological material includes cells of different sizes, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• The easily usable, mass-producible, inexpensive, and portable genetic test module accepts a sample containing nucleic acids, then using a diffusion mixer, adds and mixes the requisite reagents into the sample or into the mixtures derived from the sample, and then utilizing the module's PCR chamber amplifies the nucleic acid targets in the sample.
• The diffusion mixer utilizes a small-cross-section microchannel, increasing the efficiency of mixing. The diffusion mixer is a reliable and easily manufacturable mixer and is integral to the module's LOC device, providing for a reliable, easily assembled, and inexpensive genetic test module with a low component-count.
• The microfluidic PCR chamber is an easily manufacturable PCR cycler and is integral to the module's LOC device, in turn providing for an easily assembled and inexpensive genetic test module with a low component-count.
• GLE001.1 This aspect of the invention provides a microfluidic device for analyzing a sample fluid, the microfluidic device comprising:
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry with digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GLE001.2 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE001.3 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic device.
• GLE001.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE001.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE001.6 Preferably, the microfluidic device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE001.7 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE001.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE001.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE001.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE001.11 Preferably, the microfluidic device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE001.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE001.13 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GLE001.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE001.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE001.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE001.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE001.18 Preferably, the microfluidic device also has a supporting substrate and a cap wherein the CMOS circuitry is between the supporting substrate and the MST layer, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE001.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE001.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device with integral digital memory accepts an input fluid and processes it. The digital memory is used to store the data and control information required during the functioning of the device and the module incorporating the device. The digital memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive microfluidic system with low component-count.
• GLE002.1 This aspect of the invention provides a test module for analyzing a sample fluid, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample; and,
• digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GLE002.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE002.3 Preferably, the data stored in the digital memory is a unique identifier for the test module.
• GLE002.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE002.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE002.6 Preferably, the test module also has CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the incubation section.
• GLE002.7 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE002.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE002.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE002.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE002.11 Preferably, the test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE002.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE002.13 Preferably, the CMOS circuitry has a data interface for transmission of the hybridization data to an external device.
• GLE002.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the data interface and store the patient data in the digital memory.
• GLE002.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the control circuitry.
• GLE002.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE002.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE002.18 Preferably, the test module also has a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE002.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE002.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device with integral digital memory accepts an input fluid and processes it. The digital memory is used to store the data and control information required during the functioning of the LOC device and the module incorporating the LOC device. The information stored on the memory includes the characteristics of the module incorporating this LOC device. The digital memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE003.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry with digital memory for storing epidemiological data, and configured to download epidemiological data updates from an external source.
• GLE003.2 Preferably, the CMOS circuitry incorporates a universal serial bus (USB) device driver for operative control of a USB connection to the external source.
• GLE003.3 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE003.4 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE003.5 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.
• GLE003.6 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an enzymatic reaction temperature.
• GLE003.7 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE003.8 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE003.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE003.10 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE003.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE003.12 Preferably, the CMOS circuitry has bond-pads for connection to the USB connection and transmission of hybridization data to an external device.
• GLE003.13 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the USB connection and store the patient data in the digital memory.
• GLE003.14 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE003.15 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE003.16 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE003.17 Preferably, the LOC device also has a supporting substrate and a cap wherein the CMOS circuitry is between the supporting substrate and the MST layer, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE003.18 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE003.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GLE003.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive LOC device with integral digital memory accepts an input fluid and processes it. The digital memory is used to store the data and control information required during the functioning of the LOC device and the module incorporating the LOC device. The information stored in the memory includes epidemiological updates available at the time, with the information being used for analytical and diagnostics purposes. This information provides for module's independence from outside support. The digital memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE004.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry with digital memory for storing genetic data, and configured to download genetic data updates from an external source.
• GLE004.2 Preferably, the CMOS circuitry incorporates a universal serial bus (USB) device driver for operative control of a USB connection to the external source.
• GLE004.3 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE004.4 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE004.5 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the genetic material.
• GLE004.6 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an enzymatic reaction temperature.
• GLE004.7 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE004.8 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE004.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE004.10 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE004.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE004.12 Preferably, the CMOS circuitry has bond-pads for transmission of hybridization data via the USB connection.
• GLE004.13 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE004.14 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE004.15 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE004.16 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE004.17 Preferably, the LOC device also has a supporting substrate and a cap wherein the CMOS circuitry is between the supporting substrate and the MST layer, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE004.18 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE004.19 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• GLE004.20 Preferably, the PCR section has a thermal cycle time between 0.45 seconds and 1.5 seconds.
• The easily usable, mass-producible, and inexpensive LOC device with integral digital memory accepts an input fluid and processes it. The digital memory is used to store the data and control information required during the functioning of the LOC device and the module incorporating the LOC device. The information stored in the memory includes genetic information updates available at the time, with the information being used for analytical and diagnostics purposes. This information provides for module's independence from outside support. The digital memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE005.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry with digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample; wherein,
• the data is encrypted for secure communication with an external device.
• GLE005.2 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE005.3 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE005.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE005.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE005.6 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE005.7 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE005.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE005.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE005.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE005.11 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE005.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE005.13 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GLE005.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE005.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE005.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE005.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE005.18 Preferably, the LOC device also has a supporting substrate and a cap wherein the CMOS circuitry is between the supporting substrate and the MST layer, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE005.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE005.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device with an integral digital memory accepts a diagnostic sample and processes it. The digital memory is used to store the data and control information required during the functioning of the LOC device and the diagnostic module incorporating the LOC device. The memory also securely stores patient test result information. The capability to store patient test result information makes it possible for the diagnostic module to perform a test utilizing only a minimal external power supply, and then in conjunction with a fully featured reader, analyze the patient test results at a later time. The secure storage of patient test result information would insure that the information would not be misused through illicit channels. The digital memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE006.1 This aspect of the invention provides a test module for analyzing a sample fluid, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a supporting substrate; and,
• CMOS circuitry on the supporting substrate for operative control of the functional sections during processing and analysis of the sample.
• GLE006.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the CMOS circuitry has digital memory for storing data relating to the reagent identities.
• GLE006.3 Preferably, the data stored in the digital memory is a unique identifier for test module.
• GLE006.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE006.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE006.6 Preferably, the test module also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE006.7 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE006.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE006.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE006.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE006.11 Preferably, the test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE006.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE006.13 Preferably, the control circuitry has a data interface for transmission of the hybridization data to an external device.
• GLE006.14 Preferably, the sample is drawn from a patient and the control circuitry is configured to download patient data via the data interface and store the patient data in the digital memory.
• GLE006.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the control circuitry.
• GLE006.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE006.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE006.18 Preferably, the test module also has a LOC device that incorporates the supporting substrate and the CMOS circuitry, and has a sample inlet in fluid communication with the receptacle, a microsystems technology (MST) layer that incorporates the functional sections and a cap wherein the cap overlies the MST layer and defines the reagent reservoirs.
• GLE006.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE006.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device accepts a diagnostic sample and processes it, with the LOC device's on-chip electronics controlling all of the LOC device's functions. The control electronics being integral to the LOC device, makes the module utilizing the LOC device independent from specialized outside support and provides for the easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE007.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for installation in a test module for analyzing a sample fluid and communicating test results to an external device, the LOC device comprising:
• a supporting substrate;
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry on the supporting substrate for operative control of a communication interface in the test module for communication with the external device.
• GLE007.2 Preferably, the CMOS circuitry incorporates a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GLE007.3 Preferably, the CMOS circuitry is further configured for operative control of the functional sections during processing and analysis of the sample.
• GLE007.4 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the CMOS circuitry has digital memory for storing data relating to the reagent identities.
• GLE007.5 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE007.6 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE007.7 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE007.8 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE007.9 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE007.10 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE007.11 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE007.12 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE007.13 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE007.14 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE007.15 Preferably, the CMOS circuitry is configured for transmission of the hybridization data to an external device via the test module communications interface.
• GLE007.16 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the communications interface and store the patient data in the digital memory.
• GLE007.17 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the control circuitry.
• GLE007.18 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE007.19 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE007.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device accepts a diagnostic sample and processes it, with the LOC device's on-chip electronics controlling the data and command communications with the host. The electronics being integral to the LOC device, makes the module utilizing the LOC device independent from specialized outside support and provides for the easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE008.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for installation in a test module for analyzing a sample fluid and communicating test results to an external device, the LOC device comprising:
• a supporting substrate;
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry on the supporting substrate, the CMOS circuitry having a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with the external device.
• GLE008.2 Preferably, the CMOS circuitry is further configured for operative control of the functional sections during processing and analysis of the sample.
• GLE008.3 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the CMOS circuitry has digital memory for storing data relating to the reagent identities.
• GLE008.4 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE008.5 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE008.6 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE008.7 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE008.8 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE008.9 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE008.10 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE008.11 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE008.12 Preferably, the digital memory includes random access memory (RAM) and flash memory, the RAM being configured to store the hybridization data and the flash memory being configured to store program data to operate the functional sections and the probe identity data.
• GLE008.13 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE008.14 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE008.15 Preferably, the USB device driver is configured for transmission of the hybridization data to an external device via the USB plug.
• GLE008.16 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the USB plug and store the patient data in the digital memory.
• GLE008.17 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the control circuitry.
• GLE008.18 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE008.19 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE008.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device with integral USB device controller accepts a diagnostic sample and processes it. The USB device controller being integral to the LOC device, makes the module utilizing the LOC device independent from specialized outside support and provides for the easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE009.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a supporting substrate;
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry between the supporting substrate and the MST layer, the CMOS circuitry having a controller to control operations performed by the functional sections during processing and analysis of the sample.
• GLE009.2 Preferably, the CMOS circuitry has digital memory for storing data and operational information for use by the controller to control the functional sections during processing and analysis of the sample.
• GLE009.3 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory is a unique identifier for LOC device, the unique identifier being associated with the reagent identities.
• GLE009.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE009.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE009.6 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE009.7 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE009.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE009.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE009.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE009.11 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE009.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE009.13 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GLE009.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE009.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE009.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE009.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE009.18 Preferably, the LOC device also has a cap wherein the cap overlies the MST layer and defines the reagent reservoirs.
• GLE009.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE009.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device accepts a diagnostic sample and processes it, with the LOC device's integral controller controlling all of the LOC device's functions. The controller being integral to the LOC device, makes the module utilizing the LOC device independent from specialized outside support and provides for the easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE010.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a supporting substrate;
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry between the supporting substrate and the MST layer, the CMOS circuitry having digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GLE010.2 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GLE010.3 Preferably, the data stored in the digital memory is a unique identifier for LOC device.
• GLE010.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE010.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE010.6 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE010.7 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE010.8 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GLE010.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE010.10 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GLE010.11 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE010.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE010.13 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GLE010.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE010.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE010.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE010.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE010.18 Preferably, the LOC device also has a cap wherein the cap overlies the MST layer and defines the reagent reservoirs.
• GLE010.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE010.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive LOC device accepts a diagnostic sample and processes it, with the LOC device's integral data RAM providing for intermediate data storage. The data RAM being integral to the LOC device, makes the module utilizing the LOC device independent from specialized outside support and provides for the easily manufacturable, mass-producible, easily usable, and inexpensive module with low component-count.
• GLE011.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for analyzing a sample fluid, the LOC device comprising:
• a supporting substrate;
• a sample inlet for receiving the sample;
• a microsystems technology (MST) layer with functional sections for processing and analyzing the sample; and,
• CMOS circuitry between the supporting substrate and the MST layer, the CMOS circuitry having flash memory for storing program data to operate the functional sections during processing and analysis of the sample.
• GLE011.2 Preferably, the LOC device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the flash memory also stores data relating to the reagent identities.
• GLE011.3 Preferably, the data includes a unique identifier for LOC device.
• GLE011.4 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample, and the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GLE011.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLE011.6 Preferably, the LOC device also has a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the incubation section.
• GLE011.7 Preferably, the LOC device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE011.8 Preferably, the flash memory stores probe identity data identifying the probe at each site within the array of probes.
• GLE011.9 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE011.10 Preferably, the CMOS circuitry has random access memory (RAM) configured to store hybridization data generated from the photosensor output.
• GLE011.11 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE011.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE011.13 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GLE011.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GLE011.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE011.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE011.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE011.18 Preferably, the LOC device also has a cap wherein the cap overlies the MST layer and defines the reagent reservoirs.
• GLE011.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE011.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, and inexpensive microfluidic device with integral program and data flash memory accepts an input fluid and processes it. The flash memory is used to store the data and program required during the functioning of the device and the module incorporating the device. The flash memory being integral to the device, provides for an easily manufacturable, mass-producible, easily usable, and inexpensive microfluidic system with low component-count.
• GLE012.1 This aspect of the invention provides a test module for analyzing a sample fluid and communicating epidemiological data to a database, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the epidemiological database; and,
• a controller for operative control of the communication interface.
• GLE012.2 Preferably, the test module also has a universal serial bus (USB) plug wherein the communication interface is a device driver for operative control of the USB plug to communicate with an external device.
• GLE012.3 Preferably, the test module also has digital memory for storing epidemiological data.
• GLE012.4 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory includes the reagent identities.
• GLE012.5 Preferably, the data stored in the digital memory includes a unique identifier for test module.
• GLE012.6 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.
• GLE012.7 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an enzymatic reaction temperature.
• GLE012.8 Preferably, the test module also has CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the incubation section.
• GLE012.9 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE012.10 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to excitation, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE012.11 Preferably, the test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE012.12 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE012.13 Preferably, the CMOS circuitry is configured for communication of hybridization data with an external device.
• GLE012.14 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download and store the patient data in the digital memory.
• GLE012.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE012.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE012.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE012.18 Preferably, the test module also has a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the communication interface, the controller, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE012.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE012.20 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biochemical sample and processes and analyzes the sample, updating epidemiological databases based on the diagnostic results derived from the sample.
• The updating of epidemiological databases provides for improved science-base for the functioning of the diagnostic test modules and optimal higher-level responses to epidemiological situations. The diagnostic test module based automation of updating the databases would provide for massive quality and economic gains for health information systems.
• GLE013.1 This aspect of the invention provides a test module for analyzing a sample fluid and communicating location data to an epidemiological database, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the epidemiological database; and,
• a controller for operative control of the communication interface; wherein,
• the controller is configured to associate location data with epidemiological data sent to the communication interface for communication with the epidemiological database.
• GLE013.2 Preferably, the test module also has a universal serial bus (USB) plug wherein the communication interface is a USB device driver for operative control of the USB plug to communicate with an external device.
• GLE013.3 Preferably, the controller is configured to automatically communicate with the epidemiological database without user initiation.
• GLE013.4 Preferably, the test module also has a user interface for inputting data to the controller for communication with the epidemiological database.
• GLE013.5 Preferably, the test module also has digital memory for storing epidemiological data.
• GLE013.6 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory includes the reagent identities.
• GLE013.7 Preferably, the data stored in the digital memory includes a unique identifier for test module.
• GLE013.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.
• GLE013.9 Preferably, the test module also has CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the PCR section.
• GLE013.10 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE013.11 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to excitation, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE013.12 Preferably, the test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE013.13 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE013.14 Preferably, the CMOS circuitry is configured for communication of hybridization data to an external device.
• GLE013.15 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download and store the patient data in the digital memory.
• GLE013.16 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE013.17 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE013.18 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE013.19 Preferably, the test module also has a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the communication interface, the controller, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE013.20 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biochemical sample and processes and analyzes the sample, updating epidemiological databases based on the diagnostic results derived from the sample and the test location data.
• The updating of epidemiological databases with the diagnostics results and the location data provides for improved science-base for the functioning of the diagnostic test modules and optimal higher-level responses to epidemiological situations. The diagnostic test module based automation of updating the databases would provide for massive quality and economic gains for health information systems.
• GLE014.1 This aspect of the invention provides a test module for analyzing a sample fluid and communicating data to a medical database, the test module comprising:
• a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the medical database; and,
• a controller for operative control of the communication interface.
• GLE014.2 Preferably, the test module of claim 1 further comprising a universal serial bus (USB) plug wherein the communication interface is a USB device driver for operative control of the USB plug to communicate with an external device.
• GLE014.3 Preferably, the test module of claim 2 further comprising digital memory wherein the medical database stores electronic health records (EHR), electronic medical records (EMR) and personal health records (PHR) and, the digital memory is configured for storing data relating to EHR, EMR and PHR.
• GLE014.4 Preferably, the test module of claim 3 further comprising a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory includes the reagent identities.
• GLE014.5 Preferably, the data stored in the digital memory includes a unique identifier for test module.
• GLE014.6 Preferably, the sample is a biological sample including cells of different sizes, and one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample.
• GLE014.7 Preferably, the test module of claim 6 further comprising CMOS circuitry and a temperature sensor wherein the CMOS circuitry incorporates the digital memory and uses the temperature sensor output for feedback control of the PCR section.
• GLE014.8 Preferably, one of the functional sections is a dialysis section, the dialysis section being configured for separating cells larger than a predetermined threshold into a portion of the sample which is processed separately from the remainder of the sample containing only cells smaller than the predetermined threshold.
• GLE014.9 Preferably, one of the functional sections is a lysis section, the lysis section being configured to release nucleic acid sequences within the cells smaller than the predetermined threshold.
• GLE014.10 Preferably, the test module of claim 9 further comprising an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLE014.11 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to excitation, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLE014.12 Preferably, the test module of claim 11 further comprising a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLE014.13 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLE014.14 Preferably, the CMOS circuitry is configured to communicate hybridization data to an external device.
• GLE014.15 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GLE014.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLE014.17 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• GLE014.18 Preferably, the test module of claim 5 further comprising a LOC device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the CMOS circuitry incorporates the digital memory, the communication interface, the controller, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GLE014.19 Preferably, the reagent reservoirs each have a surface tension valve with a meniscus anchor for pinning a meniscus to retain the reagent therein, such that contact with a flow of the sample fluid removes the meniscus and the reagent combines with the sample.
• GLE014.20 Preferably, the PCR section has a thermal cycle time of less than 4 seconds.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biochemical sample and processes and analyzes the sample, updating patients' databases based on the diagnostic results derived from the sample.
• The updating of patients' databases with the diagnostics results and the location data provides for improved provision of health care for the patients, automated maintenance of patient's medical records, improved science-base for the functioning of the diagnostic test modules, and optimal higher-level responses to epidemiological situations. The diagnostic test module based automation of updating the databases would provide for massive quality and economic gains for health information systems.
• GLA001.1 This aspect of the invention provides a test module for processing and analyzing a blood sample, the test module comprising:
• an outer casing with a receptacle for receiving blood;
• functional sections for processing and analyzing the sample; and,
• a lancet for collecting the blood sample from a patient.
• GLA001.2 Preferably, the lancet is retractable into the outer casing and biased to an extended position in which a sharp end of the lancet protrudes from the outer casing.
• GLA001.3 Preferably, the lancet is spring loaded such that during use the lancet is held in a retracted position within the outer casing against the spring bias until released by user actuation.
• GLA001.4 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the blood sample.
• GLA001.5 Preferably, one of the reagent reservoirs contains anticoagulant for addition to the blood sample upstream of the functional sections.
• GLA001.6 Preferably, one of the functional sections is a dialysis section for separating cells larger than a predetermined threshold into a portion of the blood sample such that the remainder of the sample contains cells smaller than the predetermined threshold.
• GLA001.7 Preferably, one of the functional sections is a lysis chamber such that during use, cells in the blood sample are lysed to release any genetic material within.
• GLA001.8 Preferably, one of the reagent reservoirs contains a lysis reagent for lysing the cells in the lysis chamber.
• GLA001.9 Preferably, the lysis chamber has a heater for lysing the cells.
• GLA001.10 Preferably, one of the functional sections is a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences from the genetic material.
• GLA001.11 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GLA001.12 Preferably, the test module also has CMOS circuitry and a temperature sensor wherein the control circuitry uses the temperature sensor output for feedback control of the PCR section.
• GLA001.13 Preferably, the test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GLA001.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GLA001.15 Preferably, the test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GLA001.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GLA001.17 Preferably, the control circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GLA001.18 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the control circuitry.
• GLA001.19 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GLA001.20 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module with integral lancet accepts a blood sample and processes and analyzes the sample. The sterilized integral lancet obviates the logistics and cost of using an external lancet, at the same time insuring the user of the sterile quality and safety of the lancet.
• GGA001.1 This aspect of the invention provides a test module for analyzing genetic material in a biological sample, the test module comprising:
• an outer casing with a receptacle for receiving the biological sample;
• functional sections for processing and analyzing the biological sample;
• a controller for operating the functional sections; and,
• a flow-path from the receptacle to the functional sections; wherein,
• the flow-path is configured to draw the sample to be analyzed from the receptacle to the functional sections by capillary action regardless of module orientation relative to gravity.
• GGA001.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample.
• GGA001.3 Preferably, one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GGA001.4 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GGA001.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture including the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GGA001.6 Preferably, the test module also has a temperature sensor wherein the controller uses the temperature sensor output for feedback control of the PCR section.
• GGA001.7 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GGA001.8 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GGA001.9 Preferably, the test module also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GGA001.10 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GGA001.11 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GGA001.12 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GGA001.13 Preferably, the elongate heaters are independently operable.
• GGA001.14 Preferably, the test module also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GGA001.15 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GGA001.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GGA001.17 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GGA001.18 Preferably, the reagent reservoirs each have a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GGA001.19 Preferably, the test module also has a lab-on-a-chip (LOC) device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the controller is incorporated into the CMOS circuitry, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GGA001.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, inexpensive, compact, light, and portable genetic test module, with self-contained storage of required reagents, accepts a biological sample, processes the sample, analyzes the genetic material in the sample using the module's integral sensors, and provides the results electronically at its output port. The module's fluidic propulsion and reagent storage is purely capillary action based, providing for gravity-independent operation of the module, in for example spaceflight conditions.
• GGA003.1 This aspect of the invention provides a test module for analyzing genetic material in a biological sample, the test module comprising:
• an outer casing with a receptacle for receiving the biological sample;
• functional sections for processing and analyzing the biological sample;
• a controller for operating the functional sections; and,
• a flow-path from the receptacle to the functional sections; wherein,
• the flow-path is configured to draw the sample to be analyzed from the receptacle to the functional sections by capillary action regardless of module orientation.
• GGA003.2 Preferably, the test module also has a plurality of reagent reservoirs containing reagents for processing the sample.
• GGA003.3 Preferably, one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GGA003.4 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GGA003.5 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture including the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GGA003.6 Preferably, the test module also has a temperature sensor wherein the controller uses the temperature sensor output for feedback control of the PCR section.
• GGA003.7 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GGA003.8 Preferably, the PCR microchannel has a PCR inlet and a PCR outlet, and at least one section of the PCR microchannel forms an elongate PCR chamber.
• GGA003.9 Preferably, the test module also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GGA003.10 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GGA003.11 Preferably, each of the channel sections has a plurality of the elongate heaters.
• GGA003.12 Preferably, the plurality of elongate heaters are positioned end to end along the channel section.
• GGA003.13 Preferably, the elongate heaters are independently operable.
• GGA003.14 Preferably, the test module also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GGA003.15 Preferably, the PCR section has an active valve at the PCR outlet for retaining the liquid in the PCR section while the elongate heaters thermally cycle the nucleic acid sequences and a mixture of primers, dNTPs, polymerase, and buffer to amplify the nucleic acid sequences.
• GGA003.16 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor for retaining the liquid in the PCR section, the boiling-initiated valve also having a valve heater for boiling the liquid such that the meniscus unpins from the meniscus anchor and capillary driven flow out of the PCR section resumes.
• GGA003.17 Preferably, the meniscus anchor is an aperture and the valve heater is positioned at a periphery of the aperture.
• GGA003.18 Preferably, the reagent reservoirs each have a surface tension valve with an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GGA003.19 Preferably, the test module also has a lab-on-a-chip (LOC) device having a sample inlet in fluid communication with the receptacle, a supporting substrate, a microsystems technology (MST) layer, CMOS circuitry between the supporting substrate and the MST layer and a cap wherein the controller is incorporated into the CMOS circuitry, the MST layer incorporates the functional sections, and the cap overlies the MST layer and defines the reagent reservoirs.
• GGA003.20 Preferably, the PCR section is configured to complete a thermal cycle of the sample in less than 30 seconds.
• The easily usable, mass-producible, inexpensive, compact, light, and portable genetic test module, with self-contained storage of required reagents, accepts a biological sample, processes the sample, analyzes the genetic material in the sample using the module's integral sensors, and provides the results electronically at its output port. The module's fluidic propulsion and reagent storage is purely capillary action based, providing for orientation-independent operation of the module, in for example maritime conditions.
• GRE001.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to a mobile telephone, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the mobile telephone; and,
• a controller for operative control of the communication interface.
• GRE001.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE001.3 Preferably, the USB device driver is configured to draw power from the mobile telephone to power the controller and the functional sections.
• GRE001.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE001.5 Preferably, the controller is configured to download data via the mobile telephone.
• GRE001.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE001.7 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic test module.
• GRE001.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE001.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE001.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE001.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE001.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE001.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE001.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE001.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE001.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE001.17 Preferably, the controller is configured for transmission of the hybridization data to the mobile telephone.
• GRE001.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the mobile telephone and store the patient data in the digital memory.
• GRE001.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE001.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to a mobile phone/smart phone which provides the module with power, computing, communications, and user interface support. Mobile phones/smart phones are widely and inexpensively available, obviating the need for specialized, heavy, and expensive module support systems.
• GRE002.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to a laptop computer, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the laptop computer; and,
• a controller for operative control of the communication interface.
• GRE002.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE002.3 Preferably, the USB device driver is configured to draw power from the laptop computer to power the controller and the functional sections.
• GRE002.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE002.5 Preferably, the controller is configured to download data via the laptop computer.
• GRE002.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE002.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE002.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE002.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE002.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE002.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE002.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE002.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE002.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE002.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE002.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE002.17 Preferably, the controller is configured for transmission of the hybridization data to the laptop computer.
• GRE002.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the laptop computer and store the patient data in the digital memory.
• GRE002.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE002.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to a laptop/notebook which provides the module with power, computing, communications, and user interface support. Laptops/notebooks are widely and inexpensively available, obviating the need for specialized, heavy, and expensive module support systems.
• GRE003.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to a dedicated reader purpose built for operating with the microfluidic test module, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the dedicated reader; and,
• a controller for operative control of the communication interface.
• GRE003.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE003.3 Preferably, the USB device driver is configured to draw power from the dedicated reader to power the controller and the functional sections.
• GRE003.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE003.5 Preferably, the controller is configured to download data via the dedicated reader.
• GRE003.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE003.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE003.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE003.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE003.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE003.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE003.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE003.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE003.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE003.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE003.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE003.17 Preferably, the controller is configured for transmission of the hybridization data to the dedicated reader.
• GRE003.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the dedicated reader and store the patient data in the digital memory.
• GRE003.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE003.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to an inexpensive and portable dedicated reader which provides the module with power, computing, communications, and user interface support. The dedicated reader obviates the need for heavy and expensive module support systems.
• GRE004.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to a desktop computer, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the desktop computer; and,
• a controller for operative control of the communication interface.
• GRE004.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE004.3 Preferably, the USB device driver is configured to draw power from the desktop computer to power the controller and the functional sections.
• GRE004.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE004.5 Preferably, the controller is configured to download data via the desktop computer.
• GRE004.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE004.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE004.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE004.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE004.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE004.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE004.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE004.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE004.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE004.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE004.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE004.17 Preferably, the controller is configured for transmission of the hybridization data to the desktop computer.
• GRE004.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the desktop computer and store the patient data in the digital memory.
• GRE004.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE004.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to a desktop PC which provides the module with power, computing, communications, and user interface support. Desktop PCs are widely available and are inexpensive, obviating the need for specialized, heavy, and expensive module support systems.
• GRE005.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to an ebook reader, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the ebook reader; and,
• a controller for operative control of the communication interface.
• GRE005.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE005.3 Preferably, the USB device driver is configured to draw power from the ebook reader to power the controller and the functional sections.
• GRE005.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE005.5 Preferably, the controller is configured to download data via the ebook reader.
• GRE005.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE005.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE005.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE005.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE005.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE005.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE005.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE005.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE005.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE005.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE005.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE005.17 Preferably, the controller is configured for transmission of the hybridization data to the ebook reader.
• GRE005.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the ebook reader and store the patient data in the digital memory.
• GRE005.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE005.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to a ebook reader which provides the module with power, computing, communications, and user interface support. Ebook readers are widely and inexpensively available, obviating the need for specialized, heavy, and expensive module support systems.
• GRE006.1 This aspect of the invention provides a microfluidic assembly for detecting a target molecule in a sample fluid and indicating a positive or negative result, the microfluidic assembly comprising:
• a test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing the sample and detecting the target molecules;
• a communication interface a controller for transmitting an output signal from one or more of the functional sections; and,
• an indicator module for detachable engagement with the communication interface, the indicator module having an indicator for providing an indication of the target molecule being present in the fluid sample.
• GRE006.2 Preferably, the indicator module has a power supply for operating the test module.
• GRE006.3 Preferably, the indicator module provides power to the test module via the communication interface.
• GRE006.4 Preferably, the communication interface is a universal serial bus (USB) plug and the indicator module has a USB.
• GRE006.5 Preferably, the test module has a controller for operating the functional sections and providing the output signal to the communication interface.
• GRE006.6 Preferably, the test module has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE006.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE006.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE006.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE006.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE006.11 Preferably, the microfluidic assembly also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE006.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE006.13 Preferably, the test module has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE006.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE006.15 Preferably, the test module has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE006.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE006.17 Preferably, the controller is configured for transmission of the hybridization data to an external device other than the indicator module.
• GRE006.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the dedicated reader and store the patient data in the digital memory.
• GRE006.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE006.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port can optionally be interfaced to a very inexpensive and light USB power/indicator module which provides the module with power and minimal user interface support. The modules on-board digital memory securely stores test result information. The capability to store test result information makes it possible for the diagnostic module to perform a test USB power/indicator module, and then in conjunction with a fully featured reader, analyze the test results at a later time. The secure storage of test result information would insure that the information would not be misused through illicit channels. The USB power/indicator module obviates the need for specialized, heavy, and expensive module support systems.
• GRE007.1 This aspect of the invention provides a microfluidic test module for analyzing a sample fluid and communicating test results to a tablet computer, the microfluidic test module comprising:
• an outer casing with a receptacle for receiving the sample;
• functional sections for processing and analyzing the sample;
• a communication interface for communication with the tablet computer; and,
• a controller for operative control of the communication interface.
• GRE007.2 Preferably, the communication interface is a universal serial bus (USB) device driver for operative control of a USB plug in the test module for communication with an external device.
• GRE007.3 Preferably, the USB device driver is configured to draw power from the tablet computer to power the controller and the functional sections.
• GRE007.4 Preferably, the controller is further configured for operative control of the functional sections during processing and analysis of the sample.
• GRE007.5 Preferably, the controller is configured to download data via the tablet computer.
• GRE007.6 Preferably, the microfluidic test module also has a plurality of reagent reservoirs containing reagents for processing the sample and digital memory for storing data relating to the reagent identities.
• GRE007.7 Preferably, the data stored in the digital memory is a unique identifier for microfluidic test module.
• GRE007.8 Preferably, the sample is a biological sample containing genetic material and one of the functional sections is a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material.
• GRE007.9 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section and the data stored in the digital memory includes thermal cycle times and cycle numbers.
• GRE007.10 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GRE007.11 Preferably, the microfluidic test module also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GRE007.12 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GRE007.13 Preferably, the microfluidic test module also has a photosensor wherein each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the photosensor is configured for sensing the photons emitted by the probe-target hybrids.
• GRE007.14 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GRE007.15 Preferably, the microfluidic test module also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GRE007.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GRE007.17 Preferably, the controller is configured for transmission of the hybridization data to the tablet computer.
• GRE007.18 Preferably, the sample is drawn from a patient and the controller is configured to download patient data via the tablet computer and store the patient data in the digital memory.
• GRE007.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the controller.
• GRE007.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, inexpensive, compact, light, and portable Microfluidic test module, with self-contained storage of required reagents, accepts a sample and processes and analyzes the sample material using the module's integral sensors, and provides the results electronically at its output port. The module's communications port is interfaced to a tablet computer which provides the module with power, computing, communications, and user interface support. Tablet computers are widely and inexpensively available, obviating the need for specialized, heavy, and expensive module support systems.
• GCF001.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein; and,
• a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material; wherein,
• the dialysis section, the lysis section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF001.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF001.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF001.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF001.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF001.6 Preferably, the second channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF001.7 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF001.8 Preferably, the LOC device also has a plurality of reagent reservoirs wherein one of the reagent reservoirs contains a lysis reagent for chemically lysing the pathogens in the lysis section.
• GCF001.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF001.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF001.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and has a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF001.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF001.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF001.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF001.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF001.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF001.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF001.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF001.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF001.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its chemical and/or thermal lysis chambers to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The option of using chemical and thermal lysing processes simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF002.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein;
• a first nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a first portion of the sample flow from the lysis section; and,
• a second nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a second portion of the sample flow from the lysis section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF002.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF002.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF002.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF002.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first array and second array.
• GCF002.6 Preferably, the dialysis section has at least two channels with a plurality of apertures fluidically connecting the channels, the plurality of apertures being sized to correspond to the predetermined threshold.
• GCF002.7 Preferably, the at least two channels and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GCF002.8 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF002.9 Preferably, the LOC device also has a plurality of reagent reservoirs, one of the reagent reservoirs containing a lysis reagent for chemically lysing the pathogens in the lysis section.
• GCF002.10 Preferably, the lysis section has a heater for thermally lysing the pathogens while the sample is in the lysis chamber.
• GCF002.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF002.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF002.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF002.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF002.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF002.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes.
• GCF002.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF002.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF002.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF002.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its chemical and/or thermal lysis chambers to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The option of using chemical and thermal lysing processes simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF003.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein;
• a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF003.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF003.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF003.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF003.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF003.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF003.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF003.8 Preferably, the second channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF003.9 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF003.10 Preferably, the LOC device also has a plurality of reagent reservoirs wherein one of the reagent reservoirs contains a lysis reagent for chemically lysing the pathogens in the lysis section.
• GCF003.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF003.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF003.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF003.14 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF003.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF003.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF003.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF003.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF003.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the second PCR section in response to an activation signal from the CMOS circuitry.
• GCF003.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its chemical and/or thermal lysis chambers to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The option of using chemical and thermal lysing processes simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF004.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section having a lysis chamber and a heater for lysing the pathogens while the sample is in the lysis chamber; and,
• a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material; wherein,
• the dialysis section, the lysis section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF004.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF004.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF004.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF004.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF004.6 Preferably, the second channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF004.7 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF004.8 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF004.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF004.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF004.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and has a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF004.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF004.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF004.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF004.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF004.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF004.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF004.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF004.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF004.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens in its thermal lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF005.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section having a lysis chamber and a heater for lysing the pathogens while the sample is in the lysis chamber;
• a first nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a first portion of the sample flow from the lysis section; and,
• a second nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a second portion of the sample flow from the lysis section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF005.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF005.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF005.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF005.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first or second array.
• GCF005.6 Preferably, the dialysis section has at least two channels with a plurality of apertures fluidically connecting the channels, the plurality of apertures being sized to correspond to the predetermined threshold.
• GCF005.7 Preferably, the at least two channels and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GCF005.8 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF005.9 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF005.10 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF005.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF005.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF005.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF005.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF005.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF005.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes.
• GCF005.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF005.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF005.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF005.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its thermal lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF006.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section having a lysis chamber and a heater for lysing the pathogens while the sample is in the lysis chamber;
• a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF006.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF006.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF006.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF006.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF006.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF006.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF006.8 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF006.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF006.10 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF006.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF006.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF006.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF006.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF006.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF006.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF006.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF006.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF006.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the second PCR section in response to an activation signal from the CMOS circuitry.
• GCF006.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its thermal lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF007.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a first dialysis section for separating pathogens from larger constituents in the sample;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section having a lysis chamber and a heater for lysing the pathogens while the sample is in the lysis chamber;
• a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material; and,
• a second dialysis section downstream of the nucleic acid amplification section for prehybridization filtration of amplicon produced by the nucleic acid amplification section, the second dialysis section being configured to remove cell debris from the amplicon; wherein,
• the first dialysis section, the lysis section, the nucleic acid amplification section and the second dialysis section are all supported on the supporting substrate.
• GCF007.2 Preferably, the lysis section has a heater for thermally lysing the pathogens.
• GCF007.3 Preferably, the LOC device also has a hybridization section downstream of the second dialysis section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF007.4 Preferably, the first dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of first apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the first apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF007.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF007.6 Preferably, the second dialysis section has a large component channel, a small component channel and a plurality of second apertures fluidically connecting the large component channel to the small component channel, the second apertures being sized to allow nucleic acid sequences to flow from the large component channel to the small component channel while cell debris larger than the second apertures is retained in the large component channel, the small component channel being in fluid communication with the hybridization section.
• GCF007.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF007.8 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF007.9 Preferably, the nucleic acid amplification section is polymerase chain reaction (PCR) amplification section.
• GCF007.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF007.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and has a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF007.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF007.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF007.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF007.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF007.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF007.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF007.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF007.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF007.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens in its thermal lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system. The thermal lysing process simplifies assay chemistry requirements and provides for capability for a wide range of sample types.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The prehybridization filtering removes the debris resulting from the thermal lysis of the sample cells improving the effectiveness of the hybridization process and the consequent improvement in the assay sensitivity, signal-to-noise-ratio, and reliability. The prehybridization filtering also permits utilization of the thermal lysis process, with its consequent advantages, for a wider range of sample types.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF008.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the cells in the lysis section; and,
• a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material; wherein,
• the dialysis section, the lysis section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF008.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF008.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF008.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF008.5 Preferably, the at least two channels and the plurality of apertures are configured such that the sample flows though the channels and the apertures under capillary action.
• GCF008.6 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF008.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF008.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF008.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF008.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF008.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and has a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF008.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF008.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF008.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF008.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF008.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF008.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF008.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF008.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF008.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens in its chemical lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF009.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the cells in the lysis section;
• a first nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a first portion of the sample flow from the lysis section; and,
• a second nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in a second portion of the sample flow from the lysis section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF009.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF009.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF009.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF009.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first or second array.
• GCF009.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF009.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF009.8 Preferably, the second channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF009.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF009.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF009.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF009.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF009.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF009.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF009.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF009.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes.
• GCF009.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF009.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF009.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF009.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its chemical lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF010.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating pathogens from larger constituents in the sample;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the pathogens to release genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the cells in the lysis section;
• a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF010.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF010.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF010.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF010.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF010.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are larger than the pathogens and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens flow into the second channel while the larger constituents are retained in the first channel.
• GCF010.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF010.8 Preferably, the second channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF010.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF010.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF010.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF010.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF010.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF010.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF010.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF010.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes.
• GCF010.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF010.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF010.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the second PCR section in response to an activation signal from the CMOS circuitry.
• GCF010.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive pathogen detection LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate any pathogens contained in the sample, lyses the pathogens as required in its chemical lysis chamber to release the pathogens' genetic materials, amplifies any target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF011.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material; and,
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences from the genetic material; wherein,
• the dialysis section, the lysis section, the incubation section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF011.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF011.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF011.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a down stream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF011.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF011.6 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF011.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF011.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF011.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF011.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF011.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF011.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF011.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF011.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF011.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF011.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF011.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF011.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF011.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF011.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, preprocesses the sample's genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like DNA restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF012.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a first nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF012.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF012.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF012.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF012.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first and second array.
• GCF012.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a down stream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF012.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF012.8 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF012.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF012.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF012.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF012.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF012.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF012.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF012.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF012.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences.
• GCF012.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF012.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF012.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF012.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, preprocesses the sample's genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like DNA restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF013.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a first nucleic acid amplification section downstream of the incubation section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF013.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF013.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF013.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF013.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF013.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a down stream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF013.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF013.8 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF013.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF013.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF013.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF013.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF013.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF013.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF013.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF013.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF013.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF013.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF013.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF013.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, preprocesses the sample's genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like DNA restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF014.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section; and,
• a nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences from the genetic material; wherein,
• the dialysis section, the lysis section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF014.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF014.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF014.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a downstream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF014.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF014.6 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF014.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF014.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF014.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF014.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF014.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF014.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF014.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF014.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF014.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF014.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF014.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF014.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF014.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF014.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF015.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section;
• a first nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the lysis section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF015.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF015.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF015.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF015.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first and second array.
• GCF015.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a down stream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF015.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF015.8 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF015.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF015.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF015.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF015.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF015.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF015.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF015.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF015.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences.
• GCF015.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF015.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF015.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF015.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF016.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• a plurality of reagent reservoirs;
• a lysis section downstream of the dialysis section for lysing the target cells to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the target cells in the lysis section;
• a first nucleic acid amplification section downstream of the lysis section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the dialysis section, the lysis section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF016.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF016.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF016.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF016.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF016.6 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a down stream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF016.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF016.8 Preferably, the lysis section has an active valve for retaining the target cells in the lysis section during lysis such that capillary driven flow to the incubation section resumes upon opening the active valve.
• GCF016.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF016.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF016.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF016.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF016.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF016.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF016.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF016.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF016.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF016.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF016.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF016.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive genomic analysis LOC device accepts a biological sample through its sample receptacle, uses its dialysis section to separate the leukocytes contained in the sample, lyses the leukocytes in its chemical lysis chamber to release the leukocytes' genetic material, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF020.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection and genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a lysis section for lysing pathogens and leukocytes in the sample to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the pathogens and leukocytes in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material; and,
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences from the genetic material; wherein,
• the lysis section, the incubation section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF020.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF020.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample and, a photosensor for detecting hybridization of any probes within the array.
• GCF020.4 Preferably, the photosensor is less than 1600 microns from the array of probes.
• GCF020.5 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF020.6 Preferably, one of the reagent reservoirs contains adaptor primers for ligation to nucleic acid sequences in the incubation section.
• GCF020.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF020.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF020.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF020.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF020.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF020.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF020.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF020.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF020.15 Preferably, the PCR section has an active valve for retaining the sample in the PCR section during amplification of the target nucleic acid sequences such that the CMOS circuitry is configured to open the active valve after amplification to allow capillary driven flow to resume.
• GCF020.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF020.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF020.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF020.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF020.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection and genomic analysis accepts a biological sample through its sample receptacle, lyses the sample's cells in its chemical lysis chamber to release the sample's genetic material, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF021.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection and genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a lysis section for lysing pathogens and leukocytes in the sample to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the pathogens and leukocytes in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a first nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the genetic material in a first portion of the sample flow from the incubation section; and,
• a second nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the genetic material in a second portion of the sample flow from the incubation section; wherein,
• the lysis section, the incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF021.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF021.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF021.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF021.5 Preferably, the LOC device also has a first hybridization section downstream of the first PCR section that has a first array of probes for hybridization with first target nucleic acid sequences and, a second hybridization section downstream of the second PCR section that has a second array of probes for hybridization with second target nucleic acid sequences, and a photosensor for detecting hybridization of any probes within the first or second array.
• GCF021.6 Preferably, the photosensor is less than 1600 microns from the first or second array of probes.
• GCF021.7 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF021.8 Preferably, one of the reagent reservoirs contains adaptor primers for ligation to nucleic acid sequences in the incubation section.
• GCF021.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF021.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF021.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF021.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF021.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF021.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF021.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF021.16 Preferably, the LOC device also has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences.
• GCF021.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF021.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF021.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF021.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection and genomic analysis accepts a biological sample through its sample receptacle, lyses the sample's cells in its chemical lysis chamber to release the sample's genetic material, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF022.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection and genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a lysis section for lysing pathogens and leukocytes in the sample to release the genetic material therein, the lysis section being in fluid communication with one of the reagent reservoirs containing a lysis reagent for lysing the pathogens and leukocytes in the lysis section;
• an incubation section downstream of the lysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a first nucleic acid amplification section downstream of the incubation section for amplifying first nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the first nucleic acid amplification section for amplifying second nucleic acid sequences in the amplicon from the first nucleic acid amplification section; wherein,
• the lysis section, the incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF022.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF022.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF022.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF022.5 Preferably, the LOC device also has a hybridization section downstream of the second PCR section that has an array of probes for hybridization with target nucleic acid sequences and a photosensor for detecting hybridization of any probes within the array.
• GCF022.6 Preferably, the photosensor is less than 1600 microns from the array of probes.
• GCF022.7 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF022.8 Preferably, one of the reagent reservoirs contains adaptor primers for ligation to nucleic acid sequences in the incubation section.
• GCF022.9 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF022.10 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF022.11 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF022.12 Preferably, the first PCR section has a PCR microchannel for thermally cycling the sample, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF022.13 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF022.14 Preferably, the PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF022.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF022.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF022.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF022.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF022.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF022.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection and genomic analysis accepts a biological sample through its sample receptacle, lyses the sample's cells in its chemical lysis chamber to release the sample's genetic material, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF023.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection, genetic analysis and proteomic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a leukocyte dialysis section for dividing the sample into a leukocyte stream, and a pathogen and erythrocyte stream containing cells and sample constituents smaller than a first size threshold;
• a pathogen dialysis section for dividing the pathogen and erythrocyte stream into an erythrocyte stream and a pathogen stream containing cells and sample constituents smaller than a second size threshold;
• a leukocyte lysis section downstream of the leukocyte dialysis section for lysing the leukocytes with a lysis reagent to release genetic material and proteins therein;
• a leukocyte incubation section downstream of the leukocyte lysis section for enzymatic reaction of the genetic material with enzymes;
• a leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences;
• a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material and proteins therein;
• a pathogen incubation section downstream of the pathogen lysis section for enzymatic reaction of the genetic material with enzymes;
• a pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences;
• an erythrocyte lysis section downstream of the pathogen dialysis section for lysing the erythrocytes with a lysis reagent to release proteins therein; wherein,
• the leukocyte dialysis section, the pathogen dialysis section, the leukocyte lysis section, the pathogen lysis section, the erythrocyte lysis section, the leukocyte incubation section, the pathogen incubation section, the leukocyte nucleic acid amplification section and the pathogen nucleic acid amplification section are all supported on the supporting substrate.
• GCF023.2 Preferably, the leukocyte nucleic acid amplification section is a leukocyte polymerase chain reaction (PCR) section and the pathogen nucleic acid amplification section is a pathogen PCR section.
• GCF023.3 Preferably, the LOC device also has:
• a leukocyte stream hybridization section downstream of the leukocyte PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a pathogen stream hybridization section downstream of the pathogen PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a leukocyte stream protein detection section downstream of the leukocyte lysis section, the leukocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• a pathogen stream protein detection section downstream of the pathogen lysis section, the pathogen stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• an erythrocyte stream protein detection section downstream of the erythrocyte lysis section, the erythrocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins; and,
• a photosensor for detecting hybridization of any of the probes or conjugation of any of the protein probes.
• GCF023.4 Preferably, the leukocyte dialysis section has a plurality of first apertures being sized to correspond to the first size threshold.
• GCF023.5 Preferably, the pathogen dialysis section has a plurality of second apertures being sized to correspond to the second size threshold.
• GCF023.6 Preferably, the leukocyte nucleic acid amplification section is a leukocyte isothermal nucleic acid amplification section and the pathogen nucleic acid amplification section is a pathogen isothermal nucleic acid amplification section.
• GCF023.7 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the leukocyte and pathogen isothermal nucleic acid amplification sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the leukocyte and pathogen isothermal nucleic acid amplification sections.
• GCF023.8 Preferably, the LOC device also has a plurality of reagent reservoirs wherein the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF023.9 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the leukocyte and pathogen PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the leukocyte and pathogen PCR sections.
• GCF023.10 Preferably, the leukocyte lysis section has an active valve for retaining liquid in the leukocyte lysis section during leukocyte lysis and allowing flow to the incubation section in response to an activation signal from the CMOS circuitry.
• GCF023.11 Preferably, the leukocyte PCR section has a PCR microchannel for thermally cycling the sample to amplify nucleic acid sequences, the PCR microchannel defining part of a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF023.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF023.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF023.14 Preferably, the leukocyte PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF023.15 Preferably, the leukocyte incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF023.16 Preferably, the leukocyte hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences and the erythrocyte stream protein detection section has a proteomic assay chamber array for containing the protein probes within each proteomic assay chamber, each of the protein probes being configured to conjugate or hybridize with one of the target proteins.
• GCF023.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers and the proteomic assay chambers.
• GCF023.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization and conjugation data from the photosensor output and a data interface for transmission of the hybridization and conjugation data to an external device.
• GCF023.19 Preferably, the leukocyte PCR section has an active valve for retaining liquid in the leukocyte PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF023.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection, genomic analysis, and proteomic analysis accepts a blood sample through its sample receptacle, uses its dialysis section to separate the sample into pathogens, leukocytes, and erythrocyte streams, lyses the cells in the three streams in its chemical lysis chamber to release the pathogens' and the leukocytes' genetic material and also to degrade the erythrocyte structures, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, and analyzes the sample's proteomic content via a homogeneous immunoassay probe system with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF024.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection, genetic analysis and proteomic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a leukocyte dialysis section for dividing the sample into a leukocyte stream, and a pathogen and erythrocyte stream containing cells and sample constituents smaller than a first size threshold;
• a pathogen dialysis section for dividing the pathogen and erythrocyte stream into an erythrocyte stream and a pathogen stream containing cells and sample constituents smaller than a second size threshold;
• a leukocyte lysis section downstream of the leukocyte dialysis section for lysing the leukocytes with a lysis reagent to release genetic material and proteins therein;
• a leukocyte incubation section downstream of the leukocyte lysis section for enzymatic reaction of the genetic material with enzymes;
• a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material and proteins therein;
• a pathogen incubation section downstream of the pathogen lysis section for enzymatic reaction of the genetic material with enzymes;
• an erythrocyte lysis section downstream of the pathogen dialysis section for lysing the erythrocytes with a lysis reagent to release proteins therein;
• a first leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences;
• a second leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences, the leukocyte incubation section supplying the first leukocyte nucleic acid amplification section and the second leukocyte nucleic acid detection section in parallel;
• a first pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences; and,
• a second pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences, the pathogen incubation section supplying the first pathogen nucleic acid amplification section and the second pathogen nucleic acid detection section in parallel; wherein,
• the leukocyte dialysis section, the pathogen dialysis section, the leukocyte lysis section, the pathogen lysis section, the erythrocyte lysis section, the leukocyte incubation section, the pathogen incubation section, the first and second leukocyte nucleic acid amplification sections and the first and second pathogen nucleic acid amplification sections are all supported on the supporting substrate.
• GCF024.2 Preferably, the first and second leukocyte nucleic acid amplification sections are first and second leukocyte polymerase chain reaction (PCR) sections and the first and second pathogen nucleic acid amplification sections are first and second pathogen PCR sections.
• GCF024.3 Preferably, the LOC device also has:
• a first leukocyte stream hybridization section downstream of the first leukocyte PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a second leukocyte stream hybridization section downstream of the second leukocyte PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a first pathogen stream hybridization section downstream of the first pathogen PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a second pathogen stream hybridization section downstream of the second pathogen PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a leukocyte stream protein detection section downstream of the leukocyte lysis section, the leukocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• a pathogen stream protein detection section downstream of the pathogen lysis section, the pathogen stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• an erythrocyte stream protein detection section downstream of the erythrocyte lysis section, the erythrocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins; and,
• a photosensor for detecting hybridization of any of the probes or conjugation of any of the protein probes.
• GCF024.4 Preferably, the first leukocyte PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second leukocyte PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF024.5 Preferably, the first leukocyte PCR section and the second leukocyte PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF024.6 Preferably, the first and second leukocyte nucleic acid amplification sections are first and second leukocyte isothermal nucleic acid amplification sections and the first and second pathogen nucleic acid amplification sections are first and second pathogen isothermal nucleic acid amplification sections.
• GCF024.7 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second leukocyte and first and second pathogen isothermal nucleic acid amplification sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second leukocyte and the first and second pathogen isothermal nucleic acid amplification sections.
• GCF024.8 Preferably, the LOC device also has a plurality of reagent reservoirs wherein the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF024.9 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second leukocyte PCR sections and the first and second pathogen PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second leukocyte PCR sections and the first and second pathogen PCR sections.
• GCF024.10 Preferably, the leukocyte lysis section has an active valve for retaining liquid in the leukocyte lysis section during leukocyte lysis and allowing flow to the incubation section in response to an activation signal from the CMOS circuitry.
• GCF024.11 Preferably, the first leukocyte PCR section has a PCR microchannel for thermally cycling the sample to amplify nucleic acid sequences, the PCR microchannel defining part of a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF024.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF024.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF024.14 Preferably, the first leukocyte PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF024.15 Preferably, the leukocyte incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF024.16 Preferably, the first leukocyte hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences and the erythrocyte stream protein detection section has a proteomic assay chamber array for containing the protein probes such that the protein probes within each proteomic assay chamber are configured to conjugate or hybridize with one of the target proteins.
• GCF024.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers and the proteomic assay chambers.
• GCF024.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization and conjugation data from the photosensor output and a data interface for transmission of the hybridization and conjugation data to an external device.
• GCF024.19 Preferably, the leukocyte PCR section has an active valve for retaining liquid in the first leukocyte PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF024.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection, genomic analysis, and proteomic analysis accepts a blood sample through its sample receptacle, uses its dialysis section to separate the sample into pathogens, leukocytes, and erythrocyte streams, lyses the cells in the three streams in its chemical lysis chamber to release the pathogens' and the leukocytes' genetic material and also to degrade the erythrocyte structures, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, and analyzes the sample's proteomic content via a homogeneous immunoassay probe system with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF025.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection, genetic analysis and proteomic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a leukocyte dialysis section for dividing the sample into a leukocyte stream, and a pathogen and erythrocyte stream containing cells and sample constituents smaller than a first size threshold;
• a pathogen dialysis section for dividing the pathogen and erythrocyte stream into an erythrocyte stream and a pathogen stream containing cells and sample constituents smaller than a second size threshold;
• a leukocyte lysis section downstream of the leukocyte dialysis section for lysing the leukocytes with a lysis reagent to release genetic material and proteins therein;
• a leukocyte incubation section downstream of the leukocyte lysis section for enzymatic reaction of the genetic material with enzymes;
• a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material and proteins therein;
• a pathogen incubation section downstream of the pathogen lysis section for enzymatic reaction of the genetic material with enzymes;
• an erythrocyte lysis section downstream of the pathogen dialysis section for lysing the erythrocytes with a lysis reagent to release proteins therein;
• a first leukocyte nucleic acid amplification section downstream of the leukocyte incubation section for amplifying nucleic acid sequences;
• a second leukocyte nucleic acid amplification section downstream of the first leukocyte nucleic acid amplification section for amplifying nucleic acid sequences;
• a first pathogen nucleic acid amplification section downstream of the pathogen incubation section for amplifying nucleic acid sequences; and,
• a second pathogen nucleic acid amplification section downstream of the first pathogen nucleic acid amplification for amplifying nucleic acid sequences; wherein,
• the leukocyte dialysis section, the pathogen dialysis section, the leukocyte lysis section, the pathogen lysis section, the leukocyte incubation section, the pathogen incubation section, the first and second leukocyte nucleic acid amplification sections and the first and second pathogen nucleic acid amplification sections are all supported on the supporting substrate.
• GCF025.2 Preferably, the first and second leukocyte nucleic acid amplification sections are first and second leukocyte polymerase chain reaction (PCR) sections and the first and second pathogen nucleic acid amplification sections are first and second pathogen PCR sections.
• GCF025.3 Preferably, the LOC device also has:
• a leukocyte stream hybridization section downstream of the second leukocyte PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a pathogen stream hybridization section downstream of the second pathogen PCR section that has an array of probes for hybridization with target nucleic acid sequences;
• a leukocyte stream protein detection section downstream of the leukocyte lysis section, the leukocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• a pathogen stream protein detection section downstream of the pathogen lysis section, the pathogen stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins;
• an erythrocyte stream protein detection section downstream of the erythrocyte lysis section, the erythrocyte stream protein detection section having an array of protein probes for conjugation or hybridization with target proteins; and,
• a photosensor for detecting hybridization of any of the probes or conjugation of any of the protein probes.
• GCF025.4 Preferably, the first leukocyte PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second leukocyte PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF025.5 Preferably, the first leukocyte PCR section and the second leukocyte PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF025.6 Preferably, the first and second leukocyte nucleic acid amplification sections are first and second leukocyte isothermal nucleic acid amplification sections and the first and second pathogen nucleic acid amplification sections are first and second pathogen isothermal nucleic acid amplification sections.
• GCF025.7 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second leukocyte isothermal nucleic acid amplification sections and first and second pathogen isothermal nucleic acid amplification sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second leukocyte isothermal nucleic acid amplification sections and first and second pathogen isothermal nucleic acid amplification sections.
• GCF025.8 Preferably, the LOC device also has a plurality of reagent reservoirs wherein the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF025.9 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second leukocyte and the first and second pathogen PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second leukocyte and the first and second pathogen PCR sections.
• GCF025.10 Preferably, the leukocyte lysis section has an active valve for retaining liquid in the leukocyte lysis section during leukocyte lysis and allowing flow to the incubation section in response to an activation signal from the CMOS circuitry.
• GCF025.11 Preferably, the first leukocyte PCR section has a PCR microchannel for thermally cycling the sample to amplify nucleic acid sequences, the PCR microchannel defining part of a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF025.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF025.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF025.14 Preferably, the first leukocyte PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF025.15 Preferably, the leukocyte incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF025.16 Preferably, the first leukocyte hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences and the erythrocyte stream protein detection section has a proteomic assay chamber array for containing the protein probes such that the protein probes within each proteomic assay chamber are configured to conjugate or hybridize with one of the target proteins.
• GCF025.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers and the proteomic assay chambers.
• GCF025.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization and conjugation data from the photosensor output and a data interface for transmission of the hybridization and conjugation data to an external device.
• GCF025.19 Preferably, the leukocyte PCR section has an active valve for retaining liquid in the first leukocyte PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF025.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device for pathogen detection, genomic analysis, and proteomic analysis accepts a blood sample through its sample receptacle, uses its dialysis section to separate the sample into pathogens, leukocytes, and erythrocyte streams, lyses the cells in the three streams in its chemical lysis chamber to release the pathogens' and the leukocytes' genetic material and also to degrade the erythrocyte structures, preprocesses the genetic material in its incubation section, amplifies target genetic sequences, analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array, and analyzes the sample's proteomic content via a homogeneous immunoassay probe system with sensing via its integral imaging array, utilizing reagents stored in the LOC device's reagent reservoirs.
• The dialysis section functionality extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system. The dialysis section being integral to the device, provides for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• The lysing process extracts analytical and diagnostic targets from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the tandem amplification chambers allow piecewise partial optimization of the earlier cycles and later cycles of the amplification process, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF027.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for detecting pathogens in a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a pathogen dialysis section for separating pathogens from constituents smaller than a predetermined threshold;
• a pathogen lysis section downstream of the pathogen dialysis section for lysing the pathogens with a lysis reagent to release genetic material therein;
• an incubation section downstream of the pathogen dialysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material; and,
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences from the genetic material; wherein,
• the pathogen dialysis section, the pathogen lysis section, the incubation section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF027.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF027.3 Preferably, the LOC device also has a hybridization section downstream of the PCR section that has an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids and, a photosensor for detecting the probe-target hybrids.
• GCF027.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the lysis section and a plurality of apertures that are smaller than the pathogens but larger than other small constituents, the second channel being in fluid communication with the first channel via the apertures such that the small constituents flow into the second channel while the pathogens are retained in the first channel.
• GCF027.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF027.6 Preferably, the first channel is configured to draw the pathogens into the lysis section by capillary action.
• GCF027.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF027.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF027.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF027.10 Preferably, the LOC device also has CMOS circuitry positioned between the supporting substrate and the PCR section, and a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the PCR section.
• GCF027.11 Preferably, the PCR section has a PCR microchannel for thermally cycling the sample to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and has a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF027.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF027.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF027.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF027.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF027.16 Preferably, the LOC device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF027.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF027.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF027.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF027.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. This LOC device design has the advantage of enabling incubation of the sample under controlled conditions.
• GCF028.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating smaller constituents from larger constituents, the smaller constituents being smaller than a predetermined size threshold, and the larger constituents being larger than the predetermined size threshold; and,
• a nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material; wherein,
• the dialysis section and the nucleic acid amplification section are both supported on the supporting substrate.
• GCF028.2 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the nucleic acid amplification section, the hybridization section having an array of probes for hybridization with target nucleic acid sequences in amplicon generated by the nucleic acid amplification section, the probes being configured to hybridize with the target nucleic acid sequences to form probe-target hybrids wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF028.3 Preferably, the larger constituents include pathogens and cells larger than the predetermined size threshold, the pathogens and the cells larger than a predetermined threshold including target cells containing genetic material for analysis.
• GCF028.4 Preferably, the dialysis section is downstream of the nucleic acid amplification section and upstream of the hybridization section for prehybridization filtration of the amplicon, the dialysis section being configured to remove cell debris from the amplicon.
• GCF028.5 Preferably, the LOC device also has a first dialysis section upstream of the nucleic acid amplification section for separating pathogens and cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis.
• GCF028.6 Preferably, the first dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a downstream end, and a plurality of apertures that are smaller than the pathogens and the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the pathogens and the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF028.7 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF028.8 Preferably, the second dialysis section has a large constituents channel, a small constituents channel and a plurality of second apertures fluidically connecting the large constituents channel to the small constituents channel, the second apertures being sized to allow nucleic acid sequences to flow from the large constituents channel to the small constituents channel while cell debris larger than the second apertures is retained in the large constituents channel, the small constituents channel being in fluid communication with the hybridization section.
• GCF028.9 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF028.10 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF028.11 Preferably, the nucleic acid amplification section is polymerase chain reaction (PCR) amplification section.
• GCF028.12 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF028.13 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF028.14 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF028.15 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF028.16 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF028.17 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus.
• GCF028.18 Preferably, the LOC device also has an incubation section downstream of the first dialysis section and upstream of the PCR section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material.
• GCF028.19 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF028.20 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GCF029.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a dialysis section for separating cells larger than a predetermined threshold in the sample from smaller constituents, whereby the cells larger than a predetermined threshold include target cells containing genetic material for analysis;
• an incubation section downstream of the dialysis section, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material; and,
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences from the genetic material; wherein,
• the dialysis section, the incubation section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF029.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF029.3 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with target nucleic acid sequences in the sample, the probes being configured to hybridize with the target nucleic acid sequences to form probe-target hybrids wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF029.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a downstream end, and a plurality of apertures that are smaller than the target cells and larger than the smaller constituents, the second channel being in fluid communication with the first channel via the apertures such that the target cells are retained in the first channel while the smaller constituents flow into the second channel.
• GCF029.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF029.6 Preferably, the first channel is configured to draw the target cells into the incubation section by capillary action.
• GCF029.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF029.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF029.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF029.10 Preferably, the LOC device also has CMOS circuitry positioned between the supporting substrate and the PCR section, and a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the PCR section.
• GCF029.11 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF029.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF029.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF029.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF029.15 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF029.16 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF029.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF029.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF029.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF029.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. This LOC device design has the advantage of enabling incubation of the sample under controlled conditions.
• GCF030.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection and genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a dialysis section for separating pathogens and cells larger than a predetermined threshold in the sample from smaller constituents, whereby the pathogens and cells larger than the predetermined threshold contain genetic material for analysis;
• a nucleic acid amplification section downstream of the dialysis section for amplifying nucleic acid sequences from the genetic material; wherein,
• the dialysis section and the nucleic acid amplification section are all supported on the supporting substrate.
• GCF030.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF030.3 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with target nucleic acid sequences in the genetic material, the probes being configured to hybridize with the target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF030.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet at an upstream end, a second channel in fluid communication with the waste channel at a downstream end, and a plurality of apertures that are smaller than the pathogens and the cells larger than the predetermined threshold, the second channel being in fluid communication with the first channel via the apertures such that the pathogens and the cells larger than the predetermined threshold are retained in the first channel while the smaller constituents flow into the second channel.
• GCF030.5 Preferably, the first channel and the second channel are configured to fill with the sample by capillary action.
• GCF030.6 Preferably, the second channel is configured to draw the pathogens and the cells larger than the predetermined threshold into the nucleic acid amplification section by capillary action.
• GCF030.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF030.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF030.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF030.10 Preferably, the LOC device also has CMOS circuitry positioned between the supporting substrate and the PCR section, and a temperature sensor wherein the CMOS circuitry uses the temperature sensor output for feedback control of the PCR section.
• GCF030.11 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF030.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF030.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF030.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF030.15 Preferably, the PCR microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GCF030.16 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF030.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF030.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF030.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF030.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the LOC device. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GCF031.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for pathogen detection and genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample containing genetic material;
• a supporting substrate;
• a plurality of reagent reservoirs;
• an incubation section downstream of the inlet, the incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material; and,
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the genetic material; and,
• a dialysis section downstream of the nucleic acid amplification section for prehybridization filtration of amplicon produced by the nucleic acid amplification section, the dialysis section being configured to remove cell debris from the amplicon; wherein,
• the incubation section, the nucleic acid amplification section and the dialysis section are all supported on the supporting substrate.
• GCF031.2 Preferably, the incubation section has a heater for heating the genetic material and the enzymes to a predetermined enzymatic reaction temperature.
• GCF031.3 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the nucleic acid amplification section, the hybridization section having an array of hybridization chambers, each containing a different probe for hybridization with target nucleic acid sequences in the genetic material, the probes being configured to hybridize with the target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF031.4 Preferably, each of the hybridization chambers has a volume less than 900,000 cubic microns.
• GCF031.5 Preferably, each of the hybridization chambers has a volume less than 200,000 cubic microns.
• GCF031.6 Preferably, the dialysis section has a large constituents channel, a small constituents channel and a plurality of apertures fluidically connecting the large constituents channel to the small constituents channel, the apertures being sized to allow nucleic acid sequences to flow from the large constituents channel to the small constituents channel while cell debris larger than the apertures is retained in the large constituents channel, the small constituents channel being in fluid communication with the hybridization section.
• GCF031.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF031.8 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus.
• GCF031.9 Preferably, the nucleic acid amplification section is polymerase chain reaction (PCR) amplification section.
• GCF031.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF031.11 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF031.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF031.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF031.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF031.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus and the reagent flows out of the reagent reservoir.
• GCF031.16 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF031.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF031.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF031.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF031.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the LOC device. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence. This LOC device design has the advantage of enabling incubation of the sample under controlled conditions.
• GCF032.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a dialysis section for separating small constituents from larger constituents in the sample;
• a plurality of reagent reservoirs;
• a nucleic acid amplification section downstream of the dialysis section for amplifying nucleic acid sequences in the sample; wherein,
• the dialysis section and the nucleic acid amplification section are both supported on the supporting substrate.
• GCF032.2 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section.
• GCF032.3 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the PCR section, the hybridization section having an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF032.4 Preferably, the dialysis section has a first channel in fluid communication with the inlet, a second channel in fluid communication with the PCR section and a plurality of apertures that are larger than the small constituents and smaller than the larger constituents, the second channel being in fluid communication with the first channel via the apertures such that the small constituents flow into the second channel while the larger constituents are retained in the first channel.
• GCF032.5 Preferably, the first channel, the second channel and the plurality of apertures are configured to fill with the sample by capillary action.
• GCF032.6 Preferably, the second channel is configured to draw the sample to the nucleic acid amplification section by capillary action.
• GCF032.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF032.8 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus to allow the reagent to flow from the reagent reservoir.
• GCF032.9 Preferably, the LOC device also has a flow-path from the inlet to the hybridization section wherein the flow-path is configured to draw the sample from the inlet to the hybridization section by capillary action.
• GCF032.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF032.11 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF032.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF032.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF032.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF032.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus.
• GCF032.16 Preferably, the hybridization section has an array of hybridization chambers for containing the probes.
• GCF032.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF032.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF032.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF032.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GCF033.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a nucleic acid amplification section downstream of the incubation section for amplifying nucleic acid sequences in the sample; and,
• a dialysis section downstream of the nucleic acid amplification section for prehybridization filtration of amplicon produced by the nucleic acid amplification section, the dialysis section being configured to remove cell debris from the amplicon; wherein,
• the nucleic acid amplification section and the dialysis section are both supported on the supporting substrate.
• GCF033.2 Preferably, the LOC device also has a photosensor and a hybridization section downstream of the dialysis section, the hybridization section having an array of probes for hybridization with target nucleic acid sequences in the sample to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF033.3 Preferably, each of the hybridization chambers has a volume less than 900,000 cubic microns.
• GCF033.4 Preferably, each of the hybridization chambers has a volume less than 200,000 cubic microns.
• GCF033.5 Preferably, each of the hybridization chambers has a volume less than 40,000 cubic microns.
• GCF033.6 Preferably, the dialysis section has a large constituents channel, a small constituents channel and a plurality of apertures fluidically connecting the large constituents channel to the small constituents channel, the apertures being sized to allow nucleic acid sequences to flow from the large constituents channel to the small constituents channel while cell debris larger than the apertures is retained in the large constituents channel, the small constituents channel being in fluid communication with the hybridization section.
• GCF033.7 Preferably, the nucleic acid amplification section is an isothermal nucleic acid amplification section.
• GCF033.8 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for isothermal nucleic acid amplification; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus.
• GCF033.9 Preferably, the nucleic acid amplification section is polymerase chain reaction (PCR) amplification section.
• GCF033.10 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the PCR section, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the PCR section.
• GCF033.11 Preferably, the PCR section has a PCR microchannel where, during use, the sample is thermally cycled to amplify the nucleic acid sequences, the PCR microchannel defining part of the flow-path for the sample and having a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF033.12 Preferably, the LOC device also has at least one elongate heater element for heating the nucleic acid sequences within the elongate PCR microchannel, the elongate heater element extending parallel to the PCR microchannel.
• GCF033.13 Preferably, at least one section of the PCR microchannel forms an elongate PCR chamber.
• GCF033.14 Preferably, the PCR section has a plurality of the elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF033.15 Preferably, the LOC device also has a reagent reservoir for holding a reagent used for PCR; and,
• a surface tension valve having an aperture configured to pin a meniscus of the reagent such that the meniscus retains the reagent in the reagent reservoir until contact with the fluid sample removes the meniscus.
• GCF033.16 Preferably, the hybridization section has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GCF033.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GCF033.18 Preferably, the CMOS circuitry has a digital memory for storing hybridization data from the photosensor output and a data interface for transmission of the hybridization data to an external device.
• GCF033.19 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• GCF033.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• This LOC device design has the advantage of separating wanted from unwanted sample components on the basis of size, using a method superior to simple filtration because of decreased clogging. This LOC device design has the advantage of enriching the effective target concentration in the portion of the sample which is to be further processed by the LOC device. This LOC device design has the advantage of removing unwanted components of the processed mixture which may interfere with later detection of the target.
• GCF034.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample containing genetic material;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a first nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the first nucleic acid amplification section and the second nucleic acid amplification section are both supported on the supporting substrate.
• GCF034.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF034.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF034.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF034.5 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first PCR section, and a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF034.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF034.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF034.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GCF034.9 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section, and a second hybridization section downstream of the second isothermal nucleic acid amplification section, wherein the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF034.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences and the second hybridization section has a second hybridization chamber array for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GCF034.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second arrays of hybridization chambers.
• GCF034.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF034.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GCF034.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GCF034.15 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF034.16 Preferably, the first PCR section has a PCR microchannel where, during use, the sample is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF034.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF034.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF034.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF034.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample through its sample inlet and performs parallel amplification of the target genetic sequences in the sample, utilizing reagents stored in the LOC device's reagent reservoirs.
• The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF035.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample containing genetic material;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a first incubation section, the first incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a second incubation section, the second incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material in parallel with the first incubation section;
• a first nucleic acid amplification section downstream of the first incubation section for amplifying nucleic acid sequences in the genetic material; and,
• a second nucleic acid amplification section downstream of the second incubation section for amplifying nucleic acid sequences in the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the first incubation section, the second incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF035.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section and the second nucleic acid amplification section is a second PCR section.
• GCF035.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF035.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF035.5 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first PCR section, and a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF035.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section.
• GCF035.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF035.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GCF035.9 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section, and a second hybridization section downstream of the second isothermal nucleic acid amplification section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF035.10 Preferably, the first hybridization section has a first array of hybridization chambers for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences, and the second hybridization section has a second array of hybridization chambers for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GCF035.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second hybridization chamber arrays.
• GCF035.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF035.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GCF035.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GCF035.15 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF035.16 Preferably, the first PCR section has a PCR microchannel where, during use, the sample is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF035.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF035.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF035.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF035.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample through its sample inlet, performs parallel preprocessing of the sample in its parallel incubation section, and performs parallel amplification of the target genetic sequences in the sample in its parallel nucleic acid amplification section, utilizing reagents stored in the LOC device's reagent reservoirs.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction and ligation of adaptor primers, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system. Furthermore, the parallel incubation chambers allow separate nucleic acid templates or template groups to optimally undergo separate enzymatic reactions with the consequent improvement in assay versatility.
• The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF036.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample containing genetic material including DNA and RNA;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a first nucleic acid amplification section for amplifying at least some of the genetic material; and,
• a second nucleic acid amplification section for amplifying at least some the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the first nucleic acid amplification section and the second nucleic acid amplification section are both supported on the supporting substrate.
• GCF036.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second PCR section configured for amplifying the RNA in the genetic material.
• GCF036.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF036.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF036.5 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first PCR section, a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF036.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section configured for amplifying the RNA in the genetic material.
• GCF036.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF036.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GCF036.9 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section and a second hybridization section downstream of the second isothermal nucleic acid amplification section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF036.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences, and the second hybridization section has a second hybridization chamber array for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GCF036.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second arrays of hybridization chambers.
• GCF036.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF036.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GCF036.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GCF036.15 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF036.16 Preferably, the first PCR section has a PCR microchannel where, during use, the sample is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF036.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF036.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF036.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF036.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample through its sample inlet and performs parallel amplification of the target DNA and RNA sequences in the sample in its parallel nucleic acid amplification section, utilizing reagents stored in the LOC device's reagent reservoirs.
• The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GCF037.1 This aspect of the invention provides a lab-on-a-chip (LOC) device for genetic analysis of a biological sample, the LOC device comprising:
• an inlet for receiving the sample containing genetic material including DNA and RNA;
• a supporting substrate;
• a plurality of reagent reservoirs;
• a first incubation section, the first incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material;
• a second incubation section, the second incubation section being in fluid communication with one of the reagent reservoirs containing enzymes for enzymatic reaction with the genetic material in parallel with the first incubation section;
• a first nucleic acid amplification section downstream of the first incubation section for amplifying at least some of the genetic material; and,
• a second nucleic acid amplification section downstream of the second incubation section for amplifying at least some of the genetic material in parallel with the first nucleic acid amplification section; wherein,
• the first incubation section, the second incubation section, the first nucleic acid amplification section and the second nucleic acid amplification section are all supported on the supporting substrate.
• GCF037.2 Preferably, the first nucleic acid amplification section is a first polymerase chain reaction (PCR) section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second PCR section configured for amplifying the RNA in the genetic material.
• GCF037.3 Preferably, the first PCR section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second PCR section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF037.4 Preferably, the first PCR section and the second PCR section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution;
• thermal cycle time;
• thermal cycle repetitions; and,
• temperature during a particular phase of PCR.
• GCF037.5 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first PCR section, a second hybridization section downstream of the second PCR section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having a second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF037.6 Preferably, the first nucleic acid amplification section is a first isothermal nucleic acid amplification section configured for amplifying the DNA in the genetic material and the second nucleic acid amplification section is a second isothermal nucleic acid amplification section configured for amplifying the RNA in the genetic material.
• GCF037.7 Preferably, the first isothermal nucleic acid amplification section has a first set of primer pairs for annealing to a first set of complementary nucleic acid sequences in the DNA, and the second isothermal nucleic acid amplification section has a second set of primer pairs for annealing to a second set of complementary nucleic acid sequences in the RNA, the first set of complementary nucleic acid sequences being different to the second set of complementary nucleic acid sequences.
• GCF037.8 Preferably, the first isothermal nucleic acid amplification section and the second isothermal nucleic acid amplification section are configured to operate with different amplification parameters, the amplification parameters being at least one of:
• reverse-transcriptase type;
• polymerase type;
• deoxyribonucleoside triphosphate concentrations;
• buffer solution; and,
• temperature during the nucleic acid amplification.
• GCF037.9 Preferably, the LOC device also has a photosensor, a first hybridization section downstream of the first isothermal nucleic acid amplification section and a second hybridization section downstream of the second isothermal nucleic acid amplification section, the first hybridization section having a first array of probes for hybridization with first target nucleic acid sequences to form probe-target hybrids and, the second hybridization section having second array of probes for hybridization with second target nucleic acid sequences to form probe-target hybrids, wherein the photosensor is configured for detecting the probe-target hybrids.
• GCF037.10 Preferably, the first hybridization section has a first hybridization chamber array for containing the first probes such that the first probes within each hybridization chamber are configured to hybridize with one of the first target nucleic acid sequences, and the second hybridization section has a second hybridization chamber array for containing the second probes such that the second probes within each hybridization chamber are configured to hybridize with one of the second target nucleic acid sequences.
• GCF037.11 Preferably, the photosensor is an array of photodiodes positioned in registration with the first and second arrays of hybridization chambers.
• GCF037.12 Preferably, the first isothermal nucleic acid amplification section has a nucleic acid amplification microchannel for maintaining the reaction temperature of the sample, the nucleic acid amplification microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF037.13 Preferably, the nucleic acid amplification microchannel has a cross sectional area transverse to the flow less than 16,000 square microns.
• GCF037.14 Preferably, the reagent reservoirs each have a surface tension valve for retaining reagents therein, the surface tension valve having a meniscus anchor for pinning a meniscus of the reagent until contact with the sample flow removes the meniscus.
• GCF037.15 Preferably, the LOC device also has CMOS circuitry, a temperature sensor and a microsystems technology (MST) layer which incorporates the first and second PCR sections, wherein the CMOS circuitry is positioned between the supporting substrate and the MST layer, the CMOS circuitry being configured to use the temperature sensor output for feedback control of the first and second PCR sections.
• GCF037.16 Preferably, the first PCR section has a PCR microchannel where, during use, the sample is thermally cycled, the PCR microchannel defining a flow-path with a cross sectional area transverse to the flow less than 100,000 square microns.
• GCF037.17 Preferably, the PCR microchannel has at least one elongate heater element extending parallel to the PCR microchannel.
• GCF037.18 Preferably, the first PCR section has a plurality of elongate PCR chambers each formed by respective sections of the PCR microchannel, the PCR microchannel having a serpentine configuration formed by a series of wide meanders, each of the wide meanders being a channel section that forms one of the elongate PCR chambers.
• GCF037.19 Preferably, the first PCR section has an active valve for retaining liquid in the first PCR section during thermal cycling and allowing flow to the first hybridization chamber array in response to an activation signal from the CMOS circuitry.
• GCF037.20 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• The easily usable, mass-producible, and inexpensive LOC device accepts a biological sample through its sample inlet, performs parallel preprocessing of the sample in its parallel incubation section, and performs parallel amplification of the target DNA and RNA sequences in the sample in its parallel nucleic acid amplification section, utilizing reagents stored in the LOC device's reagent reservoirs.
• In the incubation section the genetic material undergo various types of preprocessing, like nucleic acid restriction, ligation of adaptor primers, and reverse transcription, to provide optimal or necessary conditions for the subsequent analytical stages, increasing the informational content of the analytical outcomes and increasing the sensitivity, signal-to-noise-ration, and reliability of the assay system. Furthermore, the parallel incubation chambers allow separate nucleic acid templates or template groups to optimally undergo separate enzymatic reactions with the consequent improvement in assay versatility.
• The amplification of target nucleic acid sequences increases the sensitivity and signal-to-noise ratio of the assay system. Furthermore, the parallel amplification chambers allow separate targets or target groups to optimally use separate primer pairs or separate groups of primer pairs and also to use separate optimal amplification parameters, with the consequent increase in assay sensitivity, signal-to-noise-ratio, and reliability.
• The reagent reservoirs, being integral to the LOC device and holding the assay's total reagent requirements, provide for the low system component-count and simple manufacturing procedures, leading into an inexpensive assay system.
• GSA001.1 This aspect of the invention provides a method of analyzing the nucleic acid content of a blood sample, the method comprising the steps of:
• providing a test module with an outer casing configured for handheld portability, the outer casing having a receptacle for receiving blood, the test module having a lysis section mounted in the outer casing for lysing cells and organisms in the blood to release the genetic material therein, a hybridization section with an array of probes for hybridization with target nucleic acid sequences in the genetic material, and circuitry for sensing which of the probes have hybridized and generating hybridization data;
• providing a test module reader for reading the hybridization data from the test module;
• inserting a blood sample in the receptacle;
• interfacing the test module with the test module reader; wherein,
• the test module reader analyses the nucleic acid content from the hybridization data.
• GSA001.2 Preferably, the test module and the test module reader are configured to interface via an electrical connection, the test module drawing operational power from the test module reader.
• GSA001.3 Preferably, the method also has:
• providing a lancet to obtain a drop of the blood from a patient.
• GSA001.4 Preferably, the lancet is retractable into the outer casing and biased to an extended position in which a sharp end of the lancet protrudes from the outer casing.
• GSA001.5 Preferably, the lancet is spring loaded such that during use the lancet is held in a retracted position within the outer casing against the spring bias until released by user actuation.
• GSA001.6 Preferably, the method also has a plurality of reagent reservoirs containing reagents for processing the blood sample.
• GSA001.7 Preferably, one of the reagent reservoirs contains anticoagulant for addition to the blood sample downstream of the receptacle.
• GSA001.8 Preferably, the method also has a dialysis section for separating constituents larger than a predetermined threshold into a portion of the blood sample such that the remainder of the sample contains constituents smaller than the predetermined threshold.
• GSA001.9 Preferably, one of the reagent reservoirs contains a lysis reagent for lysing the cells in the lysis chamber.
• GSA001.10 Preferably, the lysis chamber has a heater for lysing the cells.
• GSA001.11 Preferably, the method also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences from the genetic material.
• GSA001.12 Preferably, the method also has an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the blood sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GSA001.13 Preferably, the method also has a temperature sensor and the circuitry uses the temperature sensor output for feedback control of the PCR section.
• GSA001.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in amplicon from the PCR section, each of the probe-target hybrids being configured to emit photons in response to an input, and the circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSA001.15 Preferably, the method also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSA001.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSA001.17 Preferably, the circuitry has a digital memory for storing hybridization data from the photosensor output.
• GSA001.18 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the circuitry.
• GSA001.19 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GSA001.20 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• The easily usable, mass-producible, and inexpensive LOC device for analysis of nucleic acid content of blood samples accepts a blood sample through its sample receptacle, lyses the sample's cells in its lysis chamber to release the sample's genetic material, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array.
• The lysing process extracts the genetic material from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GSA002.1 This aspect of the invention provides a method of analyzing the nucleic acid content of a biological fluid, the method comprising the steps of:
• providing a test module with an outer casing configured for handheld portability, the outer casing having a receptacle for receiving the biological fluid, the test module having a hybridization section with an array of probes for hybridization with target nucleic acid sequences in the biological fluid, and circuitry for sensing which of the probes have hybridized and generating hybridization data;
• providing a test module reader for reading the hybridization data from the test module;
• inserting the biological fluid in the receptacle;
• interfacing the test module with the test module reader; wherein,
• the test module reader analyses the nucleic acid content from the hybridization data.
• GSA002.2 Preferably, the test module and the test module reader are configured to interface via an electrical connection, the test module drawing operational power from the test module reader.
• GSA002.3 Preferably, the biological fluid contains cells and the test module has a lysis section mounted in the outer casing for lysing the cells to release genetic material therein.
• GSA002.4 Preferably, the method also has the step of pre-processing the biological fluid prior to insertion in the receptacle.
• GSA002.5 Preferably, the biological fluid contains one or more of:
• blood;
• saliva;
• cerebrospinal fluid;
• urine;
• semen;
• amniotic fluid;
• umbilical cord blood;
• breast milk;
• sweat;
• pleural effusion;
• tears;
• pericardial fluid;
• peritoneal fluid; and,
• drinks samples.
• GSA002.6 Preferably, the biological fluid is amplicon from a polymerase chain reaction (PCR).
• GSA002.7 Preferably, the method also has a plurality of reagent reservoirs containing reagents for processing the biological fluid.
• GSA002.8 Preferably, the method also has a dialysis section for separating cells larger than a predetermined threshold into a portion of the biological fluid such that the remainder of the biological fluid contains cells smaller than the predetermined threshold.
• GSA002.9 Preferably, one of the reagent reservoirs contains a lysis reagent for lysing the cells in the lysis chamber.
• GSA002.10 Preferably, the lysis chamber has a heater for lysing the cells.
• GSA002.11 Preferably, the method also has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences from the biological fluid.
• GSA002.12 Preferably, the method also has an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the biological fluid and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GSA002.13 Preferably, the method also has a temperature sensor and the circuitry uses the temperature sensor output for feedback control of the PCR section.
• GSA002.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in amplicon from the PCR section, each of the probe-target hybrids being configured to emit photons in response to an input, and the circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSA002.15 Preferably, the method also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSA002.16 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSA002.17 Preferably, the circuitry has a digital memory for storing hybridization data from the photosensor output.
• GSA002.18 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the circuitry.
• GSA002.19 Preferably, the active valve is a boiling-initiated valve with a meniscus anchor configured to pin a meniscus that arrests capillary driven flow of the liquid, and a heater for boiling the liquid to unpin the meniscus from the meniscus anchor such that capillary driven flow resumes.
• GSA002.20 Preferably, the meniscus anchor is an aperture and the heater has an annular shape and is positioned near the aperture periphery.
• The easily usable, mass-producible, and inexpensive LOC device for analysis of nucleic acid content of biological fluid samples accepts a biological fluid sample through its sample receptacle, lyses the sample's cells in its lysis chamber to release the sample's genetic material, amplifies target genetic sequences, and analyzes the sample's nucleic acid sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array.
• The lysing process extracts the genetic material from cells in the sample and provides for follow-on processing and analysis of the targets. The lysis subunit being integral to the device, provides for simple assay procedures, low system component-count, and simple system manufacturing procedures, leading into an inexpensive assay system.
• The amplification of target genetic sequences increases the sensitivity and signal-to-noise ratio of the assay system.
• The probe hybridization section provides for analysis of the targets via hybridization. The integrated probe hybridization section provides for an easily usable, mass-producible, and inexpensive integrated solution with low system component-count.
• The integrated image sensor obviates the need for an expensive external imaging system and provides for a mass-producible inexpensive integrated solution with low system component-count that is a compact, light, and highly portable system. The integrated image sensor increases the readout sensitivity by benefiting from large angle of light collection and obviates the need for optical components in the optical collection train.
• GSE001.1 This aspect of the invention provides a microfluidic device for processing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• functional sections for processing the fluid;
• a flow-path extending from the inlet into at least one of the functional sections;
• a flow rate sensor with a conductive element positioned in proximity of the fluid flowing along the flow-path;
• CMOS circuitry configured for measuring the temperature of the conductive element; wherein,
• the CMOS circuitry is configured to provide a predetermined current through the conductive element and measure the electrical resistance of the conductive element such that a flow speed is derived from the current, the temperature sensor output and the electrical resistance, and a flow rate is derived using the flow speed and the flow-path cross section transverse to the flow direction at the conductive element.
• GSE001.2 Preferably, the conductive element is a heater element supported on an internal surface of the flow-path.
• GSE001.3 Preferably, the conductive element has a serpentine configuration.
• GSE001.4 Preferably, the flow-path is defined by a microchannel, the microchannel having a cross sectional area transverse to the flow less than 100,000 square microns.
• GSE001.5 Preferably, one of the functional sections is a polymerase chain reaction (PCR) section, and the fluid is a biological sample containing target nucleic acid sequences, the PCR section being configured for thermally cycling the sample to amplify the target nucleic acid sequences, the microchannel defining a flow-path through the PCR section.
• GSE001.6 Preferably, the microfluidic device also has at least one elongate heater element for heating the target nucleic acid sequences within the microchannel.
• GSE001.7 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer that incorporates the functional sections wherein the CMOS circuitry has digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GSE001.8 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GSE001.9 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic device.
• GSE001.10 Preferably, the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GSE001.11 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the target nucleic acid sequences.
• GSE001.12 Preferably, the microfluidic device also has an array of probes for hybridization with the target nucleic acid sequences in the amplicon from the PCR section.
• GSE001.13 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GSE001.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSE001.15 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GSE001.16 Preferably, the microfluidic device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSE001.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSE001.18 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GSE001.19 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GSE001.20 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, with aspects of the functioning of the microfluidic device related to fluidic flow rates being monitored and optimally controlled via the easily manufacturable hot-wire flow sensor.
• GSE002.1 This aspect of the invention provides a microfluidic device for processing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• functional sections for processing the fluid;
• a flow-path extending from the inlet into at least some of the functional sections;
• circuitry for operative control of the functional sections; and,
• a liquid sensor with electrodes positioned for contact with the fluid flowing along the flow-path; wherein,
• the circuitry is configured to provide a voltage across the electrodes such that current above a predetermined threshold is indicative of liquid at the electrodes.
• GSE002.2 Preferably, the microfluidic device also has a temperature sensor for sensing the temperature of the fluid in the flow-path, and a flow rate sensor with a heater element supported on an internal surface of the flow-path wherein the circuitry is configured for receiving the temperature sensor output, providing a predetermined current through the heater element and measuring electrical resistance of the conductive element such that a flow speed is derived from the current, the temperature sensor output and the electrical resistance, and a flow rate is derived using the flow speed and the flow-path cross section transverse to the flow direction at the heater element.
• GSE002.3 Preferably, the heater element has a serpentine configuration.
• GSE002.4 Preferably, the flow-path is defined by a microchannel, the microchannel having a cross sectional area transverse to the flow less than 100,000 square microns.
• GSE002.5 Preferably, one of the functional sections is a polymerase chain reaction (PCR) section, and the fluid is a biological sample containing nucleic acid sequences, the PCR section being configured for thermally cycling the sample to amplify the nucleic acid sequences, the microchannel defining a flow-path through the PCR section.
• GSE002.6 Preferably, the microfluidic device also has at least one elongate heater element for heating the nucleic acid sequences within the microchannel.
• GSE002.7 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer that incorporates the functional sections wherein the circuitry is CMOS circuitry with digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GSE002.8 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GSE002.9 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic device.
• GSE002.10 Preferably, the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GSE002.11 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GSE002.12 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GSE002.13 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GSE002.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSE002.15 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GSE002.16 Preferably, the microfluidic device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSE002.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSE002.18 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GSE002.19 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GSE002.20 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, with aspects of the functioning of the microfluidic device related to presence or absence of fluids in a given location being monitored and optimally controlled via the easily manufacturable liquid sensor.
• GSE003.1 This aspect of the invention provides a microfluidic device for processing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• functional sections for processing the fluid;
• a flow-path extending from the inlet into at least some of the functional sections;
• circuitry for operative control of the functional sections; and,
• a capillary meniscus marching velocity sensor having a plurality of liquid sensors spaced along the flow-path, each of the liquid sensors having electrodes positioned for contact with the fluid flowing along the flow-path; wherein,
• the circuitry is configured to provide a voltage across the electrodes such that current above a predetermined threshold is indicative of liquid at the electrodes and used to derive a velocity of the liquid flow front.
• GSE003.2 Preferably, the microfluidic device also has a temperature sensor for sensing the temperature of the fluid in the flow-path, and a flow rate sensor with a heater element supported on an internal surface of the flow-path wherein the circuitry is configured for receiving the temperature sensor output, providing a predetermined current through the heater element and measuring electrical resistance of the conductive element such that a flow speed is derived from the current, the temperature sensor output and the electrical resistance, and a flow rate is derived using the flow speed and the flow-path cross section transverse to the flow direction at the heater element.
• GSE003.3 Preferably, the heater element has a serpentine configuration.
• GSE003.4 Preferably, the flow-path is defined by a microchannel, the microchannel having a cross sectional area transverse to the flow less than 100,000 square microns.
• GSE003.5 Preferably, one of the functional sections is a polymerase chain reaction (PCR) section, and the fluid is a biological sample containing nucleic acid sequences, the PCR section being configured for thermally cycling the sample to amplify the nucleic acid sequences, the microchannel defining a flow-path through the PCR section.
• GSE003.6 Preferably, the microfluidic device also has at least one elongate heater element for heating the nucleic acid sequences within the microchannel.
• GSE003.7 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer that incorporates the functional sections wherein the circuitry is CMOS circuitry with digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GSE003.8 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GSE003.9 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic device.
• GSE003.10 Preferably, the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GSE003.11 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GSE003.12 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GSE003.13 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GSE003.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSE003.15 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GSE003.16 Preferably, the microfluidic device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSE003.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSE003.18 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GSE003.19 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GSE003.20 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, with aspects of the functioning of the microfluidic device related to fluidic flow rates being monitored and optimally controlled via the easily manufacturable capillary meniscus marching velocity sensor.
• GSE004.1 This aspect of the invention provides a microfluidic device for processing a fluid, the microfluidic device comprising:
• an inlet for receiving the fluid;
• functional sections for processing the fluid;
• a flow-path extending from the inlet into at least some of the functional sections;
• CMOS circuitry for operative control of the functional sections; and,
• a conductivity sensor with a first terminal and a second terminal spaced apart along the flow-path, and a first electrode and a second electrode positioned between the first terminal and the second terminal and spaced apart along the flow-path; wherein,
• the CMOS circuitry is configured to generate a current between the first terminal and the second terminal, and measure a voltage across the first electrode and the second electrode such that conductivity of the fluid in the flow-path is derived from the current and the measured voltage.
• GSE004.2 Preferably, the microfluidic device also has a temperature sensor for sensing the temperature of the fluid in the flow-path, and a flow rate sensor with a heater element supported on an internal surface of the flow-path wherein the circuitry is configured for receiving the temperature sensor output, providing a predetermined current through the heater element and measuring electrical resistance of the conductive element such that a flow speed is derived from the current, the temperature sensor output and the electrical resistance, and a flow rate is derived using the flow speed and the flow-path cross section transverse to the flow direction at the heater element.
• GSE004.3 Preferably, the heater element has a serpentine configuration.
• GSE004.4 Preferably, the flow-path is defined by a microchannel, the microchannel having a cross sectional area transverse to the flow less than 100,000 square microns.
• GSE004.5 Preferably, one of the functional sections is a polymerase chain reaction (PCR) section, and the fluid is a biological sample containing nucleic acid sequences, the PCR section being configured for thermally cycling the sample to amplify the nucleic acid sequences, the microchannel defining a flow-path through the PCR section.
• GSE004.6 Preferably, the microfluidic device also has at least one elongate heater element for heating the nucleic acid sequences within the microchannel.
• GSE004.7 Preferably, the microfluidic device also has a supporting substrate and a microsystems technology (MST) layer that incorporates the functional sections wherein the CMOS circuitry has digital memory for storing data and operational information to operatively control the functional sections during processing and analysis of the sample.
• GSE004.8 Preferably, the microfluidic device also has a plurality of reagent reservoirs containing reagents for processing the sample wherein the data stored in the digital memory relates to the reagent identities.
• GSE004.9 Preferably, the data stored in the digital memory is a unique identifier for the microfluidic device.
• GSE004.10 Preferably, the operational information stored in the digital memory relates to thermal cycle timing and duration.
• GSE004.11 Preferably, the functional sections include an incubation section upstream of the PCR section and one of the reagent reservoirs is a restriction enzyme reservoir, the incubation section having a heater for maintaining a mixture of the sample and restriction enzymes at an incubation temperature during restriction digestion of the nucleic acid sequences.
• GSE004.12 Preferably, the microfluidic device also has an array of probes for hybridization with target nucleic acid sequences in the amplicon from the PCR section.
• GSE004.13 Preferably, the data stored in the digital memory includes probe identity data identifying the probe at each site within the array of probes.
• GSE004.14 Preferably, each of the probes are configured to form a probe-target hybrid with a complementary target nucleic acid sequence contained in the amplicon, each of the probe-target hybrids being configured to emit photons in response to an input, and the CMOS circuitry incorporates a photosensor for sensing the photons emitted by the probe-target hybrids.
• GSE004.15 Preferably, the data stored in the digital memory includes hybridization data generated from the photosensor output.
• GSE004.16 Preferably, the microfluidic device also has a hybridization chamber array for containing the probes such that the probes within each hybridization chamber are configured to hybridize with one of the target nucleic acid sequences.
• GSE004.17 Preferably, the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GSE004.18 Preferably, the CMOS circuitry has bond-pads and is configured for transmission of the hybridization data to an external device.
• GSE004.19 Preferably, the sample is drawn from a patient and the CMOS circuitry is configured to download patient data via the bond-pads and store the patient data in the digital memory.
• GSE004.20 Preferably, the PCR section has an active valve for retaining liquid in the PCR section during thermal cycling and allowing flow to the hybridization chambers in response to an activation signal from the CMOS circuitry.
• The mass-producible and inexpensive microfluidic device processes and/or analyses fluids, with aspects of the functioning of the microfluidic device related to conductivity of liquid mixtures being monitored and optimally controlled via the easily manufacturable conductivity sensor.
• GDA001.1 This aspect of the invention provides a microfluidic device for simultaneous detection of multiple conditions in a patient, the microfluidic device comprising:
• an inlet for receiving a sample of biological material drawn from the patient;
• a microsystems technologies (MST) layer with a detection section having an array of probes for reaction with target molecules in the sample to form probe-target complexes, the target molecules being indicative of medical conditions in the patient; and,
• a photosensor for detecting the probe-target complexes; wherein,
• the array of probes has more than 1000 probes.
• GDA001.2 Preferably, the target molecules are target nucleic acid sequences and the array of probes has probes configured to hybridize with the target nucleic acid sequences to form probe-target hybrids.
• GDA001.3 Preferably, the target molecules are target proteins and the array of probes has probes configured to hybridize or conjugate with the target proteins to form the probe-target complexes.
• GDA001.4 Preferably, the microfluidic device also has CMOS circuitry and a supporting substrate, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the photosensor is an array of photodiodes incorporated in the CMOS circuitry.
• GDA001.5 Preferably, the microfluidic device also has an array of hybridization chambers containing the probes, wherein the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA001.6 Preferably, the microfluidic device also has a nucleic acid amplification section upstream of the array of hybridization chambers, the nucleic acid amplification section being configured for amplifying the target nucleic acid sequences.
• GDA001.7 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA001.8 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA001.9 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDA001.10 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA001.11 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA001.12 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA001.13 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA001.14 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA001.15 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GDA001.16 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA001.17 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• GDA001.18 Preferably, each of the hybridization chambers has a volume less than 9,000 cubic microns.
• GDA001.19 Preferably, the photodiodes are less than 1600 microns from the ECL probes.
• GDA001.20 Preferably, the microfluidic device also has a plurality of reagent reservoirs for different reagents required to process the fluid wherein the fluid is drawn from the inlet to the end point sensor by capillary action and without adding liquid from a source external to the microfluidic device.
• The easily usable, mass-producible, inexpensive, and portable diagnostic test module accepts a biological sample and processes and analyzes the sample, detecting and identifying combinations of diseases and conditions. The diagnostic capabilities of the test module provides reliability and speed of diagnosing diseases and there combinations, ease of use, and very low cost of diagnostics.
• GDA002.1 This aspect of the invention provides a microfluidic device for analysis of mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with an outer cellular membrane containing mitochondria and a nucleus, the mitochondria having a mitochondrial membrane containing mitochondrial DNA and the nuclei having a nuclear membrane containing nuclear DNA;
• a lysis section for lysing the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact; and,
• probes for hybridization with target nucleic acid sequences in the mitochondrial DNA to form probe-target hybrids.
• GDA002.2 Preferably, the microfluidic device also has a reagent reservoir containing a lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes.
• GDA002.3 Preferably, the lysis reagent includes a plant glycoside.
• GDA002.4 Preferably, the lysis reagent includes a non-ionic detergent.
• GDA002.5 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor configured to pin a meniscus of the reagent such that the meniscus retains the lysis reagent in the reagent reservoir until contact with the sample removes the meniscus and the lysis reagent flows out of the reagent reservoir.
• GDA002.6 Preferably, the microfluidic device also has a photosensor for detecting the probe-target hybrids.
• GDA002.7 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA002.8 Preferably, the microfluidic device also has a microsystems technologies (MST) layer which incorporates the lysis section and the hybridization chamber array, a supporting substrate and CMOS circuitry, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the array of photodiodes is incorporated in the CMOS circuitry.
• GDA002.9 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA002.10 Preferably, the microfluidic device also has a nucleic acid amplification section upstream of the array of hybridization chambers, the nucleic acid amplification section being configured for amplifying the target nucleic acid sequences.
• GDA002.11 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA002.12 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA002.13 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• 13. The microfluidic device according to claim 12 wherein the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA002.14 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA002.15 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA002.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA002.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA002.18 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GDA002.19 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA002.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• Detection of mitochondrial DNA sequences has the advantage of maintaining high sensitivity and fidelity in spite of old or degraded sample material.
• GDA003.1 This aspect of the invention provides a microfluidic device for amplifying mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with an outer cellular membrane containing mitochondria and a nucleus, the mitochondria having a mitochondrial membrane containing mitochondrial DNA and the nuclei having a nuclear membrane containing nuclear DNA;
• a lysis section for lysing the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact; and,
• a nucleic acid amplification section configured for amplifying target nucleic acid sequences in the mitochondrial DNA.
• GDA003.2 Preferably, the microfluidic device also has probes for hybridization with the target nucleic acid sequences to form probe-target hybrids.
• GDA003.3 Preferably, the microfluidic device also has a reagent reservoir containing a lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes.
• GDA003.4 Preferably, the lysis reagent includes a plant glycoside.
• GDA003.5 Preferably, the lysis reagent includes a non-ionic detergent.
• GDA003.6 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor configured to pin a meniscus of the reagent such that the meniscus retains the lysis reagent in the reagent reservoir until contact with the sample removes the meniscus and the lysis reagent flows out of the reagent reservoir.
• GDA003.7 Preferably, the microfluidic device also has a photosensor for detecting the probe-target hybrids.
• GDA003.8 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA003.9 Preferably, the microfluidic device also has a microsystems technologies (MST) layer which incorporates the lysis section and the hybridization chamber array, a supporting substrate and CMOS circuitry, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the array of photodiodes is incorporated in the CMOS circuitry.
• GDA003.10 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA003.11 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA003.12 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA003.13 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• 13. The microfluidic device according to claim 12 wherein the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA003.14 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA003.15 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA003.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA003.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA003.18 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GDA003.19 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA003.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• Detection of mitochondrial DNA sequences has the advantage of maintaining high sensitivity and fidelity in spite of old or degraded sample material. This enables more sensitive, and more specific, detection of target DNA. This LOC device has the advantages provided by sequence-specific amplification, including: sensitivity provided by amplification; broad dynamic range; and high specificity for the target DNA sequence.
• GDA004.1 This aspect of the invention provides a microfluidic device for detecting mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with mitochondria containing mitochondrial DNA;
• a lysis section for lysing the mitochondria to release the mitochondrial DNA;
• probes for hybridization with target nucleic acid sequences in the mitochondrial DNA to form probe-target hybrids; and,
• a photosensor for detecting the probe-target hybrids.
• GDA004.2 Preferably, each of the cells have nuclei containing nuclear DNA, and the lysis section is configured to lyse the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact.
• GDA004.3 Preferably, the microfluidic device also has a reagent reservoir containing a lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes.
• GDA004.4 Preferably, the lysis reagent includes a plant glycoside.
• GDA004.5 Preferably, the lysis reagent includes a non-ionic detergent.
• GDA004.6 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor configured to pin a meniscus of the reagent such that the meniscus retains the lysis reagent in the reagent reservoir until contact with the sample removes the meniscus and the lysis reagent flows out of the reagent reservoir.
• GDA004.7 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA004.8 Preferably, the microfluidic device also has a microsystems technologies (MST) layer which incorporates the lysis section and the hybridization chamber array, a supporting substrate and CMOS circuitry, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the array of photodiodes is incorporated in the CMOS circuitry.
• GDA004.9 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA004.10 Preferably, the microfluidic device also has a nucleic acid amplification section upstream of the array of hybridization chambers, the nucleic acid amplification section being configured for amplifying the target nucleic acid sequences.
• GDA004.11 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA004.12 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA004.13 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• 13. The microfluidic device according to claim 12 wherein the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA004.14 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA004.15 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA004.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA004.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA004.18 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GDA004.19 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA004.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• Detection of mitochondrial DNA sequences has the advantage of maintaining high sensitivity and fidelity in spite of old or degraded sample material. This increases the specificity of the detection of target molecules. This enables more sensitive, and more specific, detection of target DNA.
• GDA005.1 This aspect of the invention provides a microfluidic device for analysis of mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with mitochondria containing mitochondrial DNA;
• a lysis section for lysing the mitochondria to release the mitochondrial DNA;
• fluorescence resonance energy transfer (FRET) probes for hybridization with target nucleic acid sequences in the mitochondrial DNA to form probe-target hybrids; and,
• a photosensor for detecting the probe-target hybrids.
• GDA005.2 Preferably, each of the cells have nuclei containing nuclear DNA, and the lysis section is configured to lyse the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact.
• GDA005.3 Preferably, the microfluidic device also has a reagent reservoir containing a lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes.
• GDA005.4 Preferably, the lysis reagent includes a plant glycoside.
• GDA005.5 Preferably, the lysis reagent includes a non-ionic detergent.
• GDA005.6 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor configured to pin a meniscus of the reagent such that the meniscus retains the lysis reagent in the reagent reservoir until contact with the sample removes the meniscus and the lysis reagent flows out of the reagent reservoir.
• GDA005.7 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA005.8 Preferably, the microfluidic device also has a microsystems technologies (MST) layer which incorporates the lysis section and the hybridization chamber array, a supporting substrate and CMOS circuitry, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the array of photodiodes is incorporated in the CMOS circuitry.
• GDA005.9 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and a fluorophore that generates a fluorescence emission in response to an excitation light, and the hybridization chambers each have an optical window to expose the FRET probes to the excitation light.
• GDA005.10 Preferably, the microfluidic device also has a nucleic acid amplification section upstream of the array of hybridization chambers, the nucleic acid amplification section being configured for amplifying the target nucleic acid sequences.
• GDA005.11 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the FRET probes.
• GDA005.12 Preferably, the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
• GDA005.13 Preferably, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
• GDA005.14 Preferably, the fluorophore is a transition metal-ligand complex.
• GDA005.15 Preferably, the fluorophore is a lanthanide metal-ligand complex.
• GDA005.16 Preferably, the fluorophore is selected from:
• a ruthenium chelate;
• a terbium chelate; or,
• a europium chelate.
• GDA005.17 Preferably, the FRET probes each have a quencher with no native emission in response to the excitation light.
• GDA005.18 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA005.19 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA005.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• Detection of mitochondrial DNA sequences has the advantage of maintaining high sensitivity and fidelity in spite of old or degraded sample material. This increases the specificity of the detection of target molecules. This enables more sensitive, and more specific, detection of target DNA. An advantage conferred by the signal change based on complementarity is the ability to obtain a signal in a homogeneous format. No washing, additional sensitisation, or development steps are required to produce a signal whose level changes in the presence of the target.
• GDA006.1 This aspect of the invention provides a microfluidic device for analysis of mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with mitochondria containing mitochondrial DNA;
• a lysis section for lysing the mitochondria to release the mitochondrial DNA;
• electrochemiluminescent (ECL) resonance energy transfer probes for hybridization with target nucleic acid sequences in the mitochondrial DNA to form probe-target hybrids; and,
• a photosensor for detecting the probe-target hybrids.
• GDA006.2 Preferably, each of the cells have nuclei containing nuclear DNA, and the lysis section is configured to lyse the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact.
• GDA006.3 Preferably, the microfluidic device also has a reagent reservoir containing a lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes.
• GDA006.4 Preferably, the lysis reagent includes a plant glycoside.
• GDA006.5 Preferably, the lysis reagent includes a non-ionic detergent.
• GDA006.6 Preferably, the reagent reservoir has a surface tension valve with a meniscus anchor configured to pin a meniscus of the reagent such that the meniscus retains the lysis reagent in the reagent reservoir until contact with the sample removes the meniscus and the lysis reagent flows out of the reagent reservoir.
• GDA006.7 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA006.8 Preferably, the microfluidic device also has a microsystems technologies (MST) layer which incorporates the lysis section and the hybridization chamber array, a supporting substrate and CMOS circuitry, the CMOS circuitry being positioned between the MST layer and the supporting substrate wherein the array of photodiodes is incorporated in the CMOS circuitry.
• GDA006.9 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA006.10 Preferably, the microfluidic device also has a nucleic acid amplification section upstream of the array of hybridization chambers, the nucleic acid amplification section being configured for amplifying the target nucleic acid sequences.
• GDA006.11 Preferably, the nucleic acid amplification section is a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA006.12 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA006.13 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• 13. The microfluidic device according to claim 12 wherein the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA006.14 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA006.15 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA006.16 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA006.17 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• GDA006.18 Preferably, the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
• GDA006.19 Preferably, the microfluidic device also has a fluid flow-path from the PCR section to an end-point liquid sensor, the hybridization chambers being spaced along both sides of the fluid flow-path.
• GDA006.20 Preferably, the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action, and the hybridization chambers are each configured to fill with the fluid from the fluid flow-path by capillary action such that during use, the CMOS circuitry activates the hybridization heater in response to output from the liquid end point sensor indicating that the fluid has reached the liquid end point sensor.
• Detection of mitochondrial DNA sequences has the advantage of maintaining high sensitivity and fidelity in spite of old or degraded sample material. This increases the specificity of the detection of target molecules. This enables more sensitive, and more specific, detection of target DNA. An advantage conferred by the signal change based on complementarity is the ability to obtain a signal in a homogeneous format. No washing, additional sensitisation, or development steps are required to produce a signal whose level changes in the presence of the target.
• The electrochemiluminescence-based assay target detection obviates any need, of the assay system, for an excitation light source, excitation optics, and optical filter elements, in turn, providing for a more compact and more inexpensive assay system. The absence of the requirement for the rejection of any excitation light also simplifies the detector circuitry, making the assay system even more inexpensive.
• GDA007.1 This aspect of the invention provides a microfluidic device for analysis of genetic DNA and mitochondrial DNA in a sample, the microfluidic device comprising:
• an inlet for receiving a sample of biological material having cells with an outer cellular membrane containing mitochondria and a nucleus, the mitochondria has a mitochondrial membrane containing mitochondrial DNA and the nuclei have a nuclear membrane containing nuclear DNA;
• a first lysis section for lysing the outer cellular membrane and the mitochondrial membrane while the nuclear membrane remains intact;
• a second lysis section for lysing the nuclear membrane;
• mitochondrial probes downstream of the first lysis section for hybridization with target nucleic acid sequences in the mitochondrial DNA to form probe-target hybrids; and,
• nuclear probes downstream of the second lysis second for hybridization with target nucleic acid sequences in the nuclear DNA to form probe-target hybrids.
• GDA007.2 Preferably, the microfluidic device also has a first reagent reservoir containing a first lysis reagent for lysing the outer cellular membranes and the mitochondrial membranes and a second reagent reservoir containing a second lysis reagent for lysing the nuclei.
• GDA007.3 Preferably, the first lysis reagent includes a plant glycoside.
• GDA007.4 Preferably, the first lysis reagent includes a non-ionic detergent.
• GDA007.5 Preferably, the first and second reagent reservoirs each have a surface tension valve with a meniscus anchor configured to pin a meniscus that retains reagent in the reagent reservoir until contact with the sample removes the meniscus.
• GDA007.6 Preferably, the microfluidic device also has a photosensor for detecting the probe-target hybrids.
• GDA007.7 Preferably, the microfluidic device also has an array of hybridization chambers for containing the probes, wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers.
• GDA007.8 Preferably, the microfluidic device also has a first dialysis section and a second dialysis section, the first dialysis section being configured to separate pathogens and cells larger than a first size threshold from constituents smaller than the first size threshold, the second dialysis section being configured to separate constituents larger than a second size threshold, including the nuclei from the cells lysed in the first lysis section, from constituents smaller than the second size threshold including the mitochondrial DNA.
• GDA007.9 Preferably, the microfluidic device also has a first nucleic acid amplification section downstream of the second dialysis section, the first nucleic acid amplification section being configured for amplifying the target nucleic acid sequences in the mitochondrial DNA.
• GDA007.10 Preferably, the microfluidic device also has a second nucleic acid amplification section downstream of the second dialysis section, the second nucleic acid amplification section being configured for amplifying the target nucleic acid sequences in the nuclear DNA.
• GDA007.11 Preferably, the microfluidic device also has a supporting substrate, CMOS circuitry, and a microsystems technologies (MST) layer which incorporates the first and second lysis sections, the first and second dialysis sections, the first and second nucleic acid amplification sections and the hybridization chamber array, wherein the CMOS circuitry is positioned between the MST layer and the supporting substrate, and the array of photodiodes is incorporated in the CMOS circuitry.
• GDA007.12 Preferably, the probes each have a nucleic acid sequence complementary to one of the target nucleic acid sequences, and an electrochemiluminescent (ECL) luminophore, and the hybridization chambers each have electrodes for generating an excited state in the ECL luminophore in which the ECL luminophore emits photons of light.
• GDA007.13 Preferably, the first and second nucleic acid amplification sections are first and second polymerase chain reaction (PCR) sections for amplifying the target nucleic acid sequences prior to hybridization with the ECL probes.
• GDA007.14 Preferably, the MST layer has a plurality of MST channels configured to draw fluid containing the target nucleic acid sequences through the PCR section and into the hybridization chambers by capillary action.
• GDA007.15 Preferably, the CMOS circuitry has bond-pads for electrical connection to an external device wherein the CMOS circuitry is configured to convert output from the photodiodes into a signal indicative of the ECL probes that hybridized with the target nucleic acid sequences, and provide the signal to the bond-pads for transmission to the external device.
• GDA007.16 Preferably, the probes each have a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.
• GDA007.17 Preferably, the probe is configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.
• GDA007.18 Preferably, the CMOS circuitry is configured to provide an electrical pulse to the electrodes, the electrical pulse having a duration less than 0.69 seconds.
• GDA007.19 Preferably, the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.
• GDA007.20 Preferably, the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
• The easily usable, mass-producible, and inexpensive genetic analysis LOC device accepts a biochemical sample, uses dialysis sections and differential lysing to separate the nuclear DNA and mitochondrial DNA of the cells in the sample, separately amplifies the nuclear DNA and the mitochondrial DNA, and separately analyzes the sample's nuclear DNA and mitochondrial DNA sequences via hybridization with oligonucleotide probes with sensing via its integral imaging array.
• Separate analysis of the sample's nuclear DNA and mitochondrial DNA extracts additional information from the sample and increases the sensitivity, signal-to-noise ratio, and dynamic range of the assay system.
• GPK001.1 This aspect of the invention provides a microfluidic test module comprising:
• an outer casing for hand held portability; and,
• a microfluidic device mounted within the outer casing for processing a biological sample, the outer casing providing a microenvironment adjacent the microfluidic device; wherein,
• the outer casing has a membrane seal of flexible material between the microenvironment and atmosphere for reducing pressure changes in the microenvironment resulting from atmospheric pressure fluctuations.
• GPK001.2 Preferably, the outer casing has a guard for protecting the membrane seal from damaging contact.
• GPK001.3 Preferably, the microfluidic test module also has a sensor and a functional unit for adjusting the microenvironment in response to feedback from the sensor.
• GPK001.4 Preferably, the sensor and the functional unit are fabricated in the microfluidic device.
• GPK001.5 Preferably, the sensor is a humidity sensor and the functional unit is a humidifier.
• GPK001.6 Preferably, the microfluidic device is a lab-on-a-chip (LOC) device and a cap, the LOC device having a supporting substrate and a microsystems technologies (MST) layer on the supporting substrate, the MST layer incorporating MST channels and a plurality of fluidic connections for fluid communication with the cap, and the cap having a sample inlet and cap channels for fluid communication with the fluidic connections.
• GPK001.7 Preferably, the humidifier has a water reservoir and an evaporator for exposing water supplied by the water reservoir to the area encompassing the MST layer and increasing the vapor pressure of the water in the area.
• GPK001.8 Preferably, the evaporator has an aperture configured to retain the water with a meniscus pinned at the aperture, the evaporator also having a heater adjacent the aperture for raising the temperature of the water at the aperture.
• GPK001.9 Preferably, the heater is annular and positioned about the aperture.
• GPK001.10 Preferably, the evaporator has a supply channel leading from the water reservoir to the aperture, the supply channel being configured to draw water to the aperture by capillary action.
• GPK001.11 Preferably, the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
• GPK001.12 Preferably, the water reservoir is formed in the cap and the supply channel is formed in the MST layer such that the water reservoir connects to the supply channel via one of the plurality of fluidic connections, and the aperture is formed in the cap such that the supply channel connects to the aperture via another of the fluidic connections.
• GPK001.13 Preferably, the cap has a plurality of reagent reservoirs for different reagents.
• GPK001.14 Preferably, the outer casing has a receptacle for receiving an unprocessed biological sample through an opening, and a cover movable between open and closed positions, the cover exposing the opening when in the open position and closing the opening when in the closed position, the receptacle being configured for fluid communication with the sample inlet such that the biological sample flows to the sample inlet by capillary action.
• GPK001.15 Preferably, the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
• GPK001.16 Preferably, the MST layer incorporates heaters for heating fluid in the MST channels.
• GPK001.17 Preferably, the MST channels each have a cross-sectional area between 1 square micron and 400 square microns for biochemical processing of constituents within the biological sample and the cap channels each have a cross-sectional area greater than 400 square microns for receiving the biological sample and transporting cells within the biological sample to predetermined sites in the MST channels.
• GPK001.18 Preferably, the outer casing has a lancet for finger pricking a patient to obtain a blood sample for insertion into the receptacle.
• GPK001.19 Preferably, the lancet is movable between a retracted and extended position, and the outer casing has a biasing mechanism to bias the lancet into the extended position and a user actuated catch for retaining the lancet in the retracted position until user actuation.
• GPK001.20 Preferably, the LOC device has a hybridization section with probe nucleic acid sequences, the hybridization section being configured for detecting hybridization of target nucleic acid sequences in the cells of the sample fluid and the probe nucleic acid sequences.
• The easily usable, mass-producible, inexpensive, and portable microfluidic test module accepts a fluid sample and processes and analyzes the sample. The requisite fluidic propulsion on the modules fluidic devices being provided via capillary action, with pressure-relief in the module's microenvironment being provided via a flexible membrane covering a pressure relief port.
• The capillary effect propulsion maintains the low system component-count, low system complexity, and simple manufacturing procedures, further reducing the system cost. The flexible membrane pressure relief system also maintains the low system component-count, low system complexity, and simple manufacturing procedures, further reducing the system cost.
• GMO001.1 This aspect of the invention provides a fabrication system for lab-on-a-chip (LOC) devices with differing application specific functionality, the fabrication system comprising:
• a database of different functional categories, each of the functional categories having a plurality of functional section designs;
• means for selecting a compilation of the functional section designs to generate a LOC design in accordance with a specific functionality intended for LOC devices fabricated in accordance with the LOC design; and,
• a MST (microsystems technology) fabrication facility for fabricating LOC devices in accordance with the LOC design; wherein,
• the functional section designs in any one of the functional categories are functionally compatible with at least one of the functional section designs in the other functional categories.
• GMO001.2 Preferably, the LOC devices are microfluidic devices for genetic analysis of a biological sample.
• GMO001.3 Preferably, the specific functionality of the microfluidic device is one or more of:
• identifying at least one pathogen present within the biological sample;
• identifying at least one virus present within the biological sample;
• identifying at least one bacterium present within the biological sample;
• identifying at least one target nucleic acid sequence present in DNA within the biological sample;
• identifying at least one target nucleic acid sequence present in RNA within the biological sample; and,
• identifying at least one target protein present within the biological sample.
• GMO001.4 Preferably, the biological sample to be genetically analysed is:
• blood;
• saliva;
• sperm;
• amplicon from a nucleic acid amplification process; or,
• epithelial cells.
• GMO001.5 Preferably, the functional categories include one or more of:
• a dialysis category;
• a valve category;
• a lysis category;
• an incubation category;
• a sensor category;
• a reagent category;
• a probe assay category;
• an amplification category; and,
• a hybridization detection category.
• GMO001.6 Preferably, the dialysis category includes one or more of: a pathogen dialysis section for removing leukocytes from a biological sample, and a leukocyte dialysis section for removing erythrocytes and pathogens from a biological sample.
• GMO001.7 Preferably, the lysis category includes one or more of: a thermal lysis section for thermally lysing cells in a biological sample, a chemical lysis section for chemically lysing cells in a biological sample, and a combination chemical and thermal lysis section for both chemically and thermally lysing cells in a biological sample.
• GMO001.8 Preferably, the valve category includes one or more of: a boiling-initiated valve, bend actuator valve, surface tension valve, stiction valve, electroexplosive valve, thermal-bend-actuated bend-and-break valve, bubble break valve and multi-valve array designs.
• GMO001.9 Preferably, the amplification category includes one or more of: a PCR section for combined amplification of all genetic material, a tandem PCR section for separately and sequentially amplifying with different primer pair sets, a parallel PCR section for separately and simultaneously amplifying with different primer pair sets, and an isothermal amplification section.
• GMO001.10 Preferably, the hybridization detection category includes one or more of: a heated hybridization chamber array, a non-heated hybridization chamber array, a heated proteomic chamber array, a non-heated proteomic chamber array, a single photodiode per chamber, a dual photodiode per chamber, a single probe type, a positive and negative control probe chamber.
• GMO001.11 Preferably, the sensor category includes one or more of: temperature sensors, liquid sensors, end-point liquid sensors, flow rate sensors, capillary meniscus marching velocity sensors and conductivity sensors.
• GMO001.12 Preferably, the reagent category includes one or more of: anticoagulant, restriction enzymes, ligase and linker primers, lysis reagent, amplification mix including buffer, dNTPs, and primers, polymerase and reverse transcriptase.
• GMO001.13 Preferably, the probe assay category includes one or more of: fluorescent probes, electrochemiluminescent (ECL) probes, hydrolysis probes, stem-and-loop probes, primer-linked linear probes and primer-linked stem-and-loop probes.
• GMO001.14 Preferably:
• the specific functionality of the LOC device is identifying at least one pathogen present within the biological sample;
• the LOC comprises at least one valve from the valve category and at least one sensor from the sensor category; and,
• the specific functional sections of the LOC device include a section chosen from the dialysis category, a reagent chosen from the reagent category, a section chosen from the amplification section, and at least one section chosen from the hybridization detection category.
• GMO001.15 Preferably, the reagent includes: amplification mix including buffer, dNTPs, and primers; polymerase; and anticoagulant.
• GMO001.16 Preferably:
• the at least one valve is a boiling-initiated valve;
• the section chosen from the dialysis category is a pathogen dialysis section;
• the section chosen from the amplification section is a parallel PCR section for separately and simultaneously amplifying with different primer pair sets; and,
• the at least one section chosen from the hybridization detection category comprises a heated hybridization chamber array.
• GMO001.17 Preferably, the heated hybridization chamber array contains a probe assay chosen from the probe assay category.
• GMO001.18 Preferably, the probe assay is ECL probes.
• GMO001.19 Preferably, the at least one sensor includes a temperature sensor and a liquid sensor.
• GMO001.20 Preferably, the at least one section chosen from the hybridization detection category further includes a single photodiode per chamber.
• The system for fabrication of easily usable, mass-producible, and inexpensive LOC devices provides for reliable, fast, easy, and low-cost compilation of application-specific/application-optimized LOC device designs using a library of compatible microfluidic and microelectronic subsystems.
• GMV001.1 This aspect of the invention provides a reagent microvial for a reagent dispensing apparatus, the microvial comprising:
• a container for holding a volume of reagent;
• a digital memory for data relating to the reagent;
• a droplet generator for ejecting droplets of the reagent from the container; and,
• electrical contacts for connection to a control processor in the reagent dispensing apparatus to receive drive pulses for the droplet generator and provide the data to the control processor.
• GMV001.2 Preferably, the container holds between 282 microliters and 400 microliters of reagent.
• GMV001.3 Preferably, the droplet generator is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GMV001.4 Preferably, the droplet generator has a piezo-electric actuator.
• GMV001.5 Preferably, the data includes an identity transmitted to the control processor.
• GMV001.6 Preferably, the identity is a unique identity distinguishing the microvial from all other microvials.
• GMV001.7 Preferably, the data is encrypted.
• The reagent microvial with nonvolatile digital memory is used to receive a reagent, store it, and dispense it under digital control using a droplet generator. The droplet generator being part of the microvial provides for a self-contained and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, and reducing the cost of the automated manufacturing environment utilizing the microvial.
• The digital memory is used to store the information required during the functioning of the device in an automated manufacturing environment. The digital memory being part of the microvial, provides for an easily usable, reliable, safe, secure, and inexpensive approach to reagent dispensing in an automated manufacturing environment.
• GMV002.1 This aspect of the invention provides a reagent microvial for a reagent dispensing apparatus, the microvial comprising:
• a container for holding a volume of reagent;
• an integrated circuit with digital memory for data relating to an identity of the microvial; and,
• electrical contacts for connection to a control processor in the reagent dispensing apparatus to provide the data to the control processor for comparison with a list of authentic microvial identities.
• GMV002.2 Preferably, the microvial also has a droplet generator for ejecting droplets of the reagent from the container.
• GMV002.3 Preferably, the container holds between 282 microliters and 400 microliters of reagent.
• GMV002.4 Preferably, the droplet generator is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GMV002.5 Preferably, the droplet generator has a piezo-electric actuator.
• GMV002.6 Preferably, the identity is a unique identity distinguishing the microvial from all other microvials.
• GMV002.7 Preferably, the data is encrypted.
• The reagent microvial with authentication integrated circuit is used to receive a reagent, store it, and dispense it under digital control using a droplet generator. The droplet generator being part of the microvial provides for a self-contained and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, and reducing the cost of the automated manufacturing environment utilizing the microvial.
• The authentication integrated circuit is used to store the microvial authentication information used during the functioning of the device in an automated manufacturing environment. The authentication integrated circuit being part of the microvial, provides for an easily usable, reliable, safe, secure, and inexpensive approach to reagent dispensing in an automated manufacturing environment.
• GMV003.1 This aspect of the invention provides a reagent microvial for a reagent dispensing apparatus, the microvial comprising:
• a container for holding a volume of reagent;
• an integrated circuit with digital memory storing reagent data relating to specifications characterizing the reagent; and,
• electrical contacts for connection to a control processor in the reagent dispensing apparatus to provide the data to the control processor for download into devices supplied with the reagent.
• GMV003.2 Preferably, the microvial also has a droplet generator for ejecting droplets of the reagent from the container.
• GMV003.3 Preferably, the container holds between 282 microliters and 400 microliters of reagent.
• GMV003.4 Preferably, the droplet generator is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GMV003.5 Preferably, the droplet generator has a piezo-electric actuator.
• GMV003.6 Preferably, the identity is a unique identity distinguishing the microvial from all other microvials.
• GMV003.7 Preferably, the data is encrypted.
• The reagent microvial with nonvolatile digital memory is used to receive a reagent, store it, and dispense it under digital control using a droplet generator. The droplet generator being part of the microvial provides for a self-contained and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, and reducing the cost of the automated manufacturing environment utilizing the microvial.
• The digital memory is used to store the information required during the functioning of the device in an automated manufacturing environment. The information stored in the memory includes the reagent specification data written into the memory by segments of the automated manufacturing environment. This information gets read from this memory an utilized as required by other segments of the automated manufacturing environment. The digital memory being part of the microvial, provides for an easily usable, reliable, safe, secure, and inexpensive approach to reagent dispensing in an automated manufacturing environment.
• GMV004.1 This aspect of the invention provides an oligonucleotide microvial for an oligonucleotide dispensing apparatus, the microvial comprising:
• a container for holding a volume of reagent;
• an integrated circuit with digital memory storing oligonucleotide data relating to specifications characterizing the oligonucleotides; and,
• electrical contacts for connection to a control processor in the reagent dispensing apparatus to provide the data to the control processor for download into devices supplied with the oligonucleotides.
• GMV004.2 Preferably, the microvial also has a droplet generator for ejecting droplets of the oligonucleotides from the container.
• GMV004.3 Preferably, the container holds between 282 microliters and 400 microliters of reagent.
• GMV004.4 Preferably, the droplet generator is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GMV004.5 Preferably, the droplet generator has a piezo-electric actuator.
• GMV004.6 Preferably, the identity is a unique identity distinguishing the microvial from all other microvials.
• GMV004.7 Preferably, the data is encrypted.
• The oligonucleotide microvial with nonvolatile digital memory is used to receive a reagent, store it, and dispense it under digital control using a droplet generator. The droplet generator being part of the microvial provides for a self-contained and volumetrically and positionally precise oligonucleotide dispensing technique, simplifying the complexity, increasing the reliability, and reducing the cost of the automated manufacturing environment utilizing the microvial.
• The digital memory is used to store the information required during the functioning of the device in an automated manufacturing environment. The information stored in the memory includes the oligonucleotide specification data written into the memory by segments of the automated manufacturing environment. This information gets read from this memory an utilized as required by other segments of the automated manufacturing environment. The digital memory being part of the microvial, provides for an easily usable, reliable, safe, secure, and inexpensive approach to reagent dispensing in an automated manufacturing environment.
• GRD001.1 This aspect of the invention provides a reagent dispensing apparatus for loading reagents into a microfluidic device having a digital memory for data related to the reagents loaded into the microfluidic device, the reagent dispensing apparatus comprising:
• a plurality of reagent vials each of the vials having an integrated circuit with memory storing data regarding the reagent in the vial, and a droplet dispenser;
• a mounting surface for detachably mounting the microfluidic device for movement relative to the vials; and,
• a control processor for operative control of the vials and the mounting surface; wherein,
• the control processor is configured to activate the droplet dispenser of the vial selected, move the vial into registration with the microfluidic device and download the data from the integrated circuit to the digital memory of the microfluidic device.
• GRD001.2 Preferably, the reagent dispensing apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the microfluidic device.
• GRD001.3 Preferably, the vial is a microvial for holding between 282 microliters and 400 microliters.
• GRD001.4 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor and the digital memory of the microfluidic device.
• GRD001.5 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GRD001.6 Preferably, the reagent dispensing apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GRD001.7 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GRD001.8 Preferably, the microfluidic device is a lab-on-a-chip (LOC) device.
• GRD001.9 Preferably, the droplet dispenser has a piezo-electric actuator.
• GRD001.10 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GRD001.11 Preferably, the reagent dispensing apparatus also has facilities configured for applying a seal to the LOC device to close a plurality of reservoirs in which the reagents have been loaded.
• GRD001.12 Preferably, the LOC has a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• The reagent dispensing apparatus is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of microfluidic devices. The data automation provided by the reagent dispensing apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, and storage of the reagent data into the memory of the microfluidic device.
• The reagent dispensing apparatus provides for an automated and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the reagent dispensing apparatus provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GRD002.1 This aspect of the invention provides a reagent dispensing apparatus for loading reagents into a microfluidic device having a digital memory for data related to the reagents loaded into the microfluidic device, the reagent dispensing apparatus comprising:
• a plurality of reagent vials each of the vials having an integrated circuit with memory storing data regarding the reagent in the vial, and a droplet dispenser;
• a mounting surface for detachably mounting the microfluidic device for movement relative to the vials; and,
• a control processor for operative control of the vials and the mounting surface; wherein,
• the control processor is configured to automatically interrogate each of the integrated circuits to collect and store the data regarding the reagents in each of the vials.
• GRD002.2 Preferably, the control processor is configured to automatically activate the droplet dispenser of the vial selected, move the vial into registration with the microfluidic device and download information from the integrated circuit to the digital memory of the microfluidic device.
• GRD002.3 Preferably, the reagent dispensing apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the microfluidic device.
• GRD002.4 Preferably, the vial is a microvial for holding between 282 microliters and 400 microliters.
• GRD002.5 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor and the digital memory of the microfluidic device.
• GRD002.6 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GRD002.7 Preferably, the reagent dispensing apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GRD002.8 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GRD002.9 Preferably, the microfluidic device is a lab-on-a-chip (LOC) device.
• GRD002.10 Preferably, the droplet dispenser has a piezo-electric actuator.
• GRD002.11 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GRD002.12 Preferably, the LOC has a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• The reagent dispensing apparatus is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of microfluidic devices. The data automation provided by the reagent dispensing apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, and storage of the reagent data into the memory of the microfluidic device and in the reagent dispensing apparatus's computer memory.
• The reagent dispensing apparatus provides for an automated and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the reagent dispensing apparatus provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GRD003.1 This aspect of the invention provides a reagent dispensing apparatus for loading reagents into a fixed array of microfluidic devices, each microfluidic device having a digital memory for data related to the reagents loaded into the microfluidic device, the reagent dispensing apparatus comprising:
• a plurality of reagent vials each of the vials having an integrated circuit with memory storing data regarding the reagent in the vial, and a droplet dispenser;
• a mounting surface for detachably mounting the fixed array of microfluidic devices for movement relative to the vials; and,
• a control processor for operative control of the vials and the mounting surface; wherein,
• the control processor is configured to activate the droplet dispenser of the vial selected, move the vial into registration within one or more of the microfluidic devices within the fixed array and download the data from the integrated circuit to the digital memory of the one or more microfluidic devices.
• GRD003.2 Preferably, the fixed array of microfluidic devices is an array of lab-on-a-chip (LOC) devices mounted on a separable PCB (printed circuit board) wafer.
• GRD003.3 Preferably, the reagent dispensing apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the LOC device.
• GRD003.4 Preferably, the vial is a microvial for holding between 282 microliters and 400 microliters.
• GRD003.5 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor and the digital memory of the LOC device.
• GRD003.6 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GRD003.7 Preferably, the reagent dispensing apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GRD003.8 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GRD003.9 Preferably, the droplet dispenser has a piezo-electric actuator.
• GRD003.10 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GRD003.11 Preferably, the reagent dispensing apparatus also has facilities configured for applying a seal to the LOC device to close a plurality of reservoirs in which the reagents have been loaded.
• GRD003.12 Preferably, the LOC has a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• GRD003.13 Preferably, the control processor is configured to automatically interrogate each of the integrated circuits to collect and store the data regarding the reagents in each of the vials.
• The reagent dispensing apparatus is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of arrays of microfluidic devices mounted on PCB wafers. The data automation provided by the reagent dispensing apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, and storage of the reagent data into the memory of the microfluidic device.
• The reagent dispensing apparatus provides for an automated and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the reagent dispensing apparatus provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Dispensing of the reagents into the arrays of microfluidic devices mounted on PCB wafers speeds up and reduces the cost of the loading process, and by loading the reagents into the microfluidic devices after mounting the devices on the PCB wafer and soldering them, improves the chemical and physical integrity of the reagents.
• GRD004.1 This aspect of the invention provides a reagent dispensing apparatus for loading reagents into a silicon wafer on which an array of lab-on-a-chip (LOC) devices are fabricated, each LOC device having a digital memory for data related to the reagents loaded into the LOC device, the reagent dispensing apparatus comprising:
• a plurality of reagent vials each of the vials having an integrated circuit with memory storing data regarding the reagent in the vial, and a droplet dispenser;
• a mounting surface for detachably mounting the silicon wafer for movement relative to the vials; and,
• a control processor for operative control of the vials and the mounting surface; wherein,
• the control processor is configured to activate the droplet dispenser of the vial selected, move the vial into registration within one or more of the LOC devices on the silicon wafer and download the data from the integrated circuit to the digital memory of the one or more LOC devices.
• GRD004.2 Preferably, the silicon wafer is partially sawn in preparation for tessellation into individually separate LOC devices.
• GRD004.3 Preferably, the reagent dispensing apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the LOC device.
• GRD004.4 Preferably, the vial is a microvial for holding between 282 microliters and 400 microliters.
• GRD004.5 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor and the digital memory of the LOC device.
• GRD004.6 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GRD004.7 Preferably, the reagent dispensing apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GRD004.8 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GRD004.9 Preferably, the droplet dispenser has a piezo-electric actuator.
• GRD004.10 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GRD004.11 Preferably, the reagent dispensing apparatus also has facilities configured for applying a seal to the LOC device to close a plurality of reservoirs in which the reagents have been loaded.
• GRD004.12 Preferably, the LOC has a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• GRD004.13 Preferably, the control processor is configured to automatically interrogate each of the integrated circuits to collect and store the data regarding the reagents in each of the vials.
• The reagent dispensing apparatus is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of microfluidic devices on partial-depth sawn silicon wafers. The data automation provided by the reagent dispensing apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, and storage of the reagent data into the memory of the microfluidic device.
• The reagent dispensing apparatus provides for an automated and volumetrically and positionally precise reagent dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the reagent dispensing apparatus provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Dispensing of the reagents into microfluidic devices on partial-depth sawn silicon wafers speeds up the process of loading and reduces its cost.
• GPD001.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of oligonucleotide probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• a supporting substrate;
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid; and,
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively; wherein,
• the ejectors are configured to eject droplets containing the oligonucleotide probes from the corresponding reservoir onto the surface.
• GPD001.2 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD001.3 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD001.4 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD001.5 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD001.6 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD001.7 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD001.8 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD001.9 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD001.10 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD001.11 Preferably, the oligonucleotide spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD001.12 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD001.13 Preferably, the supporting substrate has a reservoir side and an ejector side opposite the reservoir side wherein the array of reservoirs is formed in the reservoir side and the array of ejectors is formed on the ejector side.
• GPD001.14 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD001.15 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface with a density greater than 1 droplet per square millimeter.
• GPD001.16 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface with a density greater than 8 droplets per square millimeter.
• GPD001.17 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface with a density greater than 60 droplets per square millimeter.
• GPD001.18 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface with a density between 500 droplets per square millimeter and 1500 droplets per square millimeter.
• GPD001.19 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface at a rate greater than 100 droplets per second.
• GPD001.20 Preferably, the array of ejectors is configured to eject droplets containing the oligonucleotide probes onto the surface at a rate greater than 1,400 droplets per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD003.1 This aspect of the invention provides a spotting device for contactless spotting of a lab-on-a-chip (LOC) device with oligonucleotide probes, the LOC device having an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, the spotting device comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid; and,
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively such that the ejectors eject droplets containing the oligonucleotide probes from the corresponding reservoir into one of the hybridization chambers; wherein,
• the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD003.2 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD003.3 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD003.4 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD003.5 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD003.6 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD003.7 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD003.8 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD003.9 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD003.10 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD003.11 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD003.12 Preferably, the spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD003.13 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD003.14 Preferably, the spotting device also has a supporting substrate having a reservoir side and an ejector side opposite the reservoir side wherein the array of reservoirs is formed in the reservoir side and the array of ejectors is formed on the ejector side.
• GPD003.15 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density greater than 1 probe spot per square millimeter.
• GPD003.16 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density greater than 8 probe spots per square millimeter.
• GPD003.17 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density greater than 60 probe spots per square millimeter.
• GPD003.18 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD003.19 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface at a rate greater than 100 probe spots per second.
• GPD003.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface at a rate greater than 1,400 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them into the hybridization chambers of LOC devices that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides into the hybridization chambers of the LOC devices, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications for storage into the memory of the LOC devices that are being spotted.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy. The large numbers of oligonucleotide reservoirs and ejectors available on the oligonucleotide spotting device also provide for a one-step spotting of each LOC device.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD004.1 This aspect of the invention provides a biochemical deposition device for contactless deposition of biochemicals on a surface, the biochemical deposition device comprising:
• an array of reservoirs for containing a plurality of biochemicals; and,
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively; wherein,
• the ejectors are configured to eject droplets containing the biochemical from the corresponding reservoir onto the surface.
• GPD004.2 Preferably, the biochemicals in the array of reservoirs are oligonucleotide probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, and the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes.
• GPD004.3 Preferably, the array of hybridization chambers is configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD004.4 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD004.5 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD004.6 Preferably, the ejector has a chamber for containing liquid with suspended oligonucleotide probes supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD004.7 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD004.8 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD004.9 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD004.10 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD004.11 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD004.12 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD004.13 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD004.14 Preferably, the biochemical deposition device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD004.15 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD004.16 Preferably, the biochemical deposition device also has a supporting substrate having a reservoir side and an ejector side opposite the reservoir side wherein the array of reservoirs is formed in the reservoir side and the array of ejectors is formed on the ejector side.
• GPD004.17 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 8 droplets per square millimeter.
• GPD004.18 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 60 droplets per square millimeter.
• GPD004.19 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density between 500 droplets per square millimeter and 1500 droplets per square millimeter.
• GPD004.20 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface at a rate greater than 100 droplets per second.
• The mass-producible and inexpensive biochemical deposition device is used as a part of a cost-effective automated mass-manufacturing environment. Biochemicals are loaded in the device's biochemical reservoirs, and the device deposits them onto a surface by ejecting the biochemicals from its biochemical reservoir onto the surfaces being deposited upon. The data automation provided by the biochemical deposition device includes automated computer-controlled dispensing of the biochemicals onto the surface being spotted, receiving the specifications of the biochemicals stored in its reservoirs, storing the biochemicals specifications in its digital memory, and transmitting of the biochemicals specifications to segments of the automated manufacturing environment.
• The biochemical deposition device provides for an automated, volumetrically and positionally precise, fast, and high-density biochemical deposition technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the biochemical deposition device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD005.1 This aspect of the invention provides a microsystems technology (MST) device for contactless spotting of oligonucleotide probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the MST device comprising:
• a monolithic substrate having a reservoir side and an ejector side opposite the reservoir side;
• an array of reservoirs formed in the reservoir side; and,
• an array of ejectors formed on the ejector side, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively; wherein,
• the ejectors are configured to eject droplets containing the oligonucleotide probes from the corresponding reservoir onto the surface.
• GPD005.2 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD005.3 Preferably, the ejector has a chamber for containing liquid with suspended oligonucleotide probes supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD005.4 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD005.5 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD005.6 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD005.7 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD005.8 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD005.9 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD005.10 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD005.11 Preferably, the MST device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD005.12 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD005.13 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs being configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD005.14 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD005.15 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD005.16 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density greater than 8 probe spots per square millimeter.
• GPD005.17 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density greater than 60 probe spots per square millimeter.
• GPD005.18 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD005.19 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface at a rate greater than 100 probe spots per second.
• GPD005.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the surface at a rate greater than 1,400 probe spots per second.
• The oligonucleotide spotting device is mass-produced inexpensively using microsystem technology (MST) and is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD006.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of oligonucleotide probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors overlaying the array of reservoirs, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively; wherein,
• the ejectors are configured to eject droplets containing the oligonucleotide probes from the corresponding reservoir onto the surface.
• GPD006.2 Preferably, the oligonucleotide spotting device also has a supporting substrate having a reservoir side and an ejector side opposite the reservoir side wherein the array of reservoirs is formed in the reservoir side and the array of ejectors is formed on the ejector side.
• GPD006.3 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD006.4 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD006.5 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD006.6 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD006.7 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD006.8 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD006.9 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD006.10 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD006.11 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD006.12 Preferably, the oligonucleotide spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD006.13 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD006.14 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD006.15 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD006.16 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD006.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD006.18 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD006.19 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD006.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device with laminar structure is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surfaces being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD007.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of oligonucleotide probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• a supporting substrate;
• an array of reservoirs on one side of the supporting substrate, the reservoirs configured for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors on the other side of the supporting substrate; and,
• a plurality of inlet channels for fluid communication between the reservoirs and the ejectors; wherein,
• the ejectors are configured to eject droplets containing the oligonucleotide probes from the corresponding reservoir onto the surface.
• GPD007.2 Preferably, each of the ejectors is configured for fluid communication with a corresponding one of the reservoirs respectively.
• GPD007.3 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD007.4 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD007.5 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD007.6 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD007.7 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD007.8 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD007.9 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD007.10 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD007.11 Preferably, each of the ejectors is in fluid communication with one of the reservoirs via more than one of the inlet channels.
• GPD007.12 Preferably, the oligonucleotide spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD007.13 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD007.14 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD007.15 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD007.16 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD007.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD007.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD007.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD007.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. It is fabricated with fluidics on both side of a silicon substrate, increasing the device integration level, reducing the device dimensions, and minimizing the device cost. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD008.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• a supporting substrate;
• an array of ejectors, each having an actuator for ejecting droplets of liquid containing the oligonucleotide probes;
• CMOS circuitry for providing each of the drop ejection actuators with a drive pulse for droplet ejection; and,
• bond-pads for electrically connecting the CMOS circuitry and an external microprocessor controller for operative control of the array of ejectors.
• GPD008.2 Preferably, the CMOS circuitry has a digital memory storing identity data for identifying the device to the external microprocessor controller.
• GPD008.3 Preferably, the oligonucleotide spotting device also has an array of reservoirs for containing the oligonucleotide probes suspended in a liquid, wherein the supporting substrate has a reservoir side and an ejector side opposite the reservoir side, the array of reservoirs being formed in the reservoir side and, the array of ejectors formed on the ejector side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively.
• GPD008.4 Preferably, the digital memory stores specification data for the oligonucleotide probes.
• GPD008.5 Preferably, each of the ejectors has a plurality of the actuators and a corresponding plurality of nozzles associated with each of the droplet ejection actuators respectively, such that actuation of one of the actuators ejects a droplet through the nozzle associated with said actuator.
• GPD008.6 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD008.7 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD008.8 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD008.9 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD008.10 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD008.11 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD008.12 Preferably, each of the ejectors have a chamber for containing the liquid for ejection from the nozzle, and a plurality of inlet channels extending from the chamber to the reservoir corresponding to the ejector.
• GPD008.13 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD008.14 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD008.15 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD008.16 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD008.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD008.18 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD008.19 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD008.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment. The spotting device performs these functions under external computer control.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD009.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• a supporting substrate;
• an array of ejectors, each having an actuator for ejecting droplets of liquid containing the oligonucleotide probes; and,
• CMOS circuitry for providing each of the drop ejection actuators with a drive pulse for droplet ejection; wherein,
• the CMOS circuitry has a digital memory for storing data related to the device.
• GPD009.2 Preferably, the CMOS circuitry has bond-pads for electrically connecting and an external microprocessor controller for operative control of the array of ejectors.
• GPD009.3 Preferably, the data includes identity data for identifying the device to the external microprocessor controller.
• GPD009.4 Preferably, the oligonucleotide spotting device also has an array of reservoirs for containing the oligonucleotide probes suspended in a liquid, wherein the supporting substrate has a reservoir side and an ejector side opposite the reservoir side, the array of reservoirs being formed in the reservoir side and, the array of ejectors formed on the ejector side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively.
• GPD009.5 Preferably, the digital memory stores specification data for the oligonucleotide probes.
• GPD009.6 Preferably, each of the ejectors has a plurality of the actuators and a corresponding plurality of nozzles associated with each of the droplet ejection actuators respectively, such that actuation of one of the actuators ejects a droplet through the nozzle associated with said actuator.
• GPD009.7 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD009.8 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD009.9 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD009.10 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD009.11 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD009.12 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD009.13 Preferably, each of the ejectors have a chamber for containing the liquid for ejection from the nozzle, and a plurality of inlet channels extending from the chamber to the reservoir corresponding to the ejector.
• GPD009.14 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD009.15 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD009.16 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD009.17 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD009.18 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD009.19 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD009.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD010.1 This aspect of the invention provides a spotting device for contactless spotting of oligonucleotide probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the spotting device comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors in fluid communication with the reservoirs, each having an actuator for ejecting droplets of liquid containing the oligonucleotide probes; and,
• CMOS circuitry for providing each of the drop ejection actuators with a drive pulse for droplet ejection; wherein,
• the CMOS circuitry has a digital memory.
• GPD010.2 Preferably, the CMOS circuitry has bond-pads for electrically connecting and an external microprocessor controller for operative control of the array of ejectors.
• GPD010.3 Preferably, the digital memory stores identity data for identifying the device to the external microprocessor controller.
• GPD010.4 Preferably, the oligonucleotide spotting device also has a supporting substrate with a reservoir side and an ejector side opposite the reservoir side, the array of reservoirs being formed in the reservoir side and, the array of ejectors formed on the ejector side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively.
• GPD010.5 Preferably, the digital memory stores specification data for the oligonucleotide probes.
• GPD010.6 Preferably, each of the ejectors has a plurality of the actuators and a corresponding plurality of nozzles associated with each of the droplet ejection actuators respectively, such that actuation of one of the actuators ejects a droplet through the nozzle associated with said actuator.
• GPD010.7 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD010.8 Preferably, the ejectors are configured to eject droplets having a volume less than 2.0 picoliters.
• GPD010.9 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD010.10 Preferably, each of the ejectors have a chamber for containing the liquid for ejection from the nozzle, and a plurality of inlet channels extending from the chamber to the reservoir corresponding to the ejector.
• GPD010.11 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD010.12 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD010.13 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD010.14 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD010.15 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD010.16 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD010.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD010.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD010.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD010.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD011.1 This aspect of the invention provides a spotting device for contactless spotting of lab-on-a-chip (LOC) devices with oligonucleotide probes, the LOC devices being held in a fixed array on a printed circuit board (PCB) and each having an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the spotting device comprising:
• a monolithic supporting substrate;
• an array of reservoirs on one side of the supporting substrate, the reservoirs containing sufficient amount of the oligonucleotide probes suspended in a liquid to spot all the LOC devices on the PCB; and,
• an array of ejectors on the other side of the supporting substrate, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively such that the ejectors eject droplets containing the oligonucleotide probes from the corresponding reservoir into one of the hybridization chambers.
• GPD011.2 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD011.3 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD011.4 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD011.5 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD011.6 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD011.7 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD011.8 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD011.9 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD011.10 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD011.11 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD011.12 Preferably, the spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD011.13 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD011.14 Preferably, the array of reservoirs is integrally formed into the one side of the monolithic supporting substrate.
• GPD011.15 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD011.16 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD011.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD011.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD011.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD011.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them into the hybridization chambers of the arrays of LOC devices mounted on PCB wafers. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled spotting with oligonucleotide of the arrays of LOC devices mounted on PCB wafers, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to the LOC devices or other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Spotting with oligonucleotide of the arrays of LOC devices mounted on PCB wafers speeds up and reduces the cost of the loading process, and spotting the LOC devices after mounting them on the PCB wafers and soldering them, improves the chemical and physical integrity of the oligonucleotide.
• GPD012.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of a silicon wafer on which an array of lab-on-a-chip (LOC) devices are fabricated, the LOC devices being configured to use the oligonucleotide probes to detect target nucleic acid sequences in a biological sample and each having an array of hybridization chambers for receiving the oligonucleotide probes, the oligonucleotide spotting device comprising:
• an array of reservoirs on one side of the supporting substrate, the reservoirs containing sufficient amount of the oligonucleotide probes suspended in a liquid to spot all the LOC devices on the wafer; and,
• an array of ejectors overlaying the array of reservoirs for fixed movement therewith, such that the ejectors eject droplets containing the oligonucleotide probes from the corresponding reservoir into one of the hybridization chambers.
• GPD012.2 Preferably, the oligonucleotide spotting device also has a supporting substrate having a reservoir side and an ejector side opposite the reservoir side wherein the array of reservoirs is formed in the reservoir side and the array of ejectors is formed on the ejector side.
• GPD012.3 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD012.4 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD012.5 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD012.6 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD012.7 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD012.8 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD012.9 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD012.10 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD012.11 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD012.12 Preferably, the oligonucleotide spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD012.13 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD012.14 Preferably, the CMOS circuitry is between the array of reservoirs and the array of ejectors.
• GPD012.15 Preferably, the LOC device has an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD012.16 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD012.17 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD012.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD012.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD012.20 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them into the hybridization chambers of the arrays of LOC devices on partial-depth sawn wafers. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled spotting with oligonucleotide of the arrays of LOC devices on partial-depth sawn wafers, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to the LOC devices or other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Spotting with oligonucleotide of the arrays of LOC devices on partial-depth sawn wafers speeds up and reduces the cost of the loading process.
• GPD013.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• a monolithic supporting substrate;
• an array of ejectors formed on one side of the supporting substrate such that the ejectors eject droplets containing the oligonucleotide probes onto the surface; wherein during use,
• each of the ejectors in the array is configured to eject droplets having a volume less than 100 picoliters.
• GPD013.2 Preferably, each of the ejectors in the array is configured to eject droplets having a volume less than 25 picoliters.
• GPD013.3 Preferably, each of the ejectors in the array is configured to eject droplets having a volume less than 6 picoliters.
• GPD013.4 Preferably, each of the ejectors in the array is configured to eject droplets having a volume between 0.1 picoliter and 1.6 picoliters.
• GPD013.5 Preferably, the monolithic supporting substrate has a reservoir side and an ejection side opposite the reservoir side, the array of ejectors being formed on the ejection side and an array of reservoirs formed in the reservoir side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively.
• GPD013.6 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD013.7 Preferably, the ejector has a chamber for containing the liquid supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD013.8 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD013.9 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD013.10 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD013.11 Preferably, the oligonucleotide spotting device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a control microprocessor operatively controlling relative movement between the nozzles and the surface to be spotted with the probes.
• GPD013.12 Preferably, the CMOS circuitry has memory for storing specification data relating to the probes in the reservoirs.
• GPD013.13 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD013.14 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD013.15 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD013.16 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD013.17 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD013.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• GPD013.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate between 300,000 probe spots per second and 1,000,000 probe spots per second.
• GPD013.20 Preferably, the array of reservoirs are integrally formed in the reservoir side of the monolithic substrate.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy. In particular, the capability of the device to spot the requisite low-volume probes provides for low probe cost, in turn, permitting the inexpensive assay system.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD014.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• an array of ejectors, each having an actuator for ejecting droplets of liquid containing the probes;
• CMOS circuitry for providing each of the actuators with a drive pulse for droplet ejection; wherein during use,
• the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD014.2 Preferably, the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD014.3 Preferably, the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• GPD014.4 Preferably, the array of ejectors spot the probes onto the surface at a rate between 300,000 probe spots per second and 1,000,000 probe spots per second.
• GPD014.5 Preferably, the CMOS circuitry has bond-pads for connection to an external control microprocessor for operative control of the array of ejectors.
• GPD014.6 Preferably, the CMOS circuitry has a digital memory storing identity data for identifying the device to the external microprocessor controller.
• GPD014.7 Preferably, the digital memory stores probe type data and probe location data, the probe type data identifying the probe types in the device and the probe location data identifying the reservoir location for each of the probe types.
• GPD014.8 Preferably, the oligonucleotide spotting device also has a supporting substrate, the supporting substrate has a reservoir side and an ejector side opposite the reservoir side, the array of ejectors being formed on the ejector side and an array of reservoirs being formed in the reservoir side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively for ejecting droplets containing the probes from the corresponding reservoir onto the surface.
• GPD014.9 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD014.10 Preferably, the ejector has a chamber for containing liquid supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD014.11 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD014.12 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD014.13 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD014.14 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD014.15 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD014.16 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD014.17 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD014.18 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• GPD014.19 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate between 300,000 probe spots per second and 1,000,000 probe spots per second.
• GPD014.20 Preferably, the array of reservoirs are integrally formed in the reservoir side of the monolithic substrate.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The high spotting rate of the device, in turn, provides a high spotting throughput and reduces the overall cost of the product assay system. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD015.1 This aspect of the invention provides a biochemical deposition device for contactless deposition of biochemicals on a surface, the biochemical deposition device comprising:
• a supporting substrate;
• an array of reservoirs on one side of the substrate, the reservoirs being configured for containing a plurality of biochemicals; and,
• an array of ejectors on the other side of the supporting substrate, each of the ejectors being configured for fluid communication with the reservoirs; wherein during use,
• the array of ejectors eject droplets containing the biochemicals onto the surface at a rate greater than 100 droplets per second.
• GPD015.2 Preferably, the array of ejectors eject droplets containing the biochemicals onto the surface at a rate greater than 1,400 droplets per second.
• GPD015.3 Preferably, the array of ejectors eject droplets containing the biochemicals onto the surface at a rate greater than 20,000 droplets per second.
• GPD015.4 Preferably, the array of ejectors eject droplets containing the biochemicals onto the surface at a rate between 300,000 droplets per second and 1,000,000 droplets per second.
• GPD015.5 Preferably, the biochemicals in the array of reservoirs are oligonucleotide probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, and the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes.
• GPD015.6 Preferably, the array of hybridization chambers is configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD015.7 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD015.8 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD015.9 Preferably, the ejector has a chamber for containing liquid with suspended oligonucleotide probes supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD015.10 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD015.11 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD015.12 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPD015.13 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPD015.14 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPD015.15 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD015.16 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD015.17 Preferably, the biochemical deposition device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD015.18 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD015.19 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 8 droplets per square millimeter.
• GPD015.20 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 60 droplets per square millimeter.
• The mass-producible and inexpensive biochemical deposition device is used as a part of a cost-effective automated mass-manufacturing environment. Biochemicals are loaded in the device's biochemical reservoirs, and the device deposits them onto a surface by ejecting the biochemicals from its biochemical reservoir onto the surfaces being deposited upon. The data automation provided by the biochemical deposition device includes automated computer-controlled dispensing of the biochemicals onto the surface being spotted, receiving the specifications of the biochemicals stored in its reservoirs, storing the biochemicals specifications in its digital memory, and transmitting of the biochemicals specifications to segments of the automated manufacturing environment.
• The biochemical deposition device provides for an automated, volumetrically and positionally precise, fast, and high-density biochemical deposition technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The high deposition rate of the device, in turn, provides a high deposition throughput and reduces the overall product costs. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy.
• The data automation provided by the biochemical deposition device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD016.1 This aspect of the invention provides an oligonucleotide spotting device for contactless spotting of probes onto a surface, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, the oligonucleotide spotting device comprising:
• an array of ejectors, each having an actuator for ejecting droplets of liquid containing the probes;
• CMOS circuitry for providing each of the actuators with a drive pulse for droplet ejection; wherein,
• the array of ejectors is configured to spot the probes onto the surface at a density more than 1 probe spot per square millimeter.
• GPD016.2 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 8 probe spots per square millimeter.
• GPD016.3 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more than 60 probe spots per square millimeter.
• GPD016.4 Preferably, the array of ejectors is configured to spot the probes onto the surface at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPD016.5 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 100 probe spots per second.
• GPD016.6 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 1,400 probe spots per second.
• GPD016.7 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate greater than 20,000 probe spots per second.
• GPD016.8 Preferably, the CMOS circuitry is configured to generate drive pulses such the array of ejectors spot the probes onto the surface at a rate between 300,000 probe spots per second and 1,000,000 probe spots per second.
• GPD016.9 Preferably, the CMOS circuitry has bond-pads for connection to an external control microprocessor for operative control of the array of ejectors.
• GPD016.10 Preferably, the CMOS circuitry has a digital memory storing identity data for identifying the device to the external microprocessor controller.
• GPD016.11 Preferably, the digital memory stores probe type data and probe location data, the probe type data identifying the probe types in the device and the probe location data identifying the reservoir location for each of the probe types.
• GPD016.12 Preferably, the oligonucleotide spotting device also has a supporting substrate, the supporting substrate has a reservoir side and an ejector side opposite the reservoir side, the array of ejectors being formed on the ejector side and an array of reservoirs being formed in the reservoir side, each of the ejectors being configured for fluid communication with a corresponding one of the probe reservoirs respectively for ejecting droplets containing the probes from the corresponding reservoir onto the surface.
• GPD016.13 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD016.14 Preferably, the ejector has a chamber for containing liquid supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD016.15 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD016.16 Preferably, the actuators in one of the ejectors are configured to actuate individually.
• GPD016.17 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD016.18 Preferably, the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes, the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD016.19 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD016.20 Preferably, the array of reservoirs are integrally formed in the reservoir side of the monolithic substrate.
• The mass-producible and inexpensive oligonucleotide spotting device is used as a part of a cost-effective automated mass-manufacturing environment. Oligonucleotides are loaded in the device's oligonucleotides reservoirs, and the device ejects them onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting device includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, receiving the specifications of the oligonucleotides stored in its reservoirs, storing the oligonucleotide specifications in its digital memory, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting device provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy. In particular, the capability of the device to spot at the requisite high-density provides for low final product dimensions, in turn, permitting the inexpensive assay system.
• The data automation provided by the oligonucleotide spotting device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPD017.1 This aspect of the invention provides a biochemical deposition device for contactless deposition of biochemicals on a surface, the biochemical deposition device comprising:
• a supporting substrate;
• an array of reservoirs on one side of the substrate, the reservoirs being configured for containing a plurality of biochemicals; and,
• an array of ejectors on the other side of the supporting substrate, the ejectors being in fluid communication with the reservoirs, and configured to eject droplets containing the biochemicals onto the surface with a density greater than 1 droplet per square millimeter.
• GPD017.2 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 8 droplets per square millimeter.
• GPD017.3 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density greater than 60 droplets per square millimeter.
• GPD017.4 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface with a density between 500 droplets per square millimeter and 1500 droplets per square millimeter.
• GPD017.5 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface at a rate greater than 100 droplets per second.
• GPD017.6 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface at a rate greater than 1,400 droplets per second.
• GPD017.7 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface at a rate greater than 20,000 droplets per second.
• GPD017.8 Preferably, the array of ejectors is configured to eject droplets containing the biochemicals onto the surface at a rate between 300,000 droplets per second and 1,000,000 droplets per second.
• GPD017.9 Preferably, the biochemicals in the array of reservoirs are oligonucleotide probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample, and the surface is a lab-on-a-chip (LOC) device having an array of hybridization chambers for receiving the oligonucleotide probes.
• GPD017.10 Preferably, the array of hybridization chambers is configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPD017.11 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPD017.12 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPD017.13 Preferably, the ejector has a chamber for containing liquid with suspended oligonucleotide probes supplied from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPD017.14 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPD017.15 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPD017.16 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPD017.17 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPD017.18 Preferably, the biochemical deposition device also has CMOS circuitry for providing the actuators with drive pulses, the CMOS circuitry having bond-pads for connection to a microprocessor controller operatively controlling relative movement between the nozzles and the surface to be spotted with the oligonucleotide probes.
• GPD017.19 Preferably, the CMOS circuitry has memory for storing specification data related to the oligonucleotide probes in the reservoir.
• GPD017.20 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• The mass-producible and inexpensive biochemical deposition device is used as a part of a cost-effective automated mass-manufacturing environment. Biochemicals are loaded in the device's biochemical reservoirs, and the device deposits them onto a surface by ejecting the biochemicals from its biochemical reservoir onto the surfaces being deposited upon. The data automation provided by the biochemical deposition device includes automated computer-controlled dispensing of the biochemicals onto the surface being spotted, receiving the specifications of the biochemicals stored in its reservoirs, storing the biochemicals specifications in its digital memory, and transmitting of the biochemicals specifications to segments of the automated manufacturing environment.
• The biochemical deposition device provides for an automated, volumetrically and positionally precise, fast, and high-density biochemical deposition technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. The device also functions as the intermediate fluidic manipulation mechanism that is required for transferring fluids from a macroscopic level of volume and positioning accuracy to a microscopic level of volume and positioning accuracy. In particular, the capability of the device to deposit biochemicals at the requisite high-density provides for low final product dimensions, in turn, permitting the inexpensive product.
• The data automation provided by the biochemical deposition device provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GAL001.1 This aspect of the invention provides a robotic system for spotting oligonucleotides comprising:
• an oligonucleotide spotting device for contactless spotting of oligonucleotides onto a surface, the oligonucleotide spotting device having an array of ejectors, each having a drop ejection actuator for ejecting droplets of liquid containing the oligonucleotides onto a surface, at least one reservoir in fluid communication with one or more of the ejectors and CMOS drive circuitry for providing each of the drop ejection actuators with a drive pulse for droplet ejection; and,
• an apparatus for loading oligonucleotides into the oligonucleotide spotting device, the apparatus having a stage for detachably mounting a plurality of the oligonucleotide spotting devices, and a plurality of oligonucleotide containers mounted for movement relative to the stage, each of the oligonucleotide containers having a droplet dispenser for ejecting droplets of fluid containing oligonucleotides into the reservoirs of the oligonucleotide spotting devices.
• GAL001.2 Preferably, the oligonucleotide spotting device has bond-pads for electrically connecting the CMOS drive circuitry and the apparatus such that the apparatus downloads oligonucleotide data to memory within the CMOS drive circuitry.
• GAL001.3 Preferably, the apparatus has a camera for optically aligning the stage relative to the droplet dispensers.
• GAL001.4 Preferably, the oligonucleotide spotting device has a supporting substrate for supporting the CMOS circuitry, the supporting substrate having a reservoir side in which the at least one reservoir is formed and an ejector side opposite the reservoir side, in which the array of ejectors are formed.
• GAL001.5 Preferably, the oligonucleotide spotting device has an array of the reservoirs in the reservoir side, each of the reservoirs being in fluid communication with two or more of the ejectors.
• GAL001.6 Preferably, the oligonucleotides are configured to be probes for hybridization with target nucleic acid sequences in a biological sample, and the liquid is a probe solution such that each of the ejectors have a respective nozzle through which the drop ejection actuator ejects a droplet of the probe solution.
• GAL001.7 Preferably, the oligonucleotide spotting device has at least one common chamber for containing the fluid to be ejected by a plurality of the nozzles.
• GAL001.8 Preferably, each of the common chambers has a plurality of chamber inlets in fluid communication with the reservoir.
• GAL001.9 Preferably, the oligonucleotide spotting device has an array of the reservoirs and an array of the common chambers respectively in fluid communication with one of the reservoirs.
• GAL001.10 Preferably, the drop ejection actuators each have a heater for generating a vapor bubble in the fluid to eject a droplet through the nozzle corresponding to that drop ejection actuator.
• GAL001.11 Preferably, the ejectors are each configured to eject a droplet of the probe solution having a volume less than 100 picoliters.
• GAL001.12 Preferably, the ejectors are each configured to eject a droplet of the probe solution having a volume less than 25 picoliters.
• GAL001.13 Preferably, the ejectors are each configured to eject a droplet of the probe solution having a volume less than 6 picoliters.
• GAL001.14 Preferably, the ejectors are each configured to eject a droplet of the probe solution having a volume between 0.1 picoliters and 1.6 picoliters.
• GAL001.15 Preferably, the containers are vials for containing an aliquot of the probe solution, each of the vials having a quality assurance chip with memory for storing data identifying the oligonucleotides.
• GAL001.16 Preferably, the vials each have a droplet dispenser and electrical contacts for receiving an actuation signal to activate the droplet dispenser.
• GAL001.17 Preferably, the droplet dispenser has a piezoelectric actuator.
• GAL001.18 Preferably, the apparatus is configured for transferring data from the quality assurance chip of the vials to the CMOS circuitry of the oligonucleotide spotting device.
• GAL001.19 Preferably, the vials are suspended on a rack adjacent the oligonucleotide spotting devices on one side of the stage, the rack being configured to establish an individual electrical connection with each of the vials.
• The apparatus for loading of oligonucleotide spotting devices is used, as part of a cost-effective automated mass-manufacturing environment, to dispense oligonucleotides contained in oligonucleotide microvials into the oligonucleotide reservoirs of oligonucleotide spotting devices. The data automation provided by the apparatus includes automated computer-controlled dispensing of the oligonucleotides into the oligonucleotide reservoirs of the oligonucleotide spotting devices, checking the oligonucleotide data stored in the memory of the microvials against the list of specifications for the oligonucleotides that have to be loaded in the oligonucleotide spotting devices, and storage of the oligonucleotide data into the memory of the oligonucleotide spotting devices.
• The apparatus for loading of oligonucleotide spotting devices provides for an automated and volumetrically and positionally precise oligonucleotide dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the apparatus for loading of oligonucleotide spotting devices provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPA001.1 This aspect of the invention provides an oligonucleotide spotting robot for spotting oligonucleotide probes into a microfluidic device having a digital memory for data related to the oligonucleotide probes loaded into the microfluidic device, the oligonucleotide dispensing robot comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively;
• a mounting surface for detachably mounting the microfluidic device for movement relative to the ejectors; and,
• a control processor for operative control of the ejectors and the mounting surface; wherein,
• the control processor is configured to activate the ejectors, move the ejectors selected for activation into registration with the microfluidic device and download the data to the digital memory.
• GPA001.2 Preferably, the oligonucleotide spotting robot also has a camera for optical feedback of the registration between the ejectors selected by the control processor and the microfluidic device.
• GPA001.3 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPA001.4 Preferably, the oligonucleotide spotting robot also has CMOS circuitry between the array of reservoirs and the array of ejectors, the CMOS circuitry being configured to drive the array of ejectors in accordance with control signals from the control microprocessor, wherein the CMOS circuitry stores the data relating to the oligonucleotide probes.
• GPA001.5 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, and the array of ejectors is mounted closely adjacent to, and facing, the stage.
• GPA001.6 Preferably, the microfluidic device is a lab-on-a-chip (LOC) device, the LOC device having an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs being configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPA001.7 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPA001.8 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPA001.9 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPA001.10 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPA001.11 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPA001.12 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPA001.13 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPA001.14 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPA001.15 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPA001.16 Preferably, the array of ejectors is configured to spot the oligonucleotide probes onto the LOC device with a density greater than 1 probe spot per square millimeter.
• GPA001.17 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 8 probe spots per square millimeter.
• GPA001.18 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 60 probe spots per square millimeter.
• GPA001.19 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPA001.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device at a rate greater than 100 probe spots per second.
• The oligonucleotide spotting robot is used as part of a cost-effective automated mass-manufacturing environment. Loaded oligonucleotide spotting devices are picked up by the robot and the robot positions the oligonucleotide spotting devices and commands them to eject the oligonucleotides onto the surfaces that are being spotted. The data automation provided by the oligonucleotide spotting robot includes automated computer-controlled dispensing of the oligonucleotides onto the surface being spotted, reading the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices, checking the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices against the relevant databases, storing the oligonucleotide specifications in the memory of the devices that are being spotted, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting robot provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the oligonucleotide spotting robot provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GPA003.1 This aspect of the invention provides an oligonucleotide spotting robot for spotting oligonucleotide probes into an array of lab-on-a-chip (LOC) devices, each having a digital memory for data related to the oligonucleotide probes loaded into that LOC device, the oligonucleotide dispensing robot comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively;
• a mounting surface for detachably mounting the array of LOC devices for movement relative to the ejectors; and,
• a control processor for operative control of the ejectors and the mounting surface; wherein,
• the control processor is configured to activate the ejectors, move the ejectors selected for activation into registration with one or more of the LOC devices and download the data specifically relevant to each of the LOC devices into the digital memory of that LOC device.
• GPA003.2 Preferably, the oligonucleotide spotting robot also has a camera for optical feedback of the registration between the ejectors selected by the control processor and the LOC devices.
• GPA003.3 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPA003.4 Preferably, the oligonucleotide spotting robot also has CMOS circuitry between the array of reservoirs and the array of ejectors, the CMOS circuitry being configured to drive the array of ejectors in accordance with control signals from the control microprocessor, wherein the CMOS circuitry stores the data relating to the oligonucleotide probes.
• GPA003.5 Preferably, the array of LOC devices are mounted to a printed circuit board (PCB) which is in turn detachably mounted to the mounting surface, the mounting surface being a stage configured for movement along two orthogonal axes, and the array of ejectors is mounted closely adjacent to, and facing, the stage.
• GPA003.6 Preferably, each of the LOC devices has an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs being configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC devices.
• GPA003.7 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPA003.8 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPA003.9 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPA003.10 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPA003.11 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPA003.12 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPA003.13 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPA003.14 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPA003.15 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPA003.16 Preferably, the array of ejectors is configured to spot the oligonucleotide probes onto the LOC device with a density greater than 1 probe spot per square millimeter.
• GPA003.17 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 8 probe spots per square millimeter.
• GPA003.18 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 60 probe spots per square millimeter.
• GPA003.19 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPA003.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device at a rate greater than 100 probe spots per second.
• The oligonucleotide spotting robot is used as part of a cost-effective automated mass-manufacturing environment. Loaded oligonucleotide spotting devices are picked up by the robot and the robot positions the oligonucleotide spotting devices and commands them to eject the oligonucleotides into the hybridization chambers of the arrays of LOC devices mounted on PCB wafers. The data automation provided by the oligonucleotide spotting robot includes automated computer-controlled dispensing of the oligonucleotides into the hybridization chambers of the arrays of LOC devices mounted on PCB wafers, reading the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices, checking the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices against the relevant databases, storing the oligonucleotide specifications in the memory of the LOC devices that are being spotted, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting robot provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the oligonucleotide spotting robot provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Spotting with oligonucleotide of the arrays of LOC devices mounted on PCB wafers speeds up and reduces the cost of the loading process, and spotting the LOC devices after mounting them on the PCB wafers and soldering them, improves the chemical and physical integrity of the oligonucleotide.
• GPA004.1 This aspect of the invention provides an oligonucleotide spotting robot for spotting oligonucleotide probes into a silicon wafer on which an array of lab-on-a-chip (LOC) devices are fabricated, each LOC device having a digital memory for data related to the reagents loaded into the LOC device, the oligonucleotide dispensing robot comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively;
• a mounting surface for detachably mounting the array of LOC devices for movement relative to the ejectors; and,
• a control processor for operative control of the ejectors and the mounting surface; wherein,
• the control processor is configured to activate the ejectors, move the ejectors selected for activation into registration with one or more of the LOC devices and download the data specifically relevant to each of the LOC devices into the digital memory of that LOC device.
• GPA004.2 Preferably, the oligonucleotide spotting robot also has a camera for optical feedback of the registration between the ejectors selected by the control processor and the LOC devices.
• GPA004.3 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPA004.4 Preferably, the oligonucleotide spotting robot also has CMOS circuitry between the array of reservoirs and the array of ejectors, the CMOS circuitry being configured to drive the array of ejectors in accordance with control signals from the control microprocessor, wherein the CMOS circuitry stores the data relating to the oligonucleotide probes.
• GPA004.5 Preferably, the silicon wafer is partially sawn in preparation for dicing into individually separate LOC devices, and the silicon wafer being detachably mounted to the mounting surface, the mounting surface being a stage configured for movement along two orthogonal axes, and the array of ejectors is mounted closely adjacent to, and facing, the stage.
• GPA004.6 Preferably, each of the LOC devices has an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs being configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC devices.
• GPA004.7 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPA004.8 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPA004.9 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPA004.10 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPA004.11 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPA004.12 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPA004.13 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPA004.14 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPA004.15 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPA004.16 Preferably, the array of ejectors is configured to spot the oligonucleotide probes onto the LOC device with a density greater than 1 probe spot per square millimeter.
• GPA004.17 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 8 probe spots per square millimeter.
• GPA004.18 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density greater than 60 probe spots per square millimeter.
• GPA004.19 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPA004.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the LOC device at a rate greater than 100 probe spots per second.
• The oligonucleotide spotting robot is used as part of a cost-effective automated mass-manufacturing environment. Loaded oligonucleotide spotting devices are picked up by the robot and the robot positions the oligonucleotide spotting devices and commands them to eject the oligonucleotides into the hybridization chambers of the arrays of LOC devices on partial-depth sawn wafers. The data automation provided by the oligonucleotide spotting robot includes automated computer-controlled dispensing of the oligonucleotides into the hybridization chambers of the arrays of LOC devices on partial-depth sawn wafers, reading the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices, checking the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices against the relevant databases, storing the oligonucleotide specifications in the memory of the LOC devices that are being spotted, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting robot provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the oligonucleotide spotting robot provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• Spotting with oligonucleotide of the arrays of LOC devices on partial-depth sawn wafers speeds up and reduces the cost of the loading process.
• GPA005.1 This aspect of the invention provides an oligonucleotide spotting robot for spotting oligonucleotide probes onto a substrate, the oligonucleotide dispensing robot comprising:
• an array of reservoirs for containing the oligonucleotide probes suspended in a liquid;
• an array of ejectors, each of the ejectors being configured for fluid communication with a corresponding one of the reservoirs respectively;
• a mounting surface for detachably mounting the substrate for movement relative to the ejectors; and,
• a control processor for operative control of the ejectors and the mounting surface; wherein,
• the array of ejectors is configured to spot the probes onto the surface at a density more than 1 probe spot per square millimeter.
• GPA005.2 Preferably, the array of ejectors is configured to spot the probes onto the substrate at a density more than 8 probe spots per square millimeter.
• GPA005.3 Preferably, the array of ejectors is configured to spot the probes onto the substrate at a density more than 60 probe spots per square millimeter.
• GPA005.4 Preferably, the array of ejectors is configured to spot the probes onto the substrate at a density more between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• GPA005.5 Preferably, the substrate is a lab-on-a-chip (LOC) device with a digital memory for data related to the oligonucleotide probes loaded into the LOC device, and the control microprocessor is configured to activate the ejectors, move the ejectors selected for activation into registration with the LOC device and download the data to the digital memory.
• GPA005.6 Preferably, the oligonucleotide spotting robot also has a camera for optical feedback of the registration between the ejectors selected by the control processor and the LOC device.
• GPA005.7 Preferably, the array of reservoirs has more than 1000 reservoirs.
• GPA005.8 Preferably, the oligonucleotide spotting robot also has CMOS circuitry between the array of reservoirs and the array of ejectors, the CMOS circuitry being configured to drive the array of ejectors in accordance with control signals from the control microprocessor, wherein the CMOS circuitry stores the data relating to the oligonucleotide probes.
• GPA005.9 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, and the array of ejectors is mounted closely adjacent to, and facing, the stage.
• GPA005.10 Preferably, the LOC device has an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs being configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device.
• GPA005.11 Preferably, each of the ejectors has a plurality of nozzles, such that the ejector is configured to eject a droplet from each nozzle respectively.
• GPA005.12 Preferably, the ejector has a chamber for containing the liquid from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle.
• GPA005.13 Preferably, the actuators are thermal actuators, each configured to generate a vapor bubble in the liquid.
• GPA005.14 Preferably, the ejectors are configured to eject droplets having a volume less than 100 picoliters.
• GPA005.15 Preferably, the ejectors are configured to eject droplets having a volume less than 25 picoliters.
• GPA005.16 Preferably, the ejectors are configured to eject droplets having a volume less than 6 picoliters.
• GPA005.17 Preferably, the ejectors are configured to eject droplets having a volume between 0.1 picoliters and 1.6 picoliters.
• GPA005.18 Preferably, the actuators in each of the ejectors are configured to actuate individually.
• GPA005.19 Preferably, each of the ejectors has a plurality of inlet channels extending from the reservoir to the chamber.
• GPA005.20 Preferably, the array of ejectors is configured to spot the oligonucleotides onto the substrate at a rate greater than 100 probe spots per second.
• The oligonucleotide spotting robot is used as part of a cost-effective automated mass-manufacturing environment. Loaded oligonucleotide spotting devices are picked up by the robot and the robot positions the oligonucleotide spotting devices and commands them to eject the oligonucleotides into the hybridization chambers of LOC devices that are being spotted. The data automation provided by the oligonucleotide spotting robot includes automated computer-controlled dispensing of the oligonucleotides into the hybridization chambers of LOC devices that are being spotted, reading the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices, checking the specifications of the oligonucleotides stored in the reservoirs of the oligonucleotide spotting devices against the relevant databases, storing the oligonucleotide specifications in the memory of the LOC devices that are being spotted, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The oligonucleotide spotting robot provides for an automated, volumetrically and positionally precise, fast, and high-density oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment. In particular, the capability of the robot to spot at the requisite high-density provides for low final LOC device dimensions, in turn, permitting the inexpensive assay system.
• The data automation provided by the oligonucleotide spotting robot provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GSS001.1 This aspect of the invention provides a system for microarray spotting and genetic analysis, the system comprising:
• containers with probes for hybridization with different target nucleic acid sequences;
• an integrated circuit secured to each of the containers respectively, each of the integrated circuits have a container digital memory storing probe specification data related to probes in that container;
• a microfluidic device for supporting an array of probes selected from the containers, the selected probes corresponding to a desired genetic test assay, the microfluidic device having a device digital memory;
• an oligonucleotide spotting robot for spotting the selected probes onto the microfluidic device to form the array of probes, the oligonucleotide spotting robot having a control microprocessor for downloading the specification data to the device digital memory; and,
• a device reader for accessing the specification data from the device digital memory in order to analyze hybridization data from the microfluidic device.
• GSS001.2 Preferably, the microfluidic device has:
• a supporting substrate;
• a microsystems technologies (MST) layer for supporting the array of selected probes; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry having a photosensor for detecting hybridization of probes within the array of selected probes.
• GSS001.3 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GSS001.4 Preferably, the device digital memory also stores device identity data for uniquely identifying the microfluidic device.
• GSS001.5 Preferably, the system also has a test module in which the microfluidic device is mounted, the test module being configured for data transmission between the device digital memory and the reader.
• GSS001.6 Preferably, the test module connects to the reader via a universal serial bus (USB) connection.
• GSS001.7 Preferably, during use, the CMOS circuitry is powered by the reader via the USB connection.
• GSS001.8 Preferably, the test module also has an excitation light source for generating a fluorescence emission from the FRET probes which have hybridized with any of the target nucleic acid sequences.
• GSS001.9 Preferably, during use, the excitation light extinguishes prior to activation of the photosensor, and the CMOS circuitry is configured to delay activation of the photosensor for a predetermined period following deactivation of the excitation light source.
• GSS001.10 Preferably, the photosensor is less than 249 microns from the probes.
• GSS001.11 Preferably, the system also has a receptacle for receiving a biological sample containing the target nucleic acid sequences, the receptacle being in fluid communication with an inlet in the microfluidic device.
• GSS001.12 Preferably, the microfluidic device has a fluid flow-path from the inlet to the end point sensor, the fluid flow-path configured to bring the target nucleic acid sequences into contact with the array of probes by capillary action.
• GSS001.13 Preferably, the microfluidic device has a plurality of reagent reservoirs for different reagents required to process the biological sample.
• GSS001.14 Preferably, each of the container digital memories stores identity data distinguishing the container from others used to spot the microfluidic device, the control microprocessor configured to download the identity data to the device digital memory of the microfluidic device being spotted.
• GSS001.15 Preferably, the containers each have a droplet generator for ejecting droplets of a liquid suspension of the probes onto the microfluidic device.
• GSS001.16 Preferably, the system also has a mounting surface for mounting the microfluidic device for movement relative to the containers such that the control microprocessor controls both the containers and the mounting section to activate the droplet generator of the container selected and moved into registration with the microfluidic device.
• GSS001.17 Preferably, the system also has a camera for optical feedback of the registration between the container selected by the control microprocessor and the microfluidic device.
• GSS001.18 Preferably, the data stored in the container digital memory and the device digital memory is encrypted.
• GSS001.19 Preferably, the droplet generators are configured to eject droplets having a volume less than 6 picoliters.
• GSS001.20 Preferably, the array of selected probes contains more than 1000 probes in an area less than 1500 microns by 1500 microns.
• The system for variable microarray spotting and genetic analysis provides for automated, fast, easy, and low-cost compilation of easy-to-use and inexpensive application-specific/application-optimized assay systems, using a library of LOC devices, an oligonucleotide spotting device, an apparatus for loading of oligonucleotide spotting devices, an oligonucleotide spotting robot, and a library of oligonucleotide probes stored in microvials with digital memory. All the process steps from probe acceptance through LOC device spotting are automated.
• GSL001.1 This aspect of the invention provides a system for loading reagents into a microfluidic device for genetic analysis, the system comprising:
• containers with reagents for processing a biological sample in the microfluidic device;
• an integrated circuit secured to each of the containers respectively, each of the integrated circuits having a container digital memory storing reagent specification data related to the reagent in that container;
• a microfluidic device for performing a desired genetic test assay, the microfluidic device having a device digital memory;
• a reagent dispensing apparatus for loading a selection of the reagents into the microfluidic device, the reagent dispensing apparatus having a control microprocessor for accessing the container digital memory and transmitting the specification data to the device digital memory; and,
• a device reader for accessing the reagent specification data from the device digital memory in order to analyze test assay results from the microfluidic device.
• GSL001.2 Preferably, the microfluidic device has:
• a supporting substrate;
• a microsystems technologies (MST) layer for supporting an array of oligonucleotide probes for hybridization with target nucleic acid sequences in the biological sample; and,
• CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry having a photosensor for detecting hybridization within the array of probes.
• GSL001.3 Preferably, the probes are fluorescence resonance energy transfer (FRET) probes.
• GSL001.4 Preferably, the device digital memory also stores device identity data for uniquely identifying the microfluidic device.
• GSL001.5 Preferably, the system also has a test module in which the microfluidic device is mounted, the test module being configured for data transmission between the device digital memory and the reader.
• GSL001.6 Preferably, the test module connects to the reader via a universal serial bus (USB) connection.
• GSL001.7 Preferably, during use, the CMOS circuitry is powered by the reader via the USB connection.
• GSL001.8 Preferably, the test module also has an excitation light source for generating a fluorescence emission from the FRET probes which have hybridized with any of the target nucleic acid sequences.
• GSL001.9 Preferably, during use, the excitation light extinguishes prior to activation of the photosensor, and the CMOS circuitry is configured to delay activation the photosensor for a predetermined period following deactivation of the excitation light source.
• GSL001.10 Preferably, the photosensor is less than 249 microns from the probes.
• GSL001.11 Preferably, the system also has a receptacle for receiving the biological sample containing the target nucleic acid sequences, the receptacle being in fluid communication with an inlet in the microfluidic device.
• GSL001.12 Preferably, the microfluidic device has a fluid flow-path from the inlet to the end point sensor, the fluid flow-path configured to bring the target nucleic acid sequences into contact with the array of probes by capillary action.
• GSL001.13 Preferably, the microfluidic device has a plurality of reagent reservoirs for the reagents required to process the biological sample.
• GSL001.14 Preferably, each of the container digital memories stores identity data distinguishing the container from others used to spot the microfluidic device, the control microprocessor being configured to download the identity data to the device digital memory of the microfluidic device being spotted.
• GSL001.15 Preferably, the containers each have a droplet generator for ejecting droplets of the reagent into the reagent reservoirs.
• GSL001.16 Preferably, the system also has a mounting surface for mounting the microfluidic device for movement relative to the containers such that the control microprocessor controls both the containers and the mounting section to activate the droplet generator of the container selected and moved into registration with the microfluidic device.
• GSL001.17 Preferably, the system also has a camera for optical feedback of the registration between the container selected by the control microprocessor and the microfluidic device.
• GSL001.18 Preferably, the data stored in the container digital memory and the device digital memory is encrypted.
• GSL001.19 Preferably, the droplet generators are configured to eject droplets having a volume less than 6 picoliters.
• GSL001.20 Preferably, the array of probes contains more than 1000 probes in an area less than 1500 microns by 1500 microns.
• The system for variable LOC device reagent loading and genetic analysis provides for automated, fast, easy, and low-cost compilation of easy-to-use and inexpensive application-specific/application-optimized assay systems, using a library of LOC devices, a reagent dispensing apparatus, and a library of reagents stored in microvials with digital memory. All the process steps from reagent acceptance through LOC device loading are automated.
• GCA001.1 This aspect of the invention provides an apparatus for dispensing reagents and loading oligonucleotide spotting devices, the apparatus comprising:
• a plurality of reagent vials, each with a droplet dispenser;
• a plurality of oligonucleotide vials, each with a droplet dispenser;
• a mounting surface for detachably mounting a microfluidic device for movement relative to the reagent vials, and detachably mounting an oligonucleotide spotting device; and,
• a control processor for operative control of the reagent vials and oligonucleotide vials and movement of the mounting surface relative to the reagent vials and oligonucleotide vials; wherein,
• the control processor is configured to activate any of the droplet dispensers, move the microfluidic device into registration with the reagent vials and move the oligonucleotide spotting device into registration with the oligonucleotide vials.
• GCA001.2 Preferably, each of the reagent vials has an integrated circuit storing reagent specification data, each of the oligonucleotide vials has an integrated circuit storing oligonucleotide specification data, and the control processor is configured to download the reagent specification data to digital memory in the microfluidic device and download the oligonucleotide specification data to digital memory in the oligonucleotide spotting device.
• GCA001.3 Preferably, the apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the microfluidic device.
• GCA001.4 Preferably, the reagent vials and the oligonucleotide vials are microvials with a volume between 282 microliters and 400 microliters.
• GCA001.5 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor.
• GCA001.6 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GCA001.7 Preferably, the apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GCA001.8 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GCA001.9 Preferably, the droplet dispenser has a piezo-electric actuator.
• GCA001.10 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GCA001.11 Preferably, the microfluidic device is a LOC device for genetic analysis of a biological sample, the LOC device having a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• GCA001.12 Preferably, the apparatus also has a facility for applying a film to the LOC device to cover reagent reservoirs formed in an exterior surface.
• GCA001.13 Preferably, the LOC device is one of an array of LOC devices fabricated on a silicon wafer, the stage being configured to detachably mount the silicon wafer for loading reagents into all the LOC devices in the array.
• GCA001.14 Preferably, the LOC device is one of an array of LOC devices mounted on a printed circuit board (PCB), the stage being configured to detachably mount the PCB for loading reagents into all the LOC devices in the array.
• The combined reagent dispensing apparatus and apparatus for loading of oligonucleotide spotting devices is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of microfluidic devices and to dispense oligonucleotides contained in oligonucleotide microvials into the oligonucleotide reservoirs of oligonucleotide spotting devices. The data automation provided by the apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices and dispensing of the oligonucleotides into the oligonucleotide reservoirs of the oligonucleotide spotting devices, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, checking the oligonucleotide data stored in the memory of the microvials against the list of specifications for the oligonucleotides that have to be loaded in the oligonucleotide spotting devices, storage of the reagent data into the memory of the microfluidic device, and storage of the oligonucleotide data into the memory of the oligonucleotide spotting devices.
• The combined reagent dispensing apparatus and apparatus for loading of oligonucleotide spotting devices provides for an automated and volumetrically and positionally precise reagent and oligonucleotide dispensing technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the combined reagent dispensing apparatus and apparatus for loading of oligonucleotide spotting devices provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GCA002.1 This aspect of the invention provides an apparatus for dispensing reagents, loading oligonucleotide spotting devices and spotting oligonucleotide probes, the apparatus comprising:
• a plurality of reagent vials, each with a droplet dispenser;
• a plurality of oligonucleotide vials, each with a droplet dispenser;
• a mounting surface for detachably mounting a microfluidic device for movement relative to the reagent vials, and detachably mounting an oligonucleotide spotting device;
• a chuck for detachably mounting the oligonucleotide spotting device adjacent the mounting surface; and,
• a control processor for operative control of the reagent and oligonucleotide vials, the oligonucleotide spotting device when mounted in the chuck and movement of the mounting surface relative to the reagent and oligonucleotide vials, and the oligonucleotide spotting device; wherein,
• the control processor is configured to activate any of the droplet dispensers, move the microfluidic device into registration with the reagent vials and move the oligonucleotide spotting device into registration with the oligonucleotide vials.
• GCA002.2 Preferably, the control processor is configured to operate the oligonucleotide spotting device when in the chuck to spot oligonucleotide probes into the microfluidic device on the mounting surface.
• GCA002.3 Preferably, each of the reagent vials has an integrated circuit storing reagent specification data, each of the oligonucleotide vials has an integrated circuit storing oligonucleotide specification data, and the control processor is configured to download the reagent specification data to digital memory in the microfluidic device and download the oligonucleotide specification data to digital memory in the oligonucleotide spotting device.
• GCA002.4 Preferably, the apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the microfluidic device.
• GCA002.5 Preferably, the reagent vials and the oligonucleotide vials are microvials with a volume between 282 microliters and 400 microliters.
• GCA002.6 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor.
• GCA002.7 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GCA002.8 Preferably, the apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GCA002.9 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GCA002.10 Preferably, the droplet dispenser has a piezo-electric actuator.
• GCA002.11 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GCA002.12 Preferably, the microfluidic device is a LOC device for genetic analysis of a biological sample, the LOC device having a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• GCA002.13 Preferably, the apparatus also has a facility for applying a film to the LOC device to cover reagent reservoirs formed in an exterior surface.
• GCA002.14 Preferably, the LOC device is one of an array of LOC devices fabricated on a silicon wafer, the stage being configured to detachably mount the silicon wafer for loading reagents into all the LOC devices in the array.
• GCA002.15 Preferably, the LOC device is one of an array of LOC devices mounted on a printed circuit board (PCB), the stage being configured to detachably mount the PCB for loading reagents into all the LOC devices in the array.
• GCA002.16 Preferably, the oligonucleotide spotting device has an array of reservoirs for containing the oligonucleotide probes and an array of ejectors, and the LOC device has an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device, the control processor being configured to operate the ejectors to correctly spot the hybridization chamber array and download an association between the specification data for the oligonucleotide probes from each of the reservoirs, and array location data locating the hybridization chamber spotted by each of the reservoirs.
• GCA002.17 Preferably, each of the ejectors has a plurality of nozzles, a chamber for containing liquid with a suspension of the oligonucleotide probes from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle, the control processor being configured to operate each of the actuators individually.
• GCA002.18 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density greater than 8 probe spots per square millimeter.
• GCA002.19 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density greater than 60 probe spots per square millimeter.
• GCA002.20 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• The combined apparatus for reagent dispensing, loading of oligonucleotide spotting devices, and oligonucleotide spotting is used, as part of a cost-effective automated mass-manufacturing environment, to dispense reagents contained in reagent microvials into the reagent reservoirs of microfluidic devices, to dispense oligonucleotides contained in oligonucleotide microvials into the oligonucleotide reservoirs of oligonucleotide spotting devices, and to eject the oligonucleotides onto the surfaces that are being spotted. The data automation provided by the apparatus includes automated computer-controlled dispensing of the reagents into the reagent reservoirs of the microfluidic devices, dispensing of the oligonucleotides into the oligonucleotide reservoirs of the oligonucleotide spotting devices, dispensing of the oligonucleotides onto the surface being spotted, checking the reagent data stored in the memory of the microvials against the list of specifications for the reagents that have to be loaded in the microfluidic device, checking the oligonucleotide data stored in the memory of the microvials against the list of specifications for the oligonucleotides that have to be loaded in the oligonucleotide spotting devices, storing the reagent data into the memory of the microfluidic device, storing the oligonucleotide specifications in the memory of the devices that are being spotted, and transmitting of the reagent and oligonucleotide specifications to other segments of the automated manufacturing environment.
• The combined apparatus for reagent dispensing, loading of oligonucleotide spotting devices, and oligonucleotide spotting provides for an automated, volumetrically and positionally precise, fast, and high-density reagent dispensing and oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the combined apparatus for reagent dispensing, loading of oligonucleotide spotting devices, and oligonucleotide spotting provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
• GCA003.1 This aspect of the invention provides an apparatus for loading oligonucleotide spotting devices and spotting oligonucleotide probes, the apparatus comprising:
• a plurality of oligonucleotide vials, each with a droplet dispenser;
• a mounting surface for detachably mounting an oligonucleotide spotting device;
• a chuck for detachably mounting the oligonucleotide spotting device adjacent the mounting surface; and,
• a control processor for operative control of the oligonucleotide vials, the oligonucleotide spotting device when mounted in the chuck and movement of the mounting surface relative to the oligonucleotide vials, and the oligonucleotide spotting device; wherein,
• the control processor is configured to activate the droplet dispensers, and move the oligonucleotide spotting device into registration with the oligonucleotide vials.
• GCA003.2 Preferably, the control processor is configured to operate the oligonucleotide spotting device when in the chuck to spot oligonucleotide probes into a microfluidic device on the mounting surface.
• GCA003.3 Preferably, each of the oligonucleotide vials has an integrated circuit storing oligonucleotide specification data, and the control processor is configured to download the oligonucleotide specification data to digital memory in the oligonucleotide spotting device.
• GCA003.4 Preferably, the apparatus also has a camera for optical feedback of the registration between the vial selected by the control processor and the oligonucleotide spotting device.
• GCA003.5 Preferably, the oligonucleotide vials are microvials with a volume between 282 microliters and 400 microliters.
• GCA003.6 Preferably, the integrated circuit for each of the microvials has a unique identifier for identifying each of the microvials individually, the unique identifier being transmitted to the control processor.
• GCA003.7 Preferably, each of the microvials has electrical contacts for receiving activation pulses for the droplet dispenser and allowing the control processor to interrogate the integrated circuit.
• GCA003.8 Preferably, the apparatus also has a rack wherein the microvials are detachably mounted to the rack for mechanical and electronic control of the microvials.
• GCA003.9 Preferably, the mounting surface is a stage configured for movement along two orthogonal axes, the rack extending parallel to one if the orthogonal axes.
• GCA003.10 Preferably, the droplet dispenser has a piezo-electric actuator.
• GCA003.11 Preferably, the droplet dispenser is configured to eject droplets with a volume between 50 picoliters and 150 picoliters.
• GCA003.12 Preferably, the apparatus also has reagent vials containing reagents for processing a biological sample wherein the microfluidic device is a LOC device for genetic analysis of the biological sample, the LOC device having a polymerase chain reaction (PCR) section and the list of reagents has one or more of:
• water;
• polymerase;
• primers;
• buffer;
• anticoagulant;
• deoxyribonucleoside triphosphates (dNTPs);
• lysis reagent;
• ligase and linkers; and,
• restriction enzymes.
• GCA003.13 Preferably, the apparatus also has a facility for applying a film to the LOC device to cover reagent reservoirs formed in an exterior surface.
• GCA003.14 Preferably, the LOC device is one of an array of LOC devices fabricated on a silicon wafer, the stage being configured to detachably mount the silicon wafer for loading reagents into all the LOC devices in the array.
• GCA003.15 Preferably, the LOC device is one of an array of LOC devices mounted on a printed circuit board (PCB), the stage being configured to detachably mount the PCB for loading reagents into all the LOC devices in the array.
• GCA003.16 Preferably, the oligonucleotide spotting device has an array of reservoirs for containing the oligonucleotide probes and an array of ejectors, and the LOC device has an array of hybridization chambers for receiving the oligonucleotide probes, the probes having nucleic acid sequences that are complementary to target nucleic acid sequences to be identified in a biological sample and the array of hybridization chambers being configured to hold a complete assay of oligonucleotide probes necessary for a predetermined analysis of the biological sample, and the array of reservoirs is configured to contain the complete assay of oligonucleotide probes necessary for the predetermined analysis to be performed by the LOC device, the control processor being configured to operate the ejectors to correctly spot the hybridization chamber array and download an association between the specification data for the oligonucleotide probes from each of the reservoirs, and array location data locating the hybridization chamber spotted by each of the reservoirs.
• GCA003.17 Preferably, each of the ejectors has a plurality of nozzles, a chamber for containing liquid with a suspension of the oligonucleotide probes from the corresponding reservoir, and a plurality of actuators, one of the actuators corresponding to each of the nozzles respectively such that the actuator ejects a droplet of the liquid from the chamber through the corresponding nozzle, the control processor being configured to operate each of the actuators individually.
• GCA003.18 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density greater than 8 probe spots per square millimeter.
• GCA003.19 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density greater than 60 probe spots per square millimeter.
• GCA003.20 Preferably, the control processor is configured to operate the array of ejectors to spot the oligonucleotides onto the LOC device with a density between 500 probe spots per square millimeter and 1500 probe spots per square millimeter.
• The combined apparatus for loading of oligonucleotide spotting devices and oligonucleotide spotting is used, as part of a cost-effective automated mass-manufacturing environment, to dispense oligonucleotides contained in oligonucleotide microvials into the oligonucleotide reservoirs of oligonucleotide spotting devices and to eject the oligonucleotides onto the surfaces that are being spotted. The data automation provided by the apparatus includes automated computer-controlled dispensing of the oligonucleotides into the oligonucleotide reservoirs of the oligonucleotide spotting devices, dispensing of the oligonucleotides onto the surface being spotted, checking the oligonucleotide data stored in the memory of the microvials against the list of specifications for the oligonucleotides that have to be loaded in the oligonucleotide spotting devices, storing the oligonucleotide specifications in the memory of the devices that are being spotted, and transmitting of the oligonucleotide specifications to other segments of the automated manufacturing environment.
• The combined apparatus for loading of oligonucleotide spotting devices and oligonucleotide spotting provides for an automated, volumetrically and positionally precise, fast, and high-density and oligonucleotide spotting technique, simplifying the complexity, increasing the reliability, increasing the security, increasing the safety, and reducing the cost of the automated manufacturing environment.
• The data automation provided by the combined apparatus for loading of oligonucleotide spotting devices and oligonucleotide spotting provides for an automated, safe, secure, and inexpensive technique of data monitoring and management in the automated manufacturing environment.
BRIEF DESCRIPTION OF THE DRAWINGS
• Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:
• FIG. 1 shows a test module and test module reader configured for fluorescence detection;
• FIG. 2 is a schematic overview of the electronic components in the test module configured for fluorescence detection;
• FIG. 3 is a schematic overview of the electronic components in the test module reader;
• FIG. 4 is a schematic representation of the architecture of the LOC device;
• FIG. 5 is a perspective of the LOC device;
• FIG. 6 is a plan view of the LOC device with features and structures from all layers superimposed on each other;
• FIG. 7 is a plan view of the LOC device with the structures of the cap shown in isolation;
• FIG. 8 is a top perspective of the cap with internal channels and reservoirs shown in dotted line;
• FIG. 9 is an exploded top perspective of the cap with internal channels and reservoirs shown in dotted line;
• FIG. 10 is a bottom perspective of the cap showing the configuration of the top channels;
• FIG. 11 is a plan view of the LOC device showing the structures of the CMOS+MST device in isolation;
• FIG. 12 is a schematic section view of the LOC device at the sample inlet;
• FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;
• FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;
• FIG. 15 is an enlarged view of Inset AE shown in FIG. 13;
• FIG. 16 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 17 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 18 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 19 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 20 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 21 is a partial perspective illustrating the laminar structure of the LOC device within Inset AE;
• FIG. 22 is schematic section view of the lysis reagent reservoir shown in FIG. 21;
• FIG. 23 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 24 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 25 is a partial perspective illustrating the laminar structure of the LOC device within Inset AI;
• FIG. 26 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 27 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 28 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 29 is a partial perspective illustrating the laminar structure of the LOC device within Inset AB;
• FIG. 30 is a schematic section view of the amplification mix reservoir and the polymerase reservoir;
• FIG. 31 show the features of a boiling-initiated valve in isolation;
• FIG. 32 is a schematic section view of the boiling-initiated valve taken through line 33-33 shown in FIG. 31;
• FIG. 33 is an enlarged view of Inset AF shown in FIG. 15;
• FIG. 34 is a schematic section view of the upstream end of the dialysis section taken through line 35-35 shown in FIG. 33;
• FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;
• FIG. 36 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 37 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 38 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 39 is a further enlarged view within Inset AK shown in FIG. 38;
• FIG. 40 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 41 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 42 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 43 is a further enlarged view within Inset AL shown in FIG. 42;
• FIG. 44 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 45 is a further enlarged view within Inset AM shown in FIG. 44;
• FIG. 46 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 47 is a further enlarged view within Inset AN shown in FIG. 46;
• FIG. 48 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 49 is a further enlarged view within Inset AC showing the amplification chamber;
• FIG. 50 is a further enlarged view within Inset AC showing the amplification section;
• FIG. 51 is a schematic section view of the amplification section;
• FIG. 52 is an enlarged plan view of the hybridization section;
• FIG. 53 is a further enlarged plan view of two hybridization chambers in isolation;
• FIG. 54 is schematic section view of a single hybridization chamber;
• FIG. 55 is an enlarged view of the humidifier illustrated in Inset AG shown in FIG. 6;
• FIG. 56 is an enlarged view of Inset AD shown in FIG. 52;
• FIG. 57 is an exploded perspective view of the LOC device within Inset AD;
• FIG. 58 is a diagram of a FRET probe in a closed configuration;
• FIG. 59 is a diagram of a FRET probe in an open and hybridized configuration;
• FIG. 60 is a graph of the intensity of an excitation light over time;
• FIG. 61 is a diagram of the excitation illumination geometry of the hybridization chamber array;
• FIG. 62 is a diagram of a Sensor Electronic Technology LED illumination geometry;
• FIG. 63 is a schematic plan view of a reagent dispensing robot;
• FIG. 64 is a perspective of a reagent microvial with inbuilt droplet generator;
• FIG. 65 is a schematic plan view of an oligonucleotide ejector robot for loading selected probes into a probe ejector chip;
• FIG. 66 is a schematic plan view of a probe spotting robot for loading probes into the LOC devices on a partial-depth sawn silicon wafer;
• FIG. 67 is an enlarged plan view of the humidity sensor shown in Inset AH of FIG. 6;
• FIG. 68 shows the features of a first variant of a thermal bend actuated valve;
• FIG. 69 is a schematic section view of the first variant of a thermal bend actuated valve taken along line 70-70 of FIG. 68;
• FIG. 70 shows the features of a second variant of a thermal bend actuated valve;
• FIG. 71 is a schematic section view of the second variant of a thermal bend actuated valve taken along line 72-72 of FIG. 70;
• FIG. 72 shows the features of a third variant of a thermal bend actuated valve;
• FIG. 73 is a schematic section view of the third variant of a thermal bend actuated valve taken along line 74-74 of FIG. 72;
• FIG. 74 shows the features of a fault tolerant valve array;
• FIG. 75 is a schematic section view of the fault tolerant valve array taken along line 76-76 of FIG. 74;
• FIG. 76 is a schematic section view of a leukocyte target dialysis section;
• FIG. 77 is a schematic showing part of the photodiode array of the photo sensor;
• FIG. 78 is plan view of a reagent reservoir with thermal bend actuated valves;
• FIG. 79 is a diagrammatic representation of the architecture of a first LOC variant;
• FIG. 80 is a diagrammatic representation of the architecture of a second LOC variant;
• FIG. 81 is a diagrammatic representation of the architecture of a fifth LOC variant;
• FIG. 82 is a diagrammatic representation of the architecture of a sixth LOC variant;
• FIG. 83 is a circuit diagram for a single photodiode;
• FIG. 84 is a timing diagram for the photodiode control signals;
• FIG. 85 shows an oligonucleotide ejector chip (ONEC);
• FIG. 86 shows an array of droplet generators from the ONEC shown in Inset AO of FIG. 85;
• FIG. 87 is a schematic section of the array of droplet generators taken along line 91-91 shown in FIG. 86;
• FIG. 88 is an enlarged view of the evaporator shown in Inset AP of FIG. 55;
• FIG. 89 is a schematic section view through a hybridization chamber with a detection photodiode and trigger photodiode;
• FIG. 90 is a diagram of linker-primed PCR;
• FIG. 91 is a schematic representation of a test module with a lancet;
• FIG. 92 shows a boiling-initiated valve array;
• FIG. 93 is a perspective of the boiling-initiated valve sectioned through line 97-97 shown in FIG. 92;
• FIG. 94 is a perspective of LOC variant VII;
• FIG. 95 is an exploded top perspective of the cap for LOC variant VII with internal channels and reservoirs shown in dotted line;
• FIG. 96 is a bottom perspective of the cap for LOC variant VII showing the cap channels;
• FIG. 97 is a plan view of LOC variant VII with features and structures from all layers superimposed on each other;
• FIG. 98 is a plan view of LOC variant VII showing the structures of the CMOS+MST device in isolation;
• FIG. 99 is a plan view of LOC variant VII with the structures of the cap shown in isolation;
• FIG. 100 is an enlarged view of Inset BA shown in FIG. 97;
• FIG. 101 is an enlarged view of Inset BB shown in FIG. 97;
• FIG. 102 is an enlarged view of Inset BC shown in FIG. 97;
• FIG. 103 is an enlarged view of Inset BD shown in FIG. 97;
• FIG. 104 is an enlarged view of Inset BE shown in FIG. 100;
• FIG. 105 is an enlarged view of Inset BF shown in FIG. 104;
• FIG. 106 is an enlarged view of Inset BG shown in FIG. 97;
• FIG. 107 is an enlarged view of the hybridization and detection section of LOC variant VII;
• FIG. 108 is a diagrammatic representation of the architecture of LOC variant VII;
• FIG. 109 is a perspective of LOC variant VIII;
• FIG. 110 is a plan view of LOC variant VIII with features and structures from all layers superimposed on each other;
• FIG. 111 is a plan view of LOC variant VIII with the structures of the cap shown in isolation;
• FIG. 112 is a bottom perspective of the cap channels for LOC variant VIII;
• FIG. 113 is a plan view of LOC variant VIII showing the structures of the CMOS+MST device in isolation;
• FIG. 114 is an enlarged view of Inset CA shown in FIG. 110;
• FIG. 115 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 116 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 117 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 118 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 119 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 120 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CA shown in FIG. 110;
• FIG. 121 is an enlarged view of Inset CB shown in FIG. 110;
• FIG. 122 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIG. 123 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIGS. 124A and 124B show a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIG. 125 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIG. 126 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIG. 127 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CB shown in FIG. 110;
• FIG. 128 is an enlarged view of Inset CC shown in FIG. 110;
• FIG. 129 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 130 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 131 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIGS. 132A and 132B show a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 133 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 134 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 135 is a partial perspective illustrating the laminar structure of LOC variant VIII within Inset CC shown in FIG. 110;
• FIG. 136 is an enlarged view of Inset CD shown in FIG. 110;
• FIG. 137 is an enlarged perspective view of Inset CD shown in FIG. 110;
• FIG. 138 is an exploded perspective view of Inset CD shown in FIG. 110;
• FIG. 139 is an enlarged view of Inset CE shown in FIG. 114;
• FIG. 140 is a diagrammatic representation of the architecture of LOC variant VIII;
• FIG. 141 is a schematic illustration of the architecture of LOC variant XIII;
• FIG. 142 is a schematic illustration of the architecture of LOC variant XIV;
• FIG. 143 is a schematic illustration of the architecture of LOC variant XV;
• FIG. 144 is a schematic illustration of the architecture of LOC variant XVI;
• FIG. 145 is a schematic illustration of the architecture of LOC variant XVII;
• FIG. 146 is a schematic illustration of the architecture of LOC variant XVIII;
• FIG. 147 is a schematic illustration of the architecture of LOC variant XIX;
• FIG. 148 is a schematic illustration of the architecture of LOC variant XX;
• FIG. 149 is a schematic illustration of the architecture of LOC variant XXI;
• FIG. 150 is a schematic illustration of the architecture of LOC variant XXII;
• FIG. 151 is a schematic illustration of the architecture of LOC variant XXIII;
• FIG. 152 is a schematic illustration of the architecture of LOC variant XXIV;
• FIG. 153 is a schematic illustration of the architecture of LOC variant XXV;
• FIG. 154 is a schematic illustration of the architecture of LOC variant XXVI;
• FIG. 155 is a schematic illustration of the architecture of LOC variant XXVII;
• FIG. 156 is a schematic illustration of the architecture of LOC variant XXVIII;
• FIG. 157 is a schematic illustration of the architecture of LOC variant XXIX;
• FIG. 158 is a schematic illustration of the architecture of LOC variant XXX;
• FIG. 159 is a schematic illustration of the architecture of LOC variant XXXI;
• FIG. 160 is a schematic illustration of the architecture of LOC variant XXXII;
• FIG. 161 is a schematic illustration of the architecture of LOC variant XXXIII;
• FIG. 162 is a schematic illustration of the architecture of LOC variant XXXIV;
• FIG. 163 is a schematic illustration of the architecture of LOC variant XXXV;
• FIG. 164 is a schematic illustration of the architecture of LOC variant XXXVI;
• FIG. 165 is a schematic illustration of the architecture of LOC variant XXXVII;
• FIG. 166 is a schematic illustration of the architecture of LOC variant XXXVIII;
• FIG. 167 is a schematic illustration of the architecture of LOC variant XXXIX;
• FIG. 168 is a schematic illustration of the architecture of LOC variant XL;
• FIG. 169 is a schematic illustration of the architecture of LOC variant XLI;
• FIG. 170 is a schematic illustration of the architecture of LOC variant XLII;
• FIG. 171 is a schematic illustration of the architecture of LOC variant XLIII;
• FIG. 172 is a schematic illustration of the architecture of LOC variant XLIV;
• FIG. 173 is a schematic illustration of the architecture of LOC variant XLV;
• FIG. 174 is a schematic illustration of the architecture of LOC variant XLVI;
• FIG. 175 is a schematic illustration of the architecture of LOC variant XLVII;
• FIG. 176 is a schematic illustration of the architecture of LOC variant XLVIII;
• FIG. 177 is a schematic illustration of the architecture of LOC variant XLIX;
• FIG. 178 is a diagram of a primer-linked, linear fluorescent probe during the initial round of amplification;
• FIG. 179 is a diagram of a primer-linked, linear fluorescent probe during a subsequent amplification cycle;
• FIGS. 180A to 180F diagrammatically illustrate thermal cycling of a primer-linked fluorescent stem-and-loop probe;
• FIG. 181 is a schematic illustration of the excitation LED relative to the hybridization chamber array and the photodiodes;
• FIG. 182 is a schematic illustration of the excitation LED and optical lens for directing light onto the hybridization chamber array of the LOC device;
• FIG. 183 is a schematic illustration of the excitation LED, optical lens, and optical prisms for directing light onto the hybridization chamber array of the LOC device;
• FIG. 184 is a schematic illustration of the excitation LED, optical lens and mirror arrangement for directing light onto the hybridization chamber array of the LOC device;
• FIG. 185 is a schematic plan view of a probe spotting robot for loading probes into the LOC devices on a separable PCB;
• FIG. 186 is a schematic illustration of an alternative dialysis section;
• FIG. 187 is a diagrammatic representation of the architecture of a ninth LOC variant;
• FIG. 188 is a plan view of LOC variant 1× with features and structures from all layers superimposed on each other;
• FIG. 189 is an enlarged view of Inset HA shown in FIG. 188;
• FIG. 190 is a diagrammatic representation of the architecture of LOC variant X;
• FIG. 191 is a perspective view of LOC variant X;
• FIG. 192 is a plan view of LOC variant X showing the structures of the CMOS+MST device in isolation;
• FIG. 193 is a perspective view of the underside of the cap with the reagent reservoirs shown in dotted line;
• FIG. 194 is a plan view showing only the features of the cap in isolation;
• FIG. 195 is a plan view showing all the features superimposed on each other, and showing the location of Insets DA to DK;
• FIG. 196 is an enlarged view of Inset DA shown in FIG. 195;
• FIG. 197 is an enlarged view of Inset DB shown in FIG. 195;
• FIG. 198 is an enlarged view of Inset DC shown in FIG. 195;
• FIG. 199 is an enlarged view of Inset DD shown in FIG. 195;
• FIG. 200 is an enlarged view of Inset DE shown in FIG. 195;
• FIG. 201 is an enlarged view of Inset DF shown in FIG. 195;
• FIG. 202 is an enlarged view of Inset DG shown in FIG. 195;
• FIG. 203 is an enlarged view of Inset DH shown in FIG. 195;
• FIG. 204 is an enlarged view of Inset DJ shown in FIG. 195;
• FIG. 205 is an enlarged view of Inset DK shown in FIG. 195;
• FIG. 206 is an enlarged view of Inset DL shown in FIG. 195;
• FIG. 207 is a diagrammatic representation of the architecture of LOC variant XI;
• FIG. 208 is a perspective view of LOC variant XI;
• FIG. 209 is a plan view of LOC variant XI showing all features superimposed on each other, and showing the location of Insets EA to EC;
• FIG. 210 is a plan view of LOC variant XI showing only the features of the cap in isolation;
• FIG. 211 is a plan view of LOC variant XI showing the structures of the CMOS+MST device in isolation;
• FIG. 212 is an enlarged view of Inset EA shown in FIG. 209;
• FIG. 213 is an enlarged view of Inset EB shown in FIG. 209;
• FIG. 214 is an enlarged view of Inset EC shown in FIG. 209;
• FIG. 215 is a diagrammatic representation of the architecture of LOC variant XII;
• FIG. 216 is a perspective view of LOC variant XII;
• FIG. 217 is a plan view of LOC variant XII showing all features superimposed on each other, and showing the location of Insets FA to FC;
• FIG. 218 is a plan view of LOC variant XII showing only the features of the cap in isolation;
• FIG. 219 is a plan view of LOC variant XII showing the structures of the CMOS+MST device in isolation;
• FIG. 220 is an enlarged view of Inset FA shown in FIG. 217;
• FIG. 221 is an enlarged view of Inset FB shown in FIG. 217;
• FIG. 222 is an enlarged view of Inset FC shown in FIG. 217;
• FIG. 223 is a schematic representation of an LED spectrometer illuminating a hybridization chamber to test blood glucose levels;
• FIG. 224 is a schematic representation of a laser excitation source emitting through an optical train into the hybridization chamber;
• FIG. 225 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 226 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 227 shows one embodiment of the shunt transistor for the photodiodes;
• FIG. 228 is a circuit diagram of the differential imager;
• FIG. 229 schematically illustrates a negative control fluorescent probe in its stem-and-loop configuration;
• FIG. 230 schematically illustrates the negative control fluorescent probe of FIG. 229 in its open configuration;
• FIG. 231 schematically illustrates a positive control fluorescent probe in its stem-and-loop configuration;
• FIG. 232 schematically illustrates the positive control fluorescent probe of FIG. 231 in its open configuration;
• FIG. 233 is a schematic section view of a conductivity sensor for use in the LOC device;
• FIG. 234 schematically illustrates a CMOS-controlled flow rate sensor;
• FIG. 235 schematically illustrates a CMOS-controlled mechanical bend-actuator mixer;
• FIG. 236 schematically illustrates a CMOS-controlled thermal impulse mixer;
• FIG. 237 illustrates the reactions occurring during an electrochemiluminescence (ECL) process;
• FIG. 238 schematically illustrates three different anode configurations;
• FIG. 239 is a schematic partial cross-section of the anode and cathode in the hybridization chamber;
• FIG. 240 schematically illustrates an anode in a ring geometry around the peripheral edge of a photodiode;
• FIG. 241 schematically illustrates an anode in a ring geometry within the peripheral edge of a photodiode;
• FIG. 242 schematically illustrates an anode with a series of fingers to increase the length of its lateral edges;
• FIG. 243 schematically illustrates the use of a transparent anode to maximise surface area coupling and ECL signal detection;
• FIG. 244 schematically illustrates the use of an anode affixed to the roof of the hybridization chamber to maximise surface area coupling and ECL signal detection;
• FIG. 245 schematically illustrates an anode interdigitated with a cathode;
• FIG. 246 is a plan view of an electroexplosive valve;
• FIG. 247 is a plan view of a thermal-bend-actuated bend-and-break valve;
• FIG. 248 is a plan view of a dual thermal-bend-actuated bend-and-break valve;
• FIG. 249 is a plan view of a stiction valve;
• FIG. 250 is a plan view of a stiction valve variant;
• FIG. 251 is a plan view of a bubble break valve;
• FIG. 252 shows a test module and test module reader configured for use with ECL detection;
• FIG. 253 is a schematic overview of the electronic components in the test module configured for use with ECL detection;
• FIG. 254 shows a test module and alternative test module readers;
• FIG. 255 shows a test module and test module reader along with the hosting system housing various databases;
• FIGS. 256A and 256B is a diagram illustrating binding of an aptamer to a protein to produce a detectable signal;
• FIGS. 257A and 257B are diagrams illustrating binding of two aptamers to a protein to produce a detectable signal;
• FIGS. 258A and 258B are diagrams illustrating binding of two antibodies to a protein to produce a detectable signal;
• FIG. 259 is a schematic side view of a reagent spotting robot;
• FIG. 260 is a schematic representation of an electrochemiluminescence-based test module with multidevice microfluidic device;
• FIGS. 261A and 261B are schematic cross-sections of a segment of the LOC device after the MST layer fabrication process using process variant I;
• FIGS. 262A and 262B are schematic cross-sections of a segment of the LOC device after the MST layer fabrication process using process variant II;
• FIG. 263 is a diagrammatic representation of the architecture of LOC variant L with ECL detection;
• FIG. 264 is a perspective view of LOC variant L;
• FIG. 265 is a plan view of LOC variant L showing the structures of the CMOS+MST device in isolation;
• FIG. 266 is a perspective view of the underside of the cap of LOC variant L with the reagent reservoirs shown in dotted lines;
• FIG. 267 is a plan view of LOC variant L showing the features of the cap in isolation;
• FIG. 268 is a plan view of LOC variant L showing all the features superimposed on each other and showing the locations of Insets GA to GL;
• FIG. 269 is an enlarged view of Inset GA shown in FIG. 268;
• FIG. 270 is an enlarged view of Inset GB shown in FIG. 268;
• FIG. 271 is an enlarged view of Inset GC shown in FIG. 268;
• FIG. 272 is an enlarged view of Inset GD shown in FIG. 268;
• FIG. 273 is an enlarged view of Inset GE shown in FIG. 268;
• FIG. 274 is an enlarged view of Inset GF shown in FIG. 268;
• FIG. 275 is an enlarged view of Inset GG shown in FIG. 268;
• FIG. 276 is an enlarged view of Inset GH shown in FIG. 268;
• FIG. 277 is an enlarged view of Inset GJ shown in FIG. 268;
• FIG. 278 is an enlarged view of Inset GK shown in FIG. 268;
• FIG. 279 is an enlarged view of Inset GL shown in FIG. 268;
• FIG. 280 is a diagrammatic representation of a LOC device with thermal insulation trench;
• FIG. 281 is a diagram of an electrochemiluminescence resonance energy transfer probe in a closed configuration;
• FIG. 282 is a diagram of an electrochemiluminescence resonance energy transfer probe in an open and hybridized configuration;
• FIG. 283 is a diagram of a primer-linked, luminescent linear probe during the initial round of amplification;
• FIG. 284 is a diagram of a primer-linked, luminescent linear probe during a subsequent amplification cycle;
• FIGS. 285A to 285F diagrammatically illustrate thermal cycling of a luminescent primer-linked stem-and-loop probe;
• FIG. 286 schematically illustrates a negative control luminescent probe in its stem-and-loop configuration;
• FIG. 287 schematically illustrates the negative control luminescent probe of FIG. 286 in its open configuration;
• FIG. 288 schematically illustrates a positive control luminescent probe in its stem-and-loop configuration;
• FIG. 289 schematically illustrates the positive control luminescent probe of FIG. 288 in its open configuration;
• FIG. 290 is a diagrammatic representation of the architecture of a microfluidic device with dialysis device, LOC device, interconnecting cap and electrochemiluminescence (ECL) detection;
• FIG. 291 is a perspective view of a microfluidic device with dialysis device, LOC device and interconnecting cap;
• FIG. 292 is a plan view showing the features of the dialysis device of the microfluidic device and showing the location of Inset JA;
• FIG. 293 is an enlarged view of Inset JA shown in FIG. 292;
• FIG. 294 is a plan view showing the features of the LOC device of the microfluidic device and showing the locations of Insets JB to JJ;
• FIG. 295 is an enlarged view of Inset JB shown in FIG. 294;
• FIG. 296 is an enlarged view of Inset JC shown in FIG. 294;
• FIG. 297 is an enlarged view of Inset JD shown in FIG. 294;
• FIG. 298 is an enlarged view of Inset JE shown in FIG. 294;
• FIG. 299 is an enlarged view of Inset JF shown in FIG. 294;
• FIG. 300 is an enlarged view of Inset JG shown in FIG. 294;
• FIG. 301 is an enlarged view of Inset JH shown in FIG. 294;
• FIG. 302 is an enlarged view of Inset JJ shown in FIG. 294;
• FIG. 303 is an enlarged view of the hybridization chamber of LOC variant L;
• FIG. 304 is an enlarged view of the hybridization chamber array of LOC variant L showing the distribution of calibration chambers;
• FIG. 305 is a flow-chart demonstrating the process of analysing DNA;
• FIG. 306 is a flow-chart demonstrating the process of analysing RNA;
• FIG. 307 is a flow-chart demonstrating the process of analysing micro-RNA;
• FIG. 308 is a flow-chart demonstrating the process of analysing small interfering RNA;
• FIG. 309 is a flow-chart demonstrating the process of analysing small-activating RNA;
• FIG. 310 is a flow-chart demonstrating the process of analysing mitochondrial DNA;
• FIG. 311 is a flow-chart demonstrating the process of analysing proteins by DNA tag amplification;
• FIG. 312 is a flow-chart demonstrating the process of analysing proteins by direct detection;
• FIG. 313 is a flow-chart demonstrating the process of analysing antibodies (immunoglobulins);
• FIG. 314 is a flow-chart demonstrating the process of analysing antigens;
• FIG. 315 is a flow-chart demonstrating the process of analysing antigens by homogeneous enzyme-linked assay;
• FIG. 316 is a flow-chart demonstrating the process of analysing sugars;
• FIG. 317 is a flow-chart demonstrating the process of analysing salts by discrete LED spectroscopy, selective precipitation and/or conductivity measurement;
• FIG. 318 is a flow-chart demonstrating the process of analysing alcohols;
• FIG. 319 is a flow-chart demonstrating the process of analysing illicit drugs;
• FIG. 320 is a flow-chart demonstrating the process of analysing pharmaceuticals;
• FIG. 321 is a flow-chart demonstrating the process of analysing toxicants;
• FIG. 322 is a flow-chart demonstrating the process of analysing metals;
• FIG. 323 is a flow-chart demonstrating the process of analysing blood, blood products and blood cultures;
• FIG. 324 is a flow-chart demonstrating the process of analysing saliva;
• FIG. 325 is a flow-chart demonstrating the process of analysing cerebrospinal fluid;
• FIG. 326 is a flow-chart demonstrating the process of analysing urine;
• FIG. 327 is a flow-chart demonstrating the process of analysing feces;
• FIG. 328 is a flow-chart demonstrating the process of analysing buccal cells;
• FIG. 329 is a flow-chart demonstrating the process of analysing skin;
• FIG. 330 is a flow-chart demonstrating the process of analysing semen;
• FIG. 331 is a flow-chart demonstrating the process of analysing solid tissue biopsies;
• FIG. 332 is a flow-chart demonstrating the process of analysing fingerprints;
• FIG. 333 is a flow-chart demonstrating the process of analysing hair;
• FIG. 334 is a flow-chart demonstrating the process of analysing nails;
• FIG. 335 is a flow-chart demonstrating the process of analysing synovial fluid;
• FIG. 336 is a flow-chart demonstrating the process of analysing a vaginal swab;
• FIG. 337 is a flow-chart demonstrating the process of analysing a cervical swab;
• FIG. 338 is a flow-chart demonstrating the process of analysing vesicle aspirate;
• FIG. 339 is a flow-chart demonstrating the process of analysing bone marrow;
• FIG. 340 is a flow-chart demonstrating the process of analysing vomitus;
• FIG. 341 is a flow-chart demonstrating the process of analysing amniotic fluid;
• FIG. 342 is a flow-chart demonstrating the process of analysing umbilical cord blood;
• FIG. 343 is a flow-chart demonstrating the process of analysing breast milk;
• FIG. 344 is a flow-chart demonstrating the process of analysing sweat;
• FIG. 345 is a flow-chart demonstrating the process of analysing fetal/embryonic tissue;
• FIG. 346 is a flow-chart demonstrating the process of analysing placental tissue;
• FIG. 347 is a flow-chart demonstrating the process of analysing vitreous humor;
• FIG. 348 is a flow-chart demonstrating the process of analysing pleural effusion;
• FIG. 349 is a flow-chart demonstrating the process of analysing tears;
• FIG. 350 is a flow-chart demonstrating the process of analysing drainage material from wounds and ulcers;
• FIG. 351 is a flow-chart demonstrating the process of analysing gastric fluid;
• FIG. 352 is a flow-chart demonstrating the process of analysing pericardial fluid;
• FIG. 353 is a flow-chart demonstrating the process of analysing peritoneal fluid;
• FIG. 354 is a flow-chart demonstrating the process of analysing sputum;
• FIG. 355 is a flow-chart demonstrating the process of analysing exhaled breath condensate;
• FIG. 356 is a flow-chart demonstrating the process of analysing teeth;
• FIG. 357 is a flow-chart demonstrating the process of analysing water;
• FIG. 358 is a flow-chart demonstrating the process of analysing food;
• FIG. 359 is a flow-chart demonstrating the process of analysing drinks;
• FIG. 360 is a flow-chart demonstrating the process of analysing plant sap;
• FIG. 361 is a flow-chart demonstrating the process of analysing plant matter;
• FIG. 362 is a flow-chart demonstrating the process of analysing soil;
• FIG. 363 is a flow-chart demonstrating the process of analysing amplicon;
• FIG. 364 is a schematic section view of a monolithic chip with MST and electronics;
• FIG. 365 is a schematic section view of separately fabricated MST and electronic chips that are then bonded together;
• FIG. 366 is a schematic section view of an image sensor chip coupled to a MST chip;
• FIG. 367 is a schematic section view showing an etched oxide chamber;
• FIG. 368 is a schematic section view showing a suspended heater;
• FIG. 369 is a schematic section view showing a bonded heater;
• FIG. 370 is a schematic section view showing a resist chamber;
• FIG. 371 is a schematic section view showing the sacrificial material;
• FIG. 372 is a schematic representation showing an external detector with optics;
• FIG. 373 is a schematic section view of a thermal valve with droplet ejection initiation;
• FIG. 374 is a schematic section view of a thermal valve with combined surface tension control and droplet ejection initiation;
• FIG. 375 is a schematic section view of a magnetic valve;
• FIG. 376 is a schematic section view of an externally actuated membrane deformation valve;
• FIG. 377 is a schematic section view of an electrowetting valve;
• FIG. 378 is a schematic representation of an excitation method using Xenon, or other noble gas, flash tube with filter;
• FIG. 379 is a flow-chart demonstrating the process of flow-through PCR;
• FIG. 380 is a schematic representation of a mixing device based on AC electrophoresis;
• FIG. 381 is a schematic representation of a rotating mixer;
• FIG. 382 is a schematic section view of in reservoirs etched in substrate;
• FIG. 383 is a schematic section view of in reservoirs in a base which connects to channels etched in substrate;
• FIG. 384 is a flow-chart demonstrating the in-situ synthesis of oligonucleotide probes in the hybridization chambers;
• FIG. 385 is a flow-chart demonstrating an analysis of the sample(s) for the target(s) of interest;
• FIG. 386 is a schematic section view of an under-chamber heater;
• FIG. 387 is a schematic section view of an in-chamber heater;
• FIG. 388 is a schematic section view of a chamber with adjacent heaters;
• FIG. 389 is a schematic representation of a micropositioned array of microvials with droplet generators for spotting into a PCB array of LOC devices;
• FIG. 390 is a schematic representation of a pipelined array of individual microvials with droplet generators for spotting into LOC devices;
• FIG. 391 is a schematic representation of an array of microvials with droplet generators for spotting into micro-positioned LOC devices;
• FIG. 392 is a schematic representation of a microprobe array;
• FIG. 393 is a schematic section view of an external pump;
• FIG. 394 is a schematic section view of a downstream jet-pump;
• FIG. 395 is a schematic section view of an on-chip peristaltic flexure array;
• FIG. 396 is a schematic section view of an on-chip rotary pump;
• FIG. 397 is a schematic section view of an external flexure peristaltic pump;
• FIG. 398 is a schematic section view of an on-chip bubble peristaltic pump;
• FIG. 399 is a schematic section view of a bubble squeeze pump;
• FIG. 400 is a schematic section view of fluid propulsion via electrophoresis;
• FIG. 401 is a schematic representation of a dialysis section for sorting pathogens optimally selected by shape;
• FIG. 402 is a schematic representation of a dialysis section for sorting nucleated cells;
• FIG. 403 is a schematic section view showing the use of poly(dimethylsiloxane) for the walls and roof;
• FIG. 404 is a flow-chart demonstrating the resetting of a photosensor by charge transfer;
• FIG. 405 is a flow-chart demonstrating the resetting of a photosensor by interline charge transfer; and
• FIG. 406 is a schematic overview of the electronic components of a LOC device with an external controller.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Overview
• This overview identifies the main components of a molecular diagnostic system that incorporates embodiments of the present invention. Comprehensive details of the system architecture and operation are set out later in the specification.
• Referring to FIGS. 1, 2, 3, 252 and 253, the system has the following top level components:
• Test modules 10 and 11 are the size of a typical USB memory key and very cheap to produce. Test modules 10 and 11 each contain a microfluidic device, typically in the form of a lab-on-a-chip (LOC) device 30 preloaded with reagents and typically more than 1000 probes for the molecular diagnostic assay (see FIGS. 1 and 252). Test module 10 schematically shown in FIG. 1 uses a fluorescence-based detection technique to identify target molecules, while test module 11 in FIG. 252 uses an electrochemiluminescence-based detection technique. The LOC device 30 has an integrated photosensor 44 for fluorescence or electrochemiluminescence detection (described in detail below). Both test modules 10 and 11 use a standard Micro-USB plug 14 for power, data and control, both have a printed circuit board (PCB) 57, and both have external power supply capacitors 32 and an inductor 15. The test modules 10 and 11 are both single-use only for mass production and distribution in sterile packaging ready for use.
• The outer casing 13 has a macroreceptacle 24 for receiving the biological sample and a removable sterile sealing tape 22, preferably with a low tack adhesive, to cover the macroreceptacle prior to use. A membrane seal 408 with a membrane guard 410 forms part of the outer casing 13 to reduce dehumidification within the test module while providing pressure relief from small air pressure fluctuations. The membrane guard 410 protects the membrane seal 408 from damage.
• Test module reader 12 powers the test module 10 or 11 via Micro-USB port 16. The test module reader 12 can adopt many different forms and a selection of these are described later. The version of the reader 12 shown in FIGS. 1, 3 and 252 is a smart phone embodiment. A block diagram of this reader 12 is shown in FIG. 3. Processor 42 runs application software from program storage 43. The processor 42 also interfaces with the display screen 18 and user interface (UI) touch screen 17 and buttons 19, a cellular radio 21, wireless network connection 23, and a satellite navigation system 25. The cellular radio 21 and wireless network connection 23 are used for communications. Satellite navigation system 25 is used for updating epidemiological databases with location data. The location data can, alternatively, be entered manually via the touch screen 17 or buttons 19. Data storage 27 holds genetic and diagnostic information, test results, patient information, assay and probe data for identifying each probe and its array position. Data storage 27 and program storage 43 may be shared in a common memory facility. Application software installed on the test module reader 12 provides analysis of results, along with additional test and diagnostic information.
• To conduct a diagnostic test, the test module 10 (or test module 11) is inserted into the Micro-USB port 16 on the test module reader 12. The sterile sealing tape 22 is peeled back and the biological sample (in a liquid form) is loaded into the sample macroreceptacle 24. Pressing start button 20 initiates testing via the application software. The sample flows into the LOC device 30 and the on-board assay extracts, incubates, amplifies and hybridizes the sample nucleic acids (the target) with presynthesized hybridization-responsive oligonucleotide probes. In the case of test module 10 (which uses fluorescence-based detection), the probes are fluorescently labelled and the LED 26 housed in the casing 13 provides the necessary excitation light to induce fluorescence emission from the hybridized probes (see FIGS. 1 and 2). In test module 11 (which uses electrochemiluminescence (ECL) detection), the LOC device 30 is loaded with ECL probes (discussed above) and the LED 26 is not necessary for generating the luminescent emission. Instead, electrodes 860 and 870 provide the excitation electrical current (see FIG. 253). The emission (fluorescent or luminescent) is detected using a photosensor 44 integrated into CMOS circuitry of each LOC device. The detected signal is amplified and converted to a digital output which is analyzed by the test module reader 12. The reader then displays the results.
• The data may be saved locally and/or uploaded to a network server containing patient records. The test module 10 or 11 is removed from the test module reader 12 and disposed of appropriately.
• FIGS. 1, 3 and 252 show the test module reader 12 configured as a mobile phone/smart phone 28. In other forms, the test module reader is a laptop/notebook 101, a dedicated reader 103, an ebook reader 107, a tablet computer 109 or desktop computer 105 for use in hospitals, private practices or laboratories (see FIG. 254). The reader can interface with a range of additional applications such as patient records, billing, online databases and multi-user environments. It can also be interfaced with a range of local or remote peripherals such as printers and patient smart cards.
• Referring to FIG. 255, the data generated by the test module 10 can be used to update, via the reader 12 and network 125, the epidemiological databases hosted on the hosting system for epidemiological data 111, the genetic databases hosted on the hosting system for genetic data 113, the electronic health records hosted on the hosting system for electronic health records (EHR) 115, the electronic medical records hosted on the hosting system for electronic medical records (EMR) 121, and the personal health records hosted on the hosting system for personal health records (PHR) 123. Conversely, the epidemiological data hosted on the hosting system for epidemiological data 111, the genetic data hosted on the hosting system for genetic data 113, the electronic health records hosted on the hosting system for electronic health records (EHR) 115, the electronic medical records hosted on the hosting system for electronic medical records (EMR) 121, and the personal health records hosted on the hosting system for personal health records (PHR) 123, can be used to update, via network 125 and the reader 12, the digital memory in the LOC 30 of the test module 10.
• Referring back to FIGS. 1, 2, 252 and 253 the reader 12 uses battery power in the mobile phone configuration. The mobile phone reader contains all test and diagnostic information preloaded. Data can also be loaded or updated via a number of wireless or contact interfaces to enable communications with peripheral devices, computers or online servers. A Micro-USB port 16 is provided for connection to a computer or mains power supply for battery recharge.
• FIG. 91 shows an embodiment of the test module 10 used for tests that only require a positive or negative result for a particular target, such as testing whether a person is infected with, for example, H1N1 Influenza A virus. Only a purpose built USB power/indicator-only module 47 is adequate. No other reader or application software is necessary. An indicator 45 on the USB power/indicator-only module 47 signals positive or negative results. This configuration is well suited to mass screening.
• Additional items supplied with the system may include a test tube containing reagents for pre-treatment of certain samples, along with spatula and lancet for sample collection. FIG. 91 shows an embodiment of the test module incorporating a spring-loaded, retractable lancet 390 and lancet release button 392 for convenience. A satellite phone can be used in remote areas.
Test Module Electronics
• FIGS. 2 and 253 are block diagrams of the electronic components in the test modules 10 and 11, respectively. The CMOS circuitry integrated in the LOC device 30 has a USB device driver 36, a controller 34, a USB-compatible LED driver 29, clock 33, power conditioner 31, RAM 38 and program and data flash memory 40. These provide the control and memory for the entire test module 10 or 11 including the photosensor 44, the temperature sensors 170, the liquid sensors 174, and the various heaters 152, 154, 182, 234, together with associated drivers 37 and 39 and registers 35 and 41. Only the LED 26 (in the case of test module 10), external power supply capacitors 32 and the Micro-USB plug 14 are external to the LOC device 30. The LOC devices 30 include bond-pads for making connections to these external components. The RAM 38 and the program and data flash memory 40 have the application software and the diagnostic and test information (Flash/Secure storage, e.g. via encryption) for over 1000 probes. In the case of test module 11 configured for ECL detection, there is no LED 26 (see FIGS. 252 and 253). Data is encrypted by the LOC device 30 for secure storage and secure communication with an external device. The LOC devices 30 are loaded with electrochemiluminescent probes and the hybridization chambers each have a pair of ECL excitation electrodes 860 and 870.
• Many types of test modules 10 are manufactured in a number of test forms, ready for off-the-shelf use. The differences between the test forms lie in the on board assay of reagents and probes.
• Some examples of infectious diseases rapidly identified with this system include:
• • Influenza—Influenza virus A, B, C, Isavirus, Thogotovirus
  • Pneumonia—respiratory syncytial virus (RSV), adenovirus, metapneumovirus, Streptococcus pneumoniae, Staphylococcus aureus
  • Tuberculosis—Mycobacterium tuberculosis, bovis, africanum, canetti, and microti
  • Plasmodium falciparum, Toxoplasma gondii and other protozoan parasites
  • Typhoid—Salmonella enterica serovar typhi
  • Ebola virus
  • Human immunodeficiency virus (HIV)
  • Dengue Fever—Flavivirus
  • Hepatitis (A through E)
  • Hospital acquired infections—for example Clostridium difficile, Vancomycin resistant Enterococcus, and Methicillin resistant Staphylococcus aureus
  • Herpes simplex virus (HSV)
  • Cytomegalovirus (CMV)
  • Epstein-Ban virus (EBV)
  • Encephalitis—Japanese Encephalitis virus, Chandipura virus
  • Whooping cough—Bordetella pertussis
  • Measles—paramyxovirus
  • Meningitis—Streptococcus pneumoniae and Neisseria meningitidis
  • Anthrax—Bacillus anthracis
• Some examples of genetic disorders identified with this system include:
• • Cystic fibrosis
  • Haemophilia
  • Sickle cell disease
  • Tay-Sachs disease
  • Haemochromatosis
  • Cerebral arteriopathy
  • Crohn's disease
  • Polycistic kidney disease
  • Congential heart disease
  • Rett syndrome
• A small selection of cancers identified by the diagnostic system include:
• • Ovarian
  • Colon carcinoma
  • Multiple endocrine neoplasia
  • Retinoblastoma
  • Turcot syndrome
• The above lists are not exhaustive and the diagnostic system can be configured to detect a much greater variety of diseases and conditions using nucleic acid and proteomic analysis.
Detailed Architecture of System Components LOC Device
• The LOC device 30 is central to the diagnostic system. It rapidly performs the four major steps of a nucleic acid based molecular diagnostic assay, i.e. sample preparation, nucleic acid extraction, nucleic acid amplification, and detection, using a microfluidic platform. The LOC device also has alternative uses, and these are detailed later. As discussed above, test modules 10 and 11 can adopt many different configurations to detect different targets Likewise, the LOC device 30 has numerous different embodiments tailored to the target(s) of interest. One form of the LOC device 30 is LOC device 301 for fluorescent detection of target nucleic acid sequences in the pathogens of a whole blood sample. For the purposes of illustration, the structure and operation of LOC device 301 is now described in detail with reference to FIGS. 4 to 26 and 27 to 57.
• FIG. 4 is a schematic representation of the architecture of the LOC device 301. For convenience, process stages shown in FIG. 4 are indicated with the reference numeral corresponding to the functional sections of the LOC device 301 that perform that process stage. The process stages associated with each of the major steps of a nucleic acid based molecular diagnostic assay are also indicated: sample input and preparation 288, extraction 290, incubation 291, amplification 292 and detection 294. The various reservoirs, chambers, valves and other components of the LOC device 301 will be described in more detail later.
• FIG. 5 is a perspective view of the LOC device 301. It is fabricated using high volume CMOS and MST (microsystems technology) manufacturing techniques. The laminar structure of the LOC device 301 is illustrated in the schematic (not to scale) partial section view of FIG. 12. The LOC device 301 has a silicon substrate 84 which supports the CMOS+MST chip 48, comprising CMOS circuitry 86 and an MST layer 87, with a cap 46 overlaying the MST layer 87. For the purposes of this patent specification, the term ‘MST layer’ is a reference to a collection of structures and layers that process the sample with various reagents. Accordingly, these structures and components are configured to define flow-paths with characteristic dimensions that will support capillary driven flow of liquids with physical characteristics similar to those of the sample during processing. In light of this, the MST layer and components are typically fabricated using surface micromachining techniques and/or bulk micromachining techniques. However, other fabrication methods can also produce structures and components dimensioned for capillary driven flows and processing very small volumes. The specific embodiments described in this specification show the MST layer as the structures and active components supported on the CMOS circuitry 86, but excluding the features of the cap 46. However, the skilled addressee will appreciate that the MST layer need not have underlying CMOS or indeed an overlying cap in order for it to process the sample.
• The overall dimensions of the LOC device shown in the following figures are 1760 μm×5824 μm. Of course, LOC devices fabricated for different applications may have different dimensions.
• FIG. 6 shows the features of the MST layer 87 superimposed with the features of the cap. Insets AA to AD, AG and AH shown in FIG. 6 are enlarged in FIGS. 13, 14, 35, 56, 55 and 67, respectively, and described in detail below for a comprehensive understanding of each structure within the LOC device 301. FIGS. 7 to 10 show the features of the cap 46 in isolation while FIG. 11 shows the CMOS+MST device 48 structures in isolation.
Laminar Structure
• FIGS. 12 and 22 are sketches that diagrammatically show the laminar structure of the CMOS+MST device 48, the cap 46 and the fluidic interaction between the two. The figures are not to scale for the purposes of illustration. FIG. 12 is a schematic section view through the sample inlet 68 and FIG. 22 is a schematic section through the reservoir 54. As best shown in FIG. 12, the CMOS+MST device 48 has a silicon substrate 84 which supports the CMOS circuitry 86 that operates the active elements within the MST layer 87 above. A passivation layer 88 seals and protects the CMOS layer 86 from the fluid flows through the MST layer 87.
• Fluid flows through both the cap channels 94 and the MST channels 90 (see for example FIGS. 7 and 16) in the cap layer 46 and MST channel layer 100, respectively. Cell transport occurs in the larger channels 94 fabricated in the cap 46, while biochemical processes are carried out in the smaller MST channels 90. Cell transport channels are sized so as to be able to transport cells in the sample to predetermined sites in the MST channels 90. Transportation of cells with sizes greater than 20 microns (for example, certain leukocytes) requires channel dimensions greater than 20 microns, and therefore a cross sectional area transverse to the flow of greater than 400 square microns. MST channels, particularly at locations in the LOC where transport of cells is not required, can be significantly smaller.
• It will be appreciated that cap channel 94 and MST channel 90 are generic references and particular MST channels 90 may also be referred to as (for example) heated microchannels or dialysis MST channels in light of their particular function. MST channels 90 are formed by etching through a MST channel layer 100 deposited on the passivation layer 88 and patterned with photoresist. The MST channels 90 are enclosed by a roof layer 66 which forms the top (with respect to the orientation shown in the figures) of the CMOS+MST device 48.
• Despite sometimes being shown as separate layers, the cap channel layer 80 and the reservoir layer 78 are formed from a unitary piece of material. Of course, the piece of material may also be non-unitary. This piece of material is etched from both sides in order to form a cap channel layer 80 in which the cap channels 94 are etched and the reservoir layer 78 in which the reservoirs 54, 56, 58, 60 and 62 are etched. Alternatively, the reservoirs and the cap channels are formed by a micromolding process. Both etching and micromolding techniques are used to produce channels with cross sectional areas transverse to the flow as large as 20,000 square microns, and as small as 8 square microns.
• At different locations in the LOC device, there can be a range of appropriate choices for the cross sectional area of the channel transverse to the flow. Where large quantities of sample, or samples with large constituents, are contained in the channel, a cross-sectional area of up to 20,000 square microns (for example, a 200 micron wide channel in a 100 micron thick layer) is suitable. Where small quantities of liquid, or mixtures without large cells present, are contained in the channel, a very small cross sectional area transverse to the flow is preferable.
• A lower seal 64 encloses the cap channels 94 and the upper seal layer 82 encloses the reservoirs 54, 56, 58, 60 and 62.
• The five reservoirs 54, 56, 58, 60 and 62 are preloaded with assay-specific reagents. In the embodiment described here, the reservoirs are preloaded with the following reagents, but other reagents can easily be substituted:
• • reservoir 54: anticoagulant with option to include erythrocyte lysis buffer
  • reservoir 56: lysis reagent
  • reservoir 58: restriction enzymes, ligase and linkers (for linker-primed PCR (see FIG. 90, extracted from T. Stachan et al., Human Molecular Genetics 2, Garland Science, NY and London, 1999))
  • reservoir 60: amplification mix (dNTPs, primers, buffer) and
  • reservoir 62: DNA polymerase.
• The cap 46 and the CMOS+MST layers 48 are in fluid communication via corresponding openings in the lower seal 64 and the roof layer 66. These openings are referred to as uptakes 96 and downtakes 92 depending on whether fluid is flowing from the MST channels 90 to the cap channels 94 or vice versa.
LOC Device Operation
• The operation of the LOC device 301 is described below in a step-wise fashion with reference to analysing pathogenic DNA in a blood sample. Of course, other types of biological or non-biological fluid are also analysed using an appropriate set, or combination, of reagents, test protocols, LOC variants and detection systems. Referring back to FIG. 4, there are five major steps involved in analysing a biological sample, comprising sample input and preparation 288, nucleic acid extraction 290, nucleic acid incubation 291, nucleic acid amplification 292 and detection and analysis 294.
• The sample input and preparation step 288 involves mixing the blood with an anticoagulant 116 and then separating pathogens from the leukocytes and erythrocytes with the pathogen dialysis section 70. As best shown in FIGS. 7 and 12, the blood sample enters the device via the sample inlet 68. Capillary action draws the blood sample along the cap channel 94 to the reservoir 54. Anticoagulant is released from the reservoir 54 as the sample blood flow opens its surface tension valve 118 (see FIGS. 15 and 22). The anticoagulant prevents the formation of clots which would block the flow.
• As best shown in FIG. 22, the anticoagulant 116 is drawn out of the reservoir 54 by capillary action and into the MST channel 90 via the downtake 92. The downtake 92 has a capillary initiation feature (CIF) 102 to shape the geometry of the meniscus such that it does not anchor to the rim of the downtake 92. Vent holes 122 in the upper seal 82 allows air to replace the anticoagulant 116 as it is drawn out of the reservoir 54.
• The MST channel 90 shown in FIG. 22 is part of a surface tension valve 118. The anticoagulant 116 fills the surface tension valve 118 and pins a meniscus 120 to the uptake 96 to a meniscus anchor 98. Prior to use, the meniscus 120 remains pinned at the uptake 96 so the anticoagulant does not flow into the cap channel 94. When the blood flows through the cap channel 94 to the uptake 96, the meniscus 120 is removed and the anticoagulant is drawn into the flow.
• FIGS. 15 to 21 show Inset AE which is a portion of Inset AA shown in FIG. 13. As shown in FIGS. 15, 16 and 17, the surface tension valve 118 has three separate MST channels 90 extending between respective downtakes 92 and uptakes 96. The number of MST channels 90 in a surface tension valve can be varied to change the flow rate of the reagent into the sample mixture. As the sample mixture and the reagents mix together by diffusion, the flow rate out of the reservoir determines the concentration of the reagent in the sample flow. Hence, the surface tension valve for each of the reservoirs is configured to match the desired reagent concentration.
• The blood passes into a pathogen dialysis section 70 (see FIGS. 4 and 15) where target cells are concentrated from the sample using an array of apertures 164 sized according to a predetermined threshold. Cells smaller than the threshold pass through the apertures while larger cells do not pass through the apertures. Unwanted cells, which may be either the larger cells withheld by the array of apertures 164 or the smaller cells that pass through the apertures, are redirected to a waste unit 76 while the target cells continue as part of the assay.
• In the pathogen dialysis section 70 described here, the pathogens from the whole blood sample are concentrated for microbial DNA analysis. The array of apertures is formed by a multitude of 3 micron diameter holes 164 fluidically connecting the input flow in the cap channel 94 to a target channel 74. The 3 micron diameter apertures 164 and the dialysis uptake holes 168 for the target channel 74 are connected by a series of dialysis MST channels 204 (best shown in FIGS. 15 and 21). Pathogens are small enough to pass through the 3 micron diameter apertures 164 and fill the target channel 74 via the dialysis MST channels 204. Cells larger than 3 microns, such as erythrocytes and leukocytes, stay in the waste channel 72 in the cap 46 which leads to a waste reservoir 76 (see FIG. 7).
• Other aperture shapes, sizes and aspect ratios can be used to isolate specific pathogens or other target cells such as leukocytes for human DNA analysis. Greater detail on the dialysis section and dialysis variants is provided later.
• Referring again to FIGS. 6 and 7, the flow is drawn through the target channel 74 to the surface tension valve 128 of the lysis reagent reservoir 56. The surface tension valve 128 has seven MST channels 90 extending between the lysis reagent reservoir 56 and the target channel 74. When the menisci are unpinned by the sample flow, the flow rate from all seven of the MST channels 90 will be greater than the flow rate from the anticoagulant reservoir 54 where the surface tension valve 118 has three MST channels 90 (assuming the physical characteristics of the fluids are roughly equivalent). Hence the proportion of lysis reagent in the sample mixture is greater than that of the anticoagulant.
• The lysis reagent and target cells mix by diffusion in the target channel 74 within the chemical lysis section 130. A boiling-initiated valve 126 stops the flow until sufficient time has passed for diffusion and lysis to take place, releasing the genetic material from the target cells (see FIGS. 6 and 7). The structure and operation of the boiling-initiated valves are described in greater detail below with reference to FIGS. 31 and 32. Other active valve types (as opposed to passive valves such as the surface tension valve 118) have also been developed by the Applicant which may be used here instead of the boiling-initiated valve. These alternative valve designs are also described later.
• When the boiling-initiated valve 126 opens, the lysed cells flow into a mixing section 131 for pre-amplification restriction digestion and linker ligation.
• Referring to FIG. 13, restriction enzymes, linkers and ligase are released from the reservoir 58 when the flow unpins the menisci at the surface tension valve 132 at the start of the mixing section 131. The mixture flows the length of the mixing section 131 for diffusion mixing. At the end of the mixing section 131 is downtake 134 leading into the incubator inlet channel 133 of the incubation section 114 (see FIG. 13). The incubator inlet channel 133 feeds the mixture into a serpentine configuration of heated microchannels 210 which provides an incubation chamber for holding the sample during restriction digestion and ligation of the linkers (see FIGS. 13 and 14).
• FIGS. 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device 301 within Inset AB of FIG. 6. Each figure shows the sequential addition of layers forming the structures of the CMOS+MST layer 48 and the cap 46. Inset AB shows the end of the incubation section 114 and the start of the amplification section 112. As best shown in FIGS. 14 and 23, the flow fills the microchannels 210 of the incubation section 114 until reaching the boiling-initiated valve 106 where the flow stops while diffusion takes place. As discussed above, the microchannel 210 upstream of the boiling-initiated valve 106 becomes an incubation chamber containing the sample, restriction enzymes, ligase and linkers. The heaters 154 are then activated and held at constant temperature for a specified time for restriction digestion and linker ligation to occur.
• The skilled worker will appreciate that this incubation step 291 (see FIG. 4) is optional and only required for some nucleic acid amplification assay types. Furthermore, in some instances, it may be necessary to have a heating step at the end of the incubation period to spike the temperature above the incubation temperature. The temperature spike inactivates the restriction enzymes and ligase prior to entering the amplification section 112. Inactivation of the restriction enzymes and ligase has particular relevance when isothermal nucleic acid amplification is being employed.
• Following incubation, the boiling-initiated valve 106 is activated (opened) and the flow resumes into the amplification section 112. Referring to FIGS. 31 and 32, the mixture fills the serpentine configuration of heated microchannels 158, which form one or more amplification chambers, until it reaches the boiling-initiated valve 108. As best shown in the schematic section view of FIG. 30, amplification mix (dNTPs, primers, buffer) is released from reservoir 60 and polymerase is subsequently released from reservoir 62 into the intermediate MST channel 212 connecting the incubation and amplification sections (114 and 112 respectively).
• FIGS. 35 to 51 show the layers of the LOC device 301 within Inset AC of FIG. 6. Each figure shows the sequential addition of layers forming the structures of the CMOS+MST device 48 and the cap 46. Inset AC is at the end of the amplification section 112 and the start of the hybridization and detection section 52. The incubated sample, amplification mix and polymerase flow through the microchannels 158 to the boiling-initiated valve 108. After sufficient time for diffusion mixing, the heaters 154 in the microchannels 158 are activated for thermal cycling or isothermal amplification. The amplification mix goes through a predetermined number of thermal cycles or a preset amplification time to amplify sufficient target DNA. After the nucleic acid amplification process, the boiling-initiated valve 108 opens and flow resumes into the hybridization and detection section 52. The operation of boiling-initiated valves is described in more detail later.
• As shown in FIG. 52, the hybridization and detection section 52 has an array of hybridization chambers 110. FIGS. 52, 53, 54 and 56 show the hybridization chamber array 110 and individual hybridization chambers 180 in detail. At the entrance to the hybridization chamber 180 is a diffusion barrier 175 which prevents diffusion of the target nucleic acid, probe strands and hybridized probes between the hybridization chambers 180 during hybridization so as to prevent erroneous hybridization detection results. The diffusion barriers 175 present a flow-path-length that is long enough to prevent the target sequences and probes diffusing out of one chamber and contaminating another chamber within the time taken for the probes and nucleic acids to hybridize and the signal to be detected, thus avoiding an erroneous result.
• Another mechanism to prevent erroneous readings is to have identical probes in a number of the hybridization chambers. The CMOS circuitry 86 derives a single result from the photodiodes 184 corresponding to the hybridization chambers 180 that contain identical probes. Anomalous results can be disregarded or weighted differently in the derivation of the single result.
• The thermal energy required for hybridization is provided by CMOS-controlled heaters 182 (described in more detail below). After the heater is activated, hybridization occurs between complementary target-probe sequences. The LED driver 29 in the CMOS circuitry 86 signals the LED 26 located in the test module 10 to illuminate. These probes only fluoresce when hybridization has occurred thereby avoiding washing and drying steps that are typically required to remove unbound strands. Hybridization forces the stem-and-loop structure of the FRET probes 186 to open, which allows the fluorophore to emit fluorescent energy in response to the LED excitation light, as discussed in greater detail later. Fluorescence is detected by a photodiode 184 in the CMOS circuitry 86 underlying each hybridization chamber 180 (see hybridization chamber description below). The photodiodes 184 for all hybridization chambers and associated electronics collectively form the photosensor 44 (see FIG. 77). In other embodiments, the photosensor may be an array of charge coupled devices (CCD array). The detected signal from the photodiodes 184 is amplified and converted to a digital output which is analyzed by the test module reader 12. Further details of the detection method are described later.
Additional Details for the LOC Device Modularity of the Design
• The LOC device 301 has many functional sections, including the reagent reservoirs 54, 56, 58, 60 and 62, the dialysis section 70, lysis section 130, incubation section 114, and amplification section 112, valve types, the humidifier and humidity sensor. In other embodiments of the LOC device, these functional sections can be omitted, additional functional sections can be added or the functional sections can be used for alternative purposes to those described above.
• For example, the incubation section 114 can be used as the first amplification section 112 of a tandem amplification assay system, with the chemical lysis reagent reservoir 56 being used to add the first amplification mix of primers, dNTPs and buffer and reagent reservoir 58 being used for adding the reverse transcriptase and/or polymerase. A chemical lysis reagent can also be added to the reservoir 56 along with the amplification mix if chemical lysis of the sample is desired or, alternatively, thermal lysis can occur in the incubation section by heating the sample for a predetermined time. In some embodiments, an additional reservoir can be incorporated immediately upstream of reservoir 58 for the mix of primers, dNTPs and buffer if there is a requirement for chemical lysis and a separation of this mix from the chemical lysis reagent is desired.
• In some circumstances it may be desirable to omit a step, such as the incubation step 291. In this case, a LOC device can be specifically fabricated to omit the reagent reservoir 58 and incubation section 114, or the reservoir can simply not be loaded with reagents or the active valves, if present, not activated to dispense the reagents into the sample flow, and the incubation section then simply becomes a channel to transport the sample from the lysis section 130 to the amplification section 112. The heaters are independently operable and therefore, where reactions are dependent on heat, such as thermal lysis, programming the heaters not to activate during this step ensures thermal lysis does not occur in LOC devices that do not require it. The dialysis section 70 can be located at the beginning of the fluidic system within the microfluidic device as shown in FIG. 4 or can be located anywhere else within the microfluidic device. For example, dialysis after the amplification phase 292 to remove cellular debris prior to the hybridization and detection step 294 may be beneficial in some circumstances. Alternatively, two or more dialysis sections can be incorporated at any location throughout the LOC device. Similarly, it is possible to incorporate additional amplification sections 112 to enable multiple targets to be amplified in parallel or in series prior to being detected in the hybridization chamber arrays 110 with specific nucleic acid probes. For analysis of samples like whole blood, in which dialysis is not required, the dialysis section 70 is simply omitted from the sample input and preparation section 288 of the LOC design. In some cases, it is not necessary to omit the dialysis section 70 from the LOC device even if the analysis does not require dialysis. If there is no geometric hindrance to the assay by the existence of a dialysis section, a LOC with the dialysis section 70 in the sample input and preparation section can still be used without a loss of the required functionality.
• Furthermore, the detection section 294 may encompass proteomic chamber arrays which are identical to the hybridization chamber arrays but are loaded with probes designed to conjugate or hybridize with sample target proteins present in non-amplified sample instead of nucleic acid probes designed to hybridize to target nucleic acid sequences.
• It will be appreciated that the LOC devices fabricated for use in this diagnostic system are different combinations of functional sections selected in accordance with the particular LOC application. The vast majority of functional sections are common to many of the LOC devices and the design of additional LOC devices for new application is a matter of compiling an appropriate combination of functional sections from the extensive selection of functional sections used in the existing LOC devices.
• Only a small number of the LOC devices are shown in this description and some more are shown schematically to illustrate the design flexibility of the LOC devices fabricated for this system. The person skilled in the art will readily recognise that the LOC devices shown in this description are not an exhaustive list and many additional LOC designs are a matter of compiling the appropriate combination of functional sections.
Sample Types
• LOC variants can accept and analyze the nucleic acid or protein content of a variety of sample types in liquid form including, but not limited to, blood and blood products, saliva, cerebrospinal fluid, urine, semen, amniotic fluid, umbilical cord blood, breast milk, sweat, pleural effusion, tear, pericardial fluid, peritoneal fluid, environmental water samples and drink samples. Amplicon obtained from macroscopic nucleic acid amplification can also be analysed using the LOC device; in this case, all the reagent reservoirs will be empty or configured not to release their contents, and the dialysis, lysis, incubation and amplification sections will be used solely to transport the sample from the sample inlet 68 to the hybridization chambers 180 for nucleic acid detection, as described above.
• For some sample types, a pre-processing step is required, for example semen may need to be liquefied and mucus may need to be pre-treated with an enzyme to reduce the viscosity prior to input into the LOC device.
Sample Input
• Referring to FIGS. 1 and 12, the sample is added to the macroreceptacle 24 of the test module 10. The macroreceptacle 24 is a truncated cone which feeds into the inlet 68 of the LOC device 301 by capillary action. Here it flows into the 64 μm wide×60 μm deep cap channel 94 where it is drawn towards the anticoagulant reservoir 54, also by capillary action.
Reagent Reservoirs
• The small volumes of reagents required by the assay systems using microfluidic devices, such as LOC device 301, permit the reagent reservoirs to contain all reagents necessary for the biochemical processing with each of the reagent reservoirs having a small volume. This volume is easily less than 1,000,000,000 cubic microns, in the vast majority of cases less than 300,000,000 cubic microns, typically less than 70,000,000 cubic microns and in the case of the LOC device 301 shown in the drawings, less than 20,000,000 cubic microns.
Dialysis Section
• Referring to FIGS. 15 to 21, 33 and 34, the pathogen dialysis section 70 is designed to concentrate pathogenic target cells from the sample. As previously described, a plurality of apertures in the form of 3 micron diameter holes 164 in the roof layer 66 filter the target cells from the bulk of the sample. As the sample flows past the 3 micron diameter apertures 164, microbial pathogens pass through the holes into a series of dialysis MST channels 204 and flow back up into the target channel 74 via 16 μm dialysis uptake holes 168 (see FIGS. 33 and 34). The remainder of the sample (erythrocytes and so on) stay in the cap channel 94. Downstream of the pathogen dialysis section 70, the cap channel 94 becomes the waste channel 72 leading to the waste reservoir 76. For biological samples of the type that generate a substantial amount of waste, a foam insert or other porous element 49 within the outer casing 13 of the test module 10 is configured to be in fluid communication with the waste reservoir 76 (see FIG. 1).
• The pathogen dialysis section 70 functions entirely on capillary action of the fluid sample. The 3 micron diameter apertures 164 at the upstream end of the pathogen dialysis section 70 have capillary initiation features (CIFs) 166 (see FIG. 33) so that the fluid is drawn down into the dialysis MST channel 204 beneath. The first uptake hole 198 for the target channel 74 also has a CIF 202 (see FIG. 15) to avoid the flow simply pinning a meniscus across the dialysis uptake holes 168.
• The small constituents dialysis section 682 schematically shown in FIG. 169 can have a similar structure to the pathogen dialysis section 70. The small constituents dialysis section separates any small target cells or molecules from a sample by sizing (and, if necessary, shaping) apertures suitable for allowing the small target cells or molecules to pass into the target channel and continue for further analysis. Larger sized cells or molecules are removed to a waste reservoir 766. Thus, the LOC device 30 (see FIGS. 1 and 252) is not limited to separating pathogens that are less than 3 μm in size, but can be used to separate cells or molecules of any size desired.
Lysis Section
• Referring back to FIGS. 7, 11 and 13, the genetic material in the sample is released from the cells by a chemical lysis process. As described above, a lysis reagent from the lysis reservoir 56 mixes with the sample flow in the target channel 74 downstream of the surface tension valve 128 for the lysis reservoir 56. However, some diagnostic assays are better suited to a thermal lysis process, or even a combination of chemical and thermal lysis of the target cells. The LOC device 301 accommodates this with the heated microchannels 210 of the incubation section 114. The sample flow fills the incubation section 114 and stops at the boiling-initiated valve 106. The incubation microchannels 210 heat the sample to a temperature at which the cellular membranes are disrupted.
• In some thermal lysis applications, an enzymatic reaction in the chemical lysis section 130 is not necessary and the thermal lysis completely replaces the enzymatic reaction in the chemical lysis section 130.
Boiling-Initiated Valve
• As discussed above, the LOC device 301 has three boiling-initiated valves 126, 106 and 108. The location of these valves is shown in FIG. 6. FIG. 31 is an enlarged plan view of the boiling-initiated valve 108 in isolation at the end of the heated microchannels 158 of the amplification section 112.
• The sample flow 119 is drawn along the heated microchannels 158 by capillary action until it reaches the boiling-initiated valve 108. The leading meniscus 120 of the sample flow pins at a meniscus anchor 98 at the valve inlet 146. The geometry of the meniscus anchor 98 stops the advancing meniscus to arrest the capillary flow. As shown in FIGS. 31 and 32, the meniscus anchor 98 is an aperture provided by an uptake opening from the MST channel 90 to the cap channel 94. Surface tension in the meniscus 120 keeps the valve closed. An annular heater 152 is at the periphery of the valve inlet 146. The annular heater 152 is CMOS-controlled via the boiling-initiated valve heater contacts 153.
• To open the valve, the CMOS circuitry 86 sends an electrical pulse to the valve heater contacts 153. The annular heater 152 resistively heats until the liquid sample 119 boils. The boiling unpins the meniscus 120 from the valve inlet 146 and initiates wetting of the cap channel 94. Once wetting the cap channel 94 begins, capillary flow resumes. The fluid sample 119 fills the cap channel 94 and flows through the valve downtake 150 to the valve outlet 148 where capillary driven flow continues along the amplification section exit channel 160 into the hybridization and detection section 52. Liquid sensors 174 are placed before and after the valve for diagnostics.
• It will be appreciated that once the boiling-initiated valves are opened, they cannot be re-closed. However, as the LOC device 301 and the test module 10 are single-use devices, re-closing the valves is unnecessary.
Incubation Section and Nucleic Acid Amplification Section
• FIGS. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubation section 114 and the amplification section 112. The incubation section 114 has a single, heated incubation microchannel 210 etched in a serpentine pattern in the MST channel layer 100 from the downtake opening 134 to the boiling-initiated valve 106 (see FIGS. 13 and 14). Control over the temperature of the incubation section 114 enables enzymatic reactions to take place with greater efficiency. Similarly, the amplification section 112 has a heated amplification microchannel 158 in a serpentine configuration leading from the boiling-initiated valve 106 to the boiling-initiated valve 108 (see FIGS. 6 and 14). These valves arrest the flow to retain the target cells in the heated incubation or amplification microchannels 210 or 158 while mixing, incubation and nucleic acid amplification takes place. The serpentine pattern of the microchannels also facilitates (to some extent) mixing of the target cells with reagents.
• In the incubation section 114 and the amplification section 112, the sample cells and the reagents are heated by the heaters 154 controlled by the CMOS circuitry 86 using pulse width modulation (PWM). Each meander of the serpentine configuration of the heated incubation microchannel 210 and amplification microchannel 158 has three separately operable heaters 154 extending between their respective heater contacts 156 (see FIG. 14) which provides for the two-dimensional control of input heat flux density. As best shown in FIG. 51, the heaters 154 are supported on the roof layer 66 and embedded in the lower seal 64. The heater material is TiAl but many other conductive metals would be suitable. The elongate heaters 154 are parallel with the longitudinal extent of each channel section that forms the wide meanders of the serpentine shape. In the amplification section 112, each of the wide meanders can operate as separate PCR chambers via individual heater control.
• The small volumes of amplicon required by the assay systems using microfluidic devices, such as LOC device 301, permit low amplification mixture volumes for amplification in amplification section 112. This volume is easily less than 400 nanoliters, in the vast majority of cases less than 170 nanoliters, typically less than 70 nanoliters and in the case of the LOC device 301, between 2 nanoliters and 30 nanoliters.
Increased Rates of Heating and Greater Diffusive Mixing
• The small cross section of each channel section increases the heating rate of the amplification fluid mix. All the fluid is kept a relatively short distance from the heater 154. Reducing the channel cross section (that is the amplification microchannel 158 cross section) to less than 100,000 square microns achieves appreciably higher heating rates than that provided by more ‘macro-scale’ equipment. Lithographic fabrication techniques allow the amplification microchannel 158 to have a cross sectional area transverse to the flow-path less than 16,000 square microns which gives substantially higher heating rates. Feature sizes on the order of 1 micron are readily achievable with lithographic techniques. If very little amplicon is needed (as is the case in the LOC device 301), the cross sectional area can be reduced to less than 2,500 square microns. For diagnostic assays with 1,000 to 2,000 probes on the LOC device, and a requirement of ‘sample-in, answer out’ in less than 1 minute, a cross sectional area transverse to the flow of between 400 square microns and 1 square micron is adequate.
• The heater element in the amplification microchannel 158 heats the nucleic acid sequences at a rate more than 80 Kelvin (K) per second, in the vast majority of cases at a rate greater than 100 K per second. Typically, the heater element heats the nucleic acid sequences at a rate more than 1,000 K per second and in many cases, the heater element heats the nucleic acid sequences at a rate more than 10,000 K per second. Commonly, based on the demands of the assay system, the heater element heats the nucleic acid sequences at a rate more than 100,000 K per second, more than 1,000,000 K per second more than 10,000,000 K per second, more than 20,000,000 K per second, more than 40,000,000 K per second, more than 80,000,000 K per second and more than 160,000,000 K per second.
• A small cross-sectional area channel is also beneficial for diffusive mixing of any reagents with the sample fluid. Before diffusive mixing is complete, diffusion of one liquid into the other is greatest near the interface between the two. Concentration decreases with distance from the interface. Using microchannels with relatively small cross sections transverse to the flow direction, keeps both fluid flows close to the interface for more rapid diffusive mixing. Reducing the channel cross section to less than 100,000 square microns achieves appreciably higher mixing rates than that provided by more ‘macro-scale’ equipment. Lithographic fabrication techniques allows microchannels with a cross sectional area transverse to the flow-path less than 16000 square microns which gives significantly higher mixing rates. If small volumes are needed (as is the case in the LOC device 301), the cross sectional area can be reduced to less than 2500 square microns. For diagnostic assays with 1000 to 2000 probes on the LOC device, and a requirement of ‘sample-in, answer out’ in less than 1 minute, a cross sectional area transverse to the flow of between 400 square microns and 1 square micron is adequate.
Short Thermal Cycle Times
• Keeping the sample mixture proximate to the heaters, and using very small fluid volumes allows rapid thermal cycling during the nucleic acid amplification process. Each thermal cycle (i.e. denaturing, annealing and primer extension) is completed in less than 30 seconds for target sequences up to 150 base pairs (bp) long. In the vast majority of diagnostic assays, the individual thermal cycle times are less than 11 seconds, and a large proportion are less than 4 seconds. LOC devices 30 with some of the most common diagnostic assays have thermal cycles time between 0.45 seconds to 1.5 seconds for target sequences up to 150 bp long. Thermal cycling at this rate allows the test module to complete the nucleic acid amplification process in much less than 10 minutes; often less than 220 seconds. For most assays, the amplification section generates sufficient amplicon in less than 80 seconds from the sample fluid entering the sample inlet. For a great many assays, sufficient amplicon is generated in 30 seconds.
• Upon completion of a preset number of amplification cycles, the amplicon is fed into the hybridization and detection section 52 via the boiling-initiated valve 108.
Hybridization Chambers
• FIGS. 52, 53, 54, 56 and 57 show the hybridization chambers 180 in the hybridization chamber array 110. The hybridization and detection section 52 has a 24×45 array 110 of hybridization chambers 180, each with hybridization-responsive FRET probes 186, heater element 182 and an integrated photodiode 184. The photodiode 184 is incorporated for detection of fluorescence resulting from the hybridization of a target nucleic acid sequence or protein with the FRET probes 186. Each photodiode 184 is independently controlled by the CMOS circuitry 86. Any material between the FRET probes 186 and the photodiode 184 must be transparent to the emitted light. Accordingly, the wall section 97 between the probes 186 and the photodiode 184 is also optically transparent to the emitted light. In the LOC device 301, the wall section 97 is a thin (approximately 0.5 micron) layer of silicon dioxide.
• Incorporation of a photodiode 184 directly beneath each hybridization chamber 180 allows the volume of probe-target hybrids to be very small while still generating a detectable fluorescence signal (see FIG. 54). The small amounts permit small volume hybridization chambers. A detectable amount of probe-target hybrid requires a quantity of probe, prior to hybridization, which is easily less than 270 picograms (corresponding to 900,000 cubic microns), in the vast majority of cases less than 60 picograms (corresponding to 200,000 cubic microns), typically less than 12 picograms (corresponding to 40,000 cubic microns) and in the case of the LOC device 301 shown in the accompanying figures, less than 2.7 picograms (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chambers allows a higher density of chambers and therefore more probes on the LOC device. In LOC device 301, the hybridization section has more than 1,000 chambers in an area of 1,500 microns by 1,500 microns (i.e. less than 2,250 square microns per chamber). Smaller volumes also reduce the reaction times so that hybridization and detection is faster. An additional advantage of the small amount of probe required in each chamber is that only very small quantities of probe solution need to be spotted into each chamber during production of the LOC device. Embodiments of the LOC device according to the invention can be spotted using a probe solution volume of 1 picoliter or less.
• After nucleic acid amplification, boiling-initiated valve 108 is activated and the amplicon flows along the flow-path 176 and into each of the hybridization chambers 180 (see FIGS. 52 and 56). An end-point liquid sensor 178 indicates when the hybridization chambers 180 are filled with amplicon and the heaters 182 can be activated.
• After sufficient hybridization time, the LED 26 (see FIG. 2) is activated. The opening in each of the hybridization chambers 180 provides an optical window 136 for exposing the FRET probes 186 to the excitation radiation (see FIGS. 52, 54 and 56). The LED 26 is illuminated for a sufficiently long time in order to induce a fluorescence signal from the probes with high intensity. During excitation, the photodiode 184 is shorted. After a pre-programmed delay 300 (see FIG. 2), the photodiode 184 is enabled and fluorescence emission is detected in the absence of the excitation light. The incident light on the active area 185 of the photodiode 184 (see FIG. 54) is converted into a photocurrent which can then be measured using CMOS circuitry 86.
• The hybridization chambers 180 are each loaded with probes for detecting a single target nucleic acid sequence. Each hybridization chambers 180 can be loaded with probes to detect over 1,000 different targets if desired. Alternatively, many or all the hybridization chambers can be loaded with the same probes to detect the same target nucleic acid repeatedly. Replicating the probes in this way throughout the hybridization chamber array 110 leads to increased confidence in the results obtained and the results can be combined by the photodiodes adjacent those hybridization chambers to provide a single result if desired. The person skilled in the art will recognise that it is possible to have from one to over 1,000 different probes on the hybridization chamber array 110, depending on the assay specification.
• Hybridization Chambers with Electrochemiluminescence Detection
• FIGS. 239, 272, 303 and 304 show the hybridization chambers 180 used in an ECL variant of the LOC device, LOC variant L 729. In this embodiment of the LOC device, a 24×45 array 110 of hybridization chambers 180, each with hybridization-responsive ECL probes 237, is positioned in registration with a corresponding array of photodiodes 184 integrated into the CMOS. In a similar fashion to the LOC devices configured for fluorescence detection, each photodiode 184 is incorporated for detection of ECL resulting from the hybridization of a target nucleic acid sequence or protein with an ECL probe 237. Each photodiode 184 is independently controlled by the CMOS circuitry 86. Again, the transparent wall section 97 between the probes 186 and the photodiode 184 is transparent to the emitted light.
• A photodiode 184 closely adjacent each hybridization chamber 180 allows the amount of probe-target hybrids to be very small while still generating a detectable ECL signal (see FIG. 239). The small amounts permit small volume hybridization chambers. A detectable amount of probe-target hybrid requires a quantity of probe, prior to hybridization, which is easily less than 270 picograms (corresponding to a chamber volume of 900,000 cubic microns), in the vast majority of cases less than 60 picograms (corresponding to 200,000 cubic microns), typically less than 12 picograms (corresponding to 40,000 cubic microns) and in the case of the LOC device shown in the drawings less than 2.7 picograms (corresponding to a chamber volume of 9,000 cubic microns). Of course, reducing the size of the hybridization chambers allows a higher density of chambers and therefore more probes on the LOC device. In the LOC device shown, the hybridization section has more than 1,000 chambers in an area of 1,500 microns by 1,500 microns (i.e. less than 2,250 square microns per chamber). Smaller volumes also reduce the reaction times so that hybridization and detection is faster. An additional advantage of the small amount of probe required in each chamber is that only very small quantities of probe solution need be spotted into each chamber during production of the LOC device. In the case of the LOC device shown in the drawings, the required amount of probe can be spotted using a solution volume of 1 picoliter or less.
• After nucleic acid amplification, the boiling-initiated valve 108 is activated and the amplicon flows along the flow-path 176 and into each of the hybridization chambers 180 (see FIGS. 52 and 304). An end-point liquid sensor 178 indicates when the hybridization chambers 180 are filled with amplicon so that the heaters 182 can be activated.
• After sufficient hybridization time, the photodiode 184 is enabled ready for collection of the ECL signal. Then the ECL excitation drivers 39 (see FIG. 253) activate the ECL electrodes 860 and 870 for a predetermined length of time. The photodiode 184 remains active for a short time after cessation of the ECL excitation current to maximize the signal-to-noise ratio. For example, if the photodiode 184 remains active for five times the decay lifetime of the luminescent emission, then the signal will have decayed to less than one percent of the initial value. The incident light on the photodiode 184 is converted into a photocurrent which can then be measured using CMOS circuitry 86.
Proteomic Assay Chambers
• Some LOC variants, such as LOC variant L 729, are configured to perform homogeneous protein assays on crude cell lysates within proteomic assay chamber arrays (see for example 124.1 to 124.3 of FIGS. 268 and 272) for the detection of host cell and/or pathogenic proteins. The proteomic assay chamber arrays 124.1-124.3 are manufactured and configured in exactly the same manner as the hybridization chamber arrays 110 (see FIGS. 52, 53, 54 and 56). Each proteomic assay chamber has a diffusion barrier 175 at the entrance to prevent diffusion of sample and reagents between chambers, thus avoiding an erroneous result (see FIGS. 198 and 199, which are insets DC and DD of FIG. 195). Where required for protein hybridization or conjugation, thermal energy is provided by CMOS-controlled heaters 182 in each chamber. In some embodiments, an end-point liquid sensor 178 is used to indicate when the proteomic assay chambers are filled with sample so that the heaters 182 can be activated. After sufficient time has elapsed, the fluorescent or electrochemiluminescent signal generated following protein recognition is detected by the photosensor 44.
Humidifier and Humidity Sensor
• Inset AG of FIG. 6 indicates the position of the humidifier 196. The humidifier prevents evaporation of the reagents and probes during operation of the LOC device 301. As best shown in the enlarged view of FIG. 55, a water reservoir 188 is fluidically connected to three evaporators 190. The water reservoir 188 is filled with molecular biology-grade water and sealed during manufacturing. As best shown in FIGS. 55 and 88, water is drawn into three downtakes 194 and along respective water supply channels 192 by capillary action to a set of three uptakes 193 at the evaporators 190. A meniscus pins at each uptake 193 to retain the water. The evaporators have annular shaped heaters 191 which encircle the uptakes 193. The annular heaters 191 are connected to the CMOS circuitry 86 by the conductive columns 376 to the top metal layer 195 (see FIG. 37). Upon activation, the annular heaters 191 heat the water causing evaporation and humidifying the device surrounds.
• The position of the humidity sensor 232 is also shown in FIG. 6. However, as best shown in the enlarged view of Inset AH in FIG. 67, the humidity sensor has a capacitive comb structure. A lithographically etched first electrode 296 and a lithographically etched second electrode 298 face each other such that their teeth are interleaved. The opposed electrodes form a capacitor with a capacitance that can be monitored by the CMOS circuitry 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, so that the capacitance also increases. The humidity sensor 232 is adjacent the hybridization chamber array 110 where humidity measurement is most important to slow evaporation from the solution containing the exposed probes.
Feedback Sensors
• Temperature and liquid sensors are incorporated throughout the LOC device 301 to provide feedback and diagnostics during device operation. Referring to FIG. 35, nine temperature sensors 170 are distributed throughout the amplification section 112 Likewise, the incubation section 114 also has nine temperature sensors 170. These sensors each use a 2×2 array of bipolar junction transistors (BJTs) to monitor the fluid temperature and provide feedback to the CMOS circuitry 86. The CMOS circuitry 86 uses this to precisely control the thermal cycling during the nucleic acid amplification process and any heating during thermal lysis and incubation.
• In the hybridization chambers 180, the CMOS circuitry 86 uses the hybridization heaters 182 as temperature sensors (see FIG. 56). The electrical resistance of the hybridization heaters 182 is temperature dependent and the CMOS circuitry 86 uses this to derive a temperature reading for each of the hybridization chambers 180.
• The LOC device 301 also has a number of MST channel liquid sensors 174 and cap channel liquid sensors 208. FIG. 35 shows a line of MST channel liquid sensors 174 at one end of every other meander in the heated microchannel 158. As best shown in FIG. 37, the MST channel liquid sensors 174 are a pair of electrodes formed by exposed areas of the top metal layer 195 in the CMOS structure 86. Liquid closes the circuit between the electrodes to indicate its presence at the sensor's location.
• FIG. 25 shows an enlarged perspective of cap channel liquid sensors 208. Opposing pairs of TiAl electrodes 218 and 220 are deposited on the roof layer 66. Between the electrodes 218 and 220 is a gap 222 to hold the circuit open in the absence of liquid. The presence of liquid closes the circuit and the CMOS circuitry 86 uses this feedback to monitor the flow.
Gravitational Independence
• The test modules 10 are orientation independent. They do not need to be secured to a flat stable surface in order to operate. Capillary driven fluid flows and a lack of external plumbing into ancillary equipment allow the modules to be truly portable and simply plugged into a similarly portable hand held reader such as a mobile telephone. Having a gravitationally independent operation means the test modules are also accelerationally independent to all practical extents. They are resistant to shock and vibration and will operate on moving vehicles or while the mobile telephone is being carried around.
Valve Alternatives Thermal Bend Actuated Valve Variant 1
• FIGS. 68 and 69 show a first variant 302 of a thermal bend actuated valve which is a thermal bend actuated pressure pulse valve. FIG. 69 is a schematic section view through line 70-70 shown in FIG. 68. The first variant thermal bend actuated valve 302 has movable member in the form of a CMOS-activated thermal bend actuator 304 of TiAl, TiN or similar resistive heater material. The sample flow enters the valve inlets 146 in the MST channel 90 but stops when a meniscus 120 pins to the aperture 306. The embodiment shown in FIGS. 68 and 69 shows the aperture external to the movable member, but it may also be defined at least partially by the movable member. In this state, the valve is closed. To open the valve, the CMOS circuitry 86 sends a series of electrical pulses to the thermal bend actuator 304. The CMOS-activated thermal bend actuator 304 is bonded to a cantilevered section 162 of the roof layer 66 fixed at the inlet end and free at the aperture end. Differential thermal expansion bends the cantilevered section 162 such that it moves rapidly towards the passivation layer 88. Fluidic drag prevents backflow of the liquid sample 119 which instead, is ejected through the aperture 306 into the cap channel 94. Reciprocating the cantilevered section 162 between the quiescent and displaced positions for a period of time ensures the meniscus 120 unpins from the aperture 306. Sample 119 accumulates in the cap channel 94 until its surfaces are wetted and capillary driven flow resumes. The sample fills the cap channel 94 and then flows to the valve outlet 148 through the valve downtake 150.
Thermal Bend Actuated Valve Variant 2
• FIGS. 70 and 71 show a second variant of the thermal bend actuated valve 308 which is a thermal bend actuated surface tension valve. FIG. 71 is a schematic section view through line 72-72 shown in FIG. 70. The second variant of the thermal bend actuated valve 308 has a CMOS-activated thermal bend actuator 304 of TiAl, TiN or similar resistive heater material. The sample flows along the MST channel 90 and into the valve inlet 146 provided by the valve uptake 151. The liquid sample 119 fills the cap channel 94 but stops when a meniscus 120 pins to the aperture 306. In this state, the valve is closed. To open the valve, the CMOS circuitry 86 sends a series of electrical pulses to the thermal bend actuator 304. The CMOS-activated thermal bend actuator 304 is bonded to a cantilevered section 162 of the roof layer 66 fixed at the inlet end and free at the aperture end. Differential thermal expansion bends the cantilevered section 162 such that the aperture 306 moves towards the passivation layer 88. The aperture 306 drags the meniscus 120 from the cap channel 94 into the MST channel 90 to re-establish capillary flow. To re-establish the capillary flow, the channel has opposing side walls that converge to a narrow section immediately downstream of the movable member such that the meniscus contacts the narrow section when the movable member moves to the actuated position. Liquid sensors 174 are placed before and after the valve for diagnostics.
Thermal Bend Actuated Valve Variant 3
• FIGS. 72 and 73 show a third variant of the thermal bend actuated valve 312, which is a thermal bend actuated surface tension valve, and is used when the liquid sample is to be retained in the cap channel 94. FIG. 73 is a schematic section view through line 74-74 shown in FIG. 72. The third variant of the thermal bend actuated valve 312 is similar to the second variant of the thermal bend actuated valve 308 with the exception that the valve inlet 146 is in a cap channel 94. The sample flows along the cap channel 94 and into the valve inlet 146 immediately upstream of the CMOS-activated thermal bend actuator 304. The liquid sample 119 fills the cap channel 94 but stops when a meniscus 120 pins to the aperture 306. In this state, the valve is closed. To open the valve, the CMOS circuitry 86 sends a series of electrical pulses to the thermal bend actuator 304. The thermal bend actuator 304 is bonded to a cantilevered section 162 of the roof layer 66 fixed at the inlet end and free at the aperture end. Differential thermal expansion bends the cantilevered section 162 such that the aperture 306 moves towards the passivation layer 88. The aperture 306 drags the meniscus 120 from the cap channel 94 into the MST channel 90 to re-establish capillary flow along the valve outlet 148. A liquid sensor 174 is placed after the valve for diagnostics.
Fault-Tolerant Multiple Valve Array
• FIG. 74 shows a fault tolerant multiple valve array 314 that may be used instead of any of the valve variants 108, 302, 308 and 312 described above. FIG. 75 is a schematic section view of the fault tolerant multiple valve array 314 taken through line 76-76 of FIG. 74.
• In the valve variants described above, there is a risk that the sample flow will fail to pin at the aperture 306 (or valve inlet 146 in the case of the boiling-initiated valves) and the capillary flow simply continues to the valve outlet 148. In effect, the valve fails to close. Conversely, the valve may fail to open (that is, the meniscus does not unpin when the valve actuates). To address this, the fault tolerant multiple valve array 314 provides fault-tolerance that will allow one or more individual valve failures.
• The fault tolerant multiple valve array 314 has four individual valves; first valve 316, second valve 318, third valve 320 and fourth valve 322. All valves are thermal bend actuated valves of the second variant 308 type (thermal bend actuated surface tension valve) described above. A first flow-path 324 and a second flow-path 326 extend between the valve inlet 146 and valve outlet 148. The first and second valves (316 and 318) are positioned along the first flow-path 324 and the third and fourth valves (320 and 322) are on the second flow-path 326.
• Liquid sensors 174 are positioned at the valve inlet 146 and the valve outlet 148, and between the two valves in each flow-path. The sensors register which, if any, of the first, second, third or fourth valves fail. If the first or the third valves (316 and 320) fail to pin a meniscus, the sample 119 flow is stopped by the second and fourth valves (318 and 322).
• Similarly, if the meniscus fails to unpin during actuation of the first and third valves, an alternative flow-path is available. The fault tolerant multiple valve array 314 only fails in the unlikely event that both valves in the first or second flow-paths (324 or 326) fail. For greater fault tolerance, the fault tolerant multiple valve array 314 is arbitrarily expandable in terms of the number of fluid flow-paths and the number of valves on each flow-path.
Boiling-Initiated Valve Array
• FIGS. 92 and 93 show another variant of the fault tolerant multiple valve array 448 in which the four individual valves are all boiling-initiated valves. As with the mechanical fault tolerant valve array 314, there is a single valve array inlet 146 and valve outlet 148. The valve inlet 146 and the valve outlet 148 are formed in the MST channel layer 100 and each have respective liquid sensors 174 providing feedback to the CMOS circuitry 86. At the valve inlet 146, the flow bifurcates into the first flow-path 324 and the second flow-path 326, both of which extend between the valve inlet 146 and valve outlet 148. The first and second boiling-initiated valves (412 and 416) are positioned along the first flow-path 324 and the third and fourth boiling-initiated valves (414 and 418) are on the second flow-path 326.
• Liquid sensors 174 are also positioned between the two boiling-initiated valves in each flow-path. The sensors register which, if any, of the first, second, third or fourth boiling-initiated valves fail. If the first or the third boiling-initiated valves (412 and 414) fail, the sample flow is most likely restrained by the second and fourth boiling-initiated valves (416 and 418). The fault tolerant multiple valve array 448 only fails in the unlikely event both boiling-initiated valves in the first flow-path 324 or second flow-paths 326 fail. For greater fault tolerance, the fault tolerant multiple valve array 448 is arbitrarily expandable in terms of the number of fluid flow-paths and the number of boiling-initiated valves on each flow-path.
• If the valve array operates without any boiling-initiated valve failure, the flow from the valve inlet 146 pins a meniscus in valve uptakes 428 and 430 in the first boiling-initiated valve 412 and the third boiling-initiated valve 414 respectively. To open the boiling-initiated valves 412 and 414, the CMOS circuitry 86 sends activation pulses to the boiling-initiated valve heater contacts 153 in both valves. The heater 152 in both boiling-initiated valves 412 and 414 heats the liquid at the meniscus above boiling temperature. Boiling unpins the menisci at both valve uptakes 428 and 430 such that capillary driven flow fills the cap channels 420 and 422 respectively.
• The valve downtakes 432 and 434 are configured so as not to pin a meniscus and arrest the capillary driven flow. The flow into the cap channels 420 and 422 continues into the first flow-path MST channel 424 and the second flow-path MST channel 426 via the valve downtakes 432 and 434 respectively.
• Capillary driven flow continues to the second and fourth boiling-initiated valves 416 and 418. Once again the flow pins a meniscus at the valve uptakes 436 and 438 of each boiling-initiated valve respectively. The liquid sensors 174 in each of the MST channels 424 and 426 provide feedback to the CMOS circuitry 86 which times the activation pulses for the valve heaters 152 accordingly. As with the first and third boiling-initiated valves 412 and 414, activating the heaters 152 in the second and fourth boiling-initiated valves 416 and 418 boils liquid at the meniscus to unpin it from the uptakes 436 and 438. Capillary driven flow into the cap channels 440 and 442 is followed by capillary driven flow through the downtakes 444 and 446 respectively. Flow along the first flow-path 324 reunites with flow in the second flow-path 326 at the valve outlet 148 where the liquid sensor 174 signals the CMOS circuitry 86 that the fault tolerant multiple valve array 448 has opened.
Electroexplosive Valve
• FIG. 246 is a plan view of an electroexplosive valve 71. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of the roof layer membrane 75 when a meniscus 120 pins to each aperture 306. In this state, the valve is closed. When the CMOS circuitry 86 forces current through the resistors 77, the point along each resistor of highest current density 79 explodes to destroy a roof layer rigid link 81 connecting the membrane 75 to fixed surroundings. With the membrane 75 unsupported and dislodged, the valve is open, and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148.
Thermal-Bend-Actuated Bend-and-Break Valve
• A thermal-bend-actuated bend-and-break valve 83 is shown in plan view in FIG. 247. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of the roof layer cantilevered section 162 and connected membrane 75 when a meniscus 120 pins at each aperture 306. In this state, the valve is closed. A series of electrical pulses from the CMOS circuitry 86 to the thermal bend actuator 304 forces the roof layer cantilevered section 162, to which the thermal bend actuator 304 is bonded, to deflect towards the passivation layer 88 by differential thermal expansion, breaking the weak roof layer rigid links 81 connecting the membrane 75 to its surroundings. With the membrane 75 unsupported and dislodged, the valve is open, and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148.
Dual Thermal-Bend-Actuated Bend-and-Break Valve
• A dual thermal-bend-actuated bend-and-break valve 85 is shown in plan view in FIG. 248. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of both roof layer cantilevered sections 162 and the connected membrane 75 when a meniscus 120 pins at each aperture 306. In this state, the valve is closed. Both thermal bend actuators 304 are bonded to a roof layer cantilevered section 162 and deflect towards the passivation layer 88 by differential thermal expansion upon receiving an electrical pulse from the CMOS circuitry 86 to break the rigid links 81 connecting the membrane 75 to its surroundings. With the membrane 75 unsupported and dislodged, the valve is open, and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148.
Stiction Valve
• A stiction valve 89 is shown in plan view in FIG. 249. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of the roof layer cantilevered section 162 and connected membrane 75 when a meniscus 120 pins at each aperture 306. In this state, the valve is closed. A series of electrical pulses from the CMOS circuitry 86 to the thermal bend actuator 304 forces the roof layer cantilevered section 162, to which the thermal bend actuator 304 is bonded, to deflect by differential thermal expansion, forcing the membrane 75 into contact with the passivation layer 88. With stiction between the membrane 75 and passivation layer 88 counterbalancing the restorative force, and deformation of the flexible link 91 accommodating the cantilevered section 162 restorative displacement, the valve remains statically open for the liquid sample 119 to flow from the valve inlet 146 to the valve outlet 148.
Stiction Valve Variant
• A stiction valve variant 93 is shown in plan view in FIG. 250. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of the roof layer cantilevered section 162 when a meniscus 120 pins at each aperture 306. In this state, the valve is closed. A series of electrical pulses from the CMOS circuitry 86 to the thermal bend actuator 304 forces the roof layer cantilevered section 162, to which the thermal bend actuator 304 is bonded, to deflect by differential thermal expansion and contact the passivation layer 88. With stiction between the cantilevered section 162 and passivation layer 88 counterbalancing the restorative force, and the length of the cantilevered section 162 sufficient so that its mechanical flexibility accommodates restorative displacement of the thermal bend actuator 304, the valve remains statically open for the liquid sample 119 to flow from the valve inlet 146 to the valve outlet 148.
Bubble Break Valve
• A bubble break valve 95 is shown in plan view in FIG. 251. The sample flows along the MST channel 90 and into the valve inlet 146 formed by the valve uptake 151. The liquid sample 119 fills the cap valve interface cavity 73, stopping one side of the roof layer membrane 75 when the meniscus 120 pins at the aperture 306. In this state, the valve is closed. The annular heater 152 is resistively heated by an electrical pulse from the CMOS circuitry 86 until a vapor bubble is generated in the liquid sample that forces the membrane 75 to rotate around and break the roof layer rigid link 81. With the membrane 75 unsupported and dislodged, the valve is open, and the liquid sample 119 flows from the valve inlet 146 to the valve outlet 148.
Other Valve Array Variants
• Any of the valve variants described above can be used to form a valve array. Furthermore, the valve array can include different types of valve.
Dialysis Variants Leukocyte Target
• The dialysis design described above in the LOC device 301 targets pathogens. FIG. 76 is a schematic section view of a dialysis section 328 designed to concentrate leukocytes from a blood sample for human DNA analysis. It will be appreciated that the structure is essentially the same as that of the pathogen target dialysis section 70 described above with the exception that apertures in the form of 7.5 micron diameter holes 165 restrict leukocytes from passing from the cap channel 94 to the dialysis MST channels 204. In situations where the sample being analysed is a blood sample, and the presence of haemoglobin from the erythrocytes interferes with the subsequent reaction steps, addition of an erythrocyte lysis buffer along with the anticoagulant in the reservoir 54 (see FIG. 22), will ensure that the majority of the lysed erythrocytes (and hence haemoglobin) will be removed from the sample during this dialysis step. A commonly used erythrocyte lysis buffer is 0.15M NH₄CL, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.2-7.4, but a person skilled in the art will recognise that any buffer which efficiently lyses erythrocytes can be used.
• Downstream of the leukocyte dialysis section 328, the cap channel 94 becomes the target channel 74 such that the leukocytes continue as part of the assay. Furthermore, in this case, the dialysis uptake holes 168 lead to a waste channel 72 so that all smaller cells and components in the sample are removed. It should be noted that this dialysis variant only reduces the concentration of the unwanted specimens in the target channel 74.
• FIG. 170 schematically illustrates a large constituents dialysis section 686 which also separates any large target constituents from a sample. The apertures in this dialysis section are fabricated with a size and shape tailored to withhold the large target constituents of interest in the target channel for further analysis. As with the leukocyte dialysis section described above, most (but not all) smaller sized cells, organisms or molecules flow to a waste reservoir 768. Thus, other embodiments of the LOC device are not limited to separating leukocytes that are larger than 7.5 μm in size, but can be used to separate cells, organisms or molecules of any size desired.
• Dialysis Section with Flow Channel to Prevent Trapped Air Bubbles
• Described below is an embodiment of the LOC device referred to as LOC variant VIII 518 and shown in FIGS. 110, 114, 115 and 139. This LOC device has a dialysis section that fills with the fluid sample without leaving air bubbles trapped in the channels. LOC variant VIII 518 also has an additional layer of material referred to as an interface layer 594. The interface layer 594 is positioned between the cap channel layer 80 and the MST channel layer 100 of the CMOS+MST device 48. The interface layer 594 allows a more complex fluidic interconnection between the reagent reservoirs and the MST layer 87 without increasing the size of the silicon substrate 84.
• Referring to FIG. 114, the bypass channel 600 is designed to introduce a time delay in the fluid sample flow from the interface waste channel 604 to the interface target channel 602. This time delay allows the fluid sample to flow through the dialysis MST channel 204 to the dialysis uptake 168 where it pins a meniscus. With a capillary initiation feature (CIF) 202 at the uptake from the bypass channel 600 to the interface target channel 602, the sample fluid fills the interface target channel 602 from a point upstream of all the dialysis uptakes 168 from the dialysis MST channels 204.
• Without the bypass channel 600, the interface target channel 602 still starts filling from the upstream end, but eventually the advancing meniscus reaches and passes over an uptake belonging to an MST channel that has not yet filled, leading into air entrapment at that point. Trapped air reduces the sample flow rate through the leukocyte dialysis section 328.
• Dialysis Section with Active Valve and Liquid Sensor
• FIG. 186 schematically illustrates an alternative dialysis section 722 with features designed to avoid trapped bubbles. The alternative dialysis section has the same structure as that shown in FIG. 114, but without the bypass channel 600. The fluid sample enters the interface waste channel 604 from the cap channel 94. Fluid sample flows into the dialysis MST channels 204 through the 3 micron downtake openings 164. Cells larger than 3 microns remain in the interface waste channel 604 and are eventually directed to the waste reservoir 76. The dialysis uptake openings 168 are surface tension valves that pin a meniscus to halt the flow of the fluid sample. Instead of a bypass channel 600 (see FIG. 114), an active valve, such as a boiling-initiated valve 724 is placed at the dialysis uptake opening 168 of the dialysis MST channel 204 at the upstream end. In the dialysis MST channel 204 at the downstream end is a liquid sensor 174. When all the dialysis MST channels 204 have filled, the liquid sensor 174 triggers the boiling-initiated valve 724. The sample flows into the upstream end of the interface target channel 602 and eventually opens the surface tension valves at each of the dialysis uptake openings 168 sequentially in the downstream direction.
• Without the boiling-initiated valve 724, the interface target channel 602 (assuming that the small constituents channel contains the target cells, such as pathogens) starts filling from the upstream end, but the advancing meniscus is prone to pass over a dialysis uptake 168 that has not yet filled, leading to air entrapment at that point. Any active valve variant can be substituted for the boiling-initiated valve 724 and in a further embodiment, all the dialysis uptakes 168 can have an active valve to ensure that the sample flows into the interface target channel 602 from all the dialysis MST channels 204.
Pre-Hybridization Filtering
• A variant of the LOC device, LOC variant XII 758, uses a small constituents dialysis section 682 placed at the outlet of the amplification section 112 (see FIGS. 215 to 222). The small constituents dialysis section 682 provides a pre-hybridization filter purification phase 293 (see FIG. 215). Pre-hybridization filtering removes cell debris remaining in the sample flow following cell lysis. Hybridization efficiency can be affected by cell debris, so it is advantageous to reduce the concentration of cell debris prior to hybridization.
• Referring to FIGS. 220, 221 and 222, the small constituents dialysis section 682 has three adjacent channels fabricated in the bottom channel layer 100; a large constituents channel 760 flanked by two small constituents channels 762. A series of stoma in the form of inverse tapered openings 764 along both sides of the large constituents channel 760 provide a fluidic connection to the small constituents channels 762. In most practical applications, the stoma will be between 1 to 8 microns wide and 1 to 8 microns high. As the sample flows down the large constituents channel 760, particles small enough to pass through the inverse tapered openings (e.g. the amplicon) flow through to the small constituents channels 762, while the larger particles (e.g. cell debris) remain in the large constituents channel which eventually terminates at a blind end 766. The smaller particles continue along the small constituents channels to opposite sides of the hybridization chamber array 110 where they both follow serpentine paths through the array to respective blind ends 768 (see FIG. 222). The small constituents amplicons fill all the individual hybridization chambers 180 prior to detection.
Nucleic Acid Amplification Variants Parallel PCR
• Several variants of the LOC device have multiple amplification sections operating in parallel. For example, LOC variant VII 492 shown in FIG. 108 has parallel amplification sections 112.1 to 112.4 which allow multiple nucleic acid amplification assays to be performed simultaneously.
• LOC variant XI 746 shown in FIG. 207 also has parallel amplification sections 112.1 to 112.4, but additionally has parallel incubation sections 114.1 to 114.4, so that the sample can be processed differently prior to amplification. Other LOC variants, such as LOC XIV 641 shown schematically in FIG. 142, demonstrate that the number of parallel amplification sections can be “X” number, which is only limited by the size of the LOC device. A larger LOC device can be made to accommodate greater numbers of parallel amplification sections.
• Separate amplification sections can be configured to run on different cycle times and/or temperatures for particular target sizes or particular amplification mix constituents. With several amplification sections running in parallel, the LOC device can operate a multiplex nucleic acid amplification process or a uniplex amplification process in each of the sections. In multiplex nucleic acid amplification, more than one target sequence is amplified using more than one pair of primers. A parallel nucleic acid amplification system with “m” chambers can run the equivalent of an n-plex amplification where n=n(1)+n(2)+ . . . +n(i)+ . . . +n(m), with n(i) being the number of different primer pairs used in the multiplex amplification being run in chamber “i”, bearing in mind that the SNR (signal-to-noise ratio) in the parallel amplification system is higher than the n-plex amplification run in a single chamber system. Under the special case where n(i)=1, the amplification in chamber “i” becomes just a uniplex amplification.
Tandem PCR
• FIGS. 143, 146, 150, 153 and 156 (amongst others) schematically illustrate LOC devices in which the amplification sections 112.1 and 112.2 operate in series. The first amplification section 112.1 includes two reagent reservoirs, 60.1 for the amplification mix and 62.1 for the polymerase. Each amplification section added after the initial section also includes two reagent reservoirs, reservoir 60.2 for the amplification mix and reservoir 62.2 for the polymerase.
• Amplification sections in series allow tandem PCR assays whereby the first amplification section 112.1 is for pre-amplification to increase the sensitivity of the subsequent nucleic acid amplification performed in section 112.2. Amplification sections in series can also be used for nested PCR reactions.
• In tandem PCR used for pre-amplification, the first amplification section 112.1 is used to amplify nucleic acid sequences in the sample which include the target sequence. This amplification need not be specific to the target sequence (for example, whole genome amplification), but does increase the target sequence concentration. After pre-amplification, the sample is mixed with reagents from the reservoirs 60.2 and 62.2 and then passes into the second amplification section 112.2. The reagents stored in reservoir 60.2 include specific primers to amplify only the target sequence in the pre-amplified sample mix. It should be noted that a similar approach, where PCR is replaced by an isothermal technique in the first or second amplification stages, can also be employed to achieve the advantages of preamplification.
• Nested PCR is a particular form of tandem PCR, with the additional advantage of high target specificity. In nested PCR, the nucleic acid amplification step in the first amplification section 112.1 amplifies a sequence larger than the final target sequence by using primers, forming part of the amplification mix reagents stored in reservoir 60.1, which are complementary to regions external to the target sequence. The reaction in the first amplification section 112.1 results in amplicons which are the target sequence plus the flanking sections. This amplified mixture is mixed with reagents from reservoir 60.2 and polymerase from 62.2. The reagents stored in reservoir 60.2 include primers which are complementary to sites at each end of the target sequence, i.e. a subsection of the amplicon from the first amplification stage. When nucleic acid amplification is performed in the second section 112.2, the chances of amplification arising from sequence locations unrelated to the target are greatly diminished, as the amplicon concentration from the first amplification stage is far greater than the concentration of the original sample molecules. The sensitivity and specificity advantages of nested PCR can also be achieved when one or both of the PCR amplification stages are replaced by a sequence-specific isothermal amplification technique.
• Storing polymerases separately and adding them independently to the sample mix has the advantage that different polymerases can be selected for the pre-amplification and final nucleic acid amplification steps. For example, this allows selection of a low-error-rate (e.g. proofreading) polymerase for the pre-amplification step to avoid the creation of target sequences containing errors, or false target sequences, while allowing the use of a higher-speed, or more thermally tolerant, polymerase for the final amplification.
Direct PCR
• Traditionally, PCR requires extensive purification of the target DNA prior to preparation of the reaction mixture. However, with appropriate changes to the chemistry and sample concentration, it is possible to perform nucleic acid amplification with minimal DNA purification, or direct amplification. When the nucleic acid amplification process is PCR, this approach is called direct PCR. In LOC devices where nucleic acid amplification is performed at a controlled, constant temperature, the approach is direct isothermal amplification. Direct nucleic acid amplification techniques have considerable advantages for use in LOC devices, particularly relating to simplification of the required fluidic design. Adjustments to the amplification chemistry for direct PCR or direct isothermal amplification include increased buffer strength, the use of polymerases which have high activity and processivity, and additives which chelate with potential polymerase inhibitors. Dilution of inhibitors present in the sample is also important.
• To take advantage of direct nucleic acid amplification techniques, the LOC device designs incorporate two additional features. The first feature is reagent reservoirs (for example reservoir 58 in FIG. 8) which are appropriately dimensioned to supply a sufficient quantity of amplification reaction mix, or diluent, so that the final concentrations of sample components which might interfere with amplification chemistry are low enough to permit successful nucleic acid amplification. The desired dilution of non-cellular sample components is in the range of 5× to 20×. Different LOC structures, for example the pathogen dialysis section 70 in FIG. 4, are used when appropriate to ensure that the concentration of target nucleic acid sequences is maintained at a high enough level for amplification and detection. In this embodiment, further illustrated in FIG. 6, a dialysis section which effectively concentrates pathogens small enough to be passed into the amplification section 292 is employed upstream of the sample extraction section 290, and rejects larger cells to a waste receptacle 76. In another embodiment, a dialysis section is used to selectively deplete proteins and salts in blood plasma while retaining cells of interest.
• The second LOC structural feature which supports direct nucleic acid amplification is design of channel aspect ratios to adjust the mixing ratio between the sample and the amplification mix components. For example, to ensure dilution of inhibitors associated with the sample in the preferred 5×-20× range through a single mixing step, the length and cross-section of the sample and reagent channels are designed such that the sample channel, upstream of the location where mixing is initiated, constitutes a flow impedance 4×-19× higher than the flow impedance of the channels through which the reagent mixture flows. Control over flow impedances in microchannels is readily achieved through control over the design geometry. The flow impedance of a microchannel increases linearly with the channel length, for a constant cross-section. Importantly for mixing designs, flow impedance in microchannels depends more strongly on the smallest cross-sectional dimension. For example, the flow impedance of a microchannel with rectangular cross-section is inversely proportional to the cube of the smallest perpendicular dimension, when the aspect ratio is far from unity.
• Reverse-Transcriptase PCR (RT-PCR)
• Where the sample nucleic acid species being analysed or extracted is RNA, such as from RNA viruses or messenger RNA, it is first necessary to reverse transcribe the RNA into complementary DNA (cDNA) prior to PCR amplification. The reverse transcription reaction can be performed in the same chamber as the PCR (one-step RT-PCR) or it can be performed as a separate, initial reaction (two-step RT-PCR). In the LOC variants described herein, a one-step RT-PCR can be performed simply by adding the reverse transcriptase to reagent reservoir 62 along with the polymerase and programming the heaters 154 to cycle firstly for the reverse transcription step and then progress onto the nucleic acid amplification step. A two-step RT-PCR could also be easily achieved by utilizing the reagent reservoir 58 to store and dispense the buffers, primers, dNTPs and reverse transcriptase and the incubation section 114 for the reverse transcription step followed by amplification in the normal way in the amplification section 112.
Isothermal Nucleic Acid Amplification
• For some applications, isothermal nucleic acid amplification is the preferred method of nucleic acid amplification, thus avoiding the need to repetitively cycle the reaction components through various temperature cycles but instead maintaining the amplification section at a constant temperature, typically around 37° C. to 41° C. A number of isothermal nucleic acid amplification methods have been described, including Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), Helicase-Dependent isothermal DNA Amplification (HDA), Rolling Circle Amplification (RCA), Ramification Amplification (RAM) and Loop-mediated Isothermal Amplification (LAMP), and any of these, or other isothermal amplification methods, can be employed in particular embodiments of the LOC device described herein.
• In order to perform isothermal nucleic acid amplification, the reagent reservoirs 60 and 62 adjoining the amplification section will be loaded with the appropriate reagents for the specified isothermal method instead of PCR amplification mix and polymerase. For example, for SDA, reagent reservoir 60 contains amplification buffer, primers and dNTPs and reagent reservoir 62 contains an appropriate nickase enzyme and Exo-DNA polymerase. For RPA, reagent reservoir 60 contains the amplification buffer, primers, dNTPs and recombinase proteins, with reagent reservoir 62 containing a strand displacing DNA polymerase such as Bsu. Similarly, for HDA, reagent reservoir 60 contains amplification buffer, primers and dNTPs and reagent reservoir 62 contains an appropriate DNA polymerase and a helicase enzyme to unwind the double stranded DNA strand instead of using heat. The skilled person will appreciate that the necessary reagents can be split between the two reagent reservoirs in any manner appropriate for the nucleic acid amplification process.
• For amplification of viral nucleic acids from RNA viruses such as HIV or hepatitis C virus, NASBA or TMA is appropriate as it is unnecessary to first transcribe the RNA to cDNA. In this example, reagent reservoir 60 is filled with amplification buffer, primers and dNTPs and reagent reservoir 62 is filled with RNA polymerase, reverse transcriptase and, optionally, RNase H.
• For some forms of isothermal nucleic acid amplification it may be necessary to have an initial denaturation cycle to separate the double stranded DNA template, prior to maintaining the temperature for the isothermal nucleic acid amplification to proceed. This is readily achievable in all embodiments of the LOC device described herein, as the temperature of the mix in the amplification section 112 can be carefully controlled by the heaters 154 in the amplification microchannels 158 (see FIG. 14).
• Isothermal nucleic acid amplification is more tolerant of potential inhibitors in the sample and, as such, is generally suitable for use where direct nucleic acid amplification from the sample is desired. Therefore, isothermal nucleic acid amplification is sometimes useful in LOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677, amongst others, shown in FIGS. 171, 172 and 175, respectively. Direct isothermal amplification may also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 as shown in FIGS. 171 and 175 and/or a pre-hybridization dialysis step 682 as indicated in FIG. 172 to help partially concentrate the target cells in the sample before nucleic acid amplification or remove unwanted cellular debris prior to the sample entering the hybridization chamber array 110, respectively. The person skilled in the art will appreciate that any combination of pre-amplification dialysis and pre-hybridization dialysis can be used.
• Isothermal nucleic acid amplification can also be performed in parallel amplification sections such as those schematically represented in FIGS. 108, 140 and 142, multiplexed and some methods of isothermal nucleic acid amplification, such as LAMP, are compatible with an initial reverse transcription step to amplify RNA.
Other Design Variants
• Reagent Reservoirs with Active (Mechanical) Valves
• Particular embodiments of the LOC device retain reagents in the reservoirs using thermal bend actuated valves (see FIGS. 68 to 73) instead of surface tension valves (see FIGS. 15 and 22). FIG. 78 shows a reagent reservoir 344 in fluid communication with the target channel 74 via three thermal bend actuated valves of the third variant design 312 shown in FIGS. 72 and 73. The reagent reservoir 344 is filled with reagent which flows into the cap channel 94 to the third variant of the thermal bend actuated valves 312 where a meniscus is formed at the apertures 306 in each of the CMOS-activated thermal bend actuators 304. The fluid sample flows along the target channel 74 past the uptake holes 96. Before the sample flow reaches the uptake holes 96, the third variant of the thermal bend actuated valves 312 open and reagent flows through the MST channels 90 to the uptake holes 96. The reagent starts to fill the target channel 74 when the sample flow reaches the uptake holes 96. The combined flow then continues along the target channel 74.
Conductivity Sensor
• FIG. 233 is a schematic section view of a conductivity sensor 810 used to detect salts and various targets via enzymatic reactions or antibody conjugations (for example) that lead to changes in conductivity. The conductivity sensor 810 measures the conductivity of liquid 812 in the channel 800 by forcing a current between the first terminal 802 and the second terminal 808 and measuring the voltage across the first electrode 804 and second electrode 806. The first and second terminals 802, 808 and first and second electrodes 804, 806, are part of the top metal layer 195 of the CMOS circuitry 86 exposed through windows in the passivation layer 88.
Flow Rate Sensor
• In addition to temperature and liquid sensors, the LOC device can also incorporate CMOS-controlled flow rate sensors 740, as schematically illustrated in FIG. 234 and in LOC Variant X 728 (see FIGS. 190 to 206). The sensors are used to determine the flow rate in two steps. In the first step, the temperature of the serpentine heater element 814 is determined by applying a low current and measuring the voltage to determine the resistance of the serpentine heater element 814, and therefore the temperature of the element 814 using the known relationship between resistance and the temperature of the heater element. At this stage, minimal heat is being dissipated in the element 814 and the temperature of the liquid in the channel is equal to the calculated temperature of the element 814. In the second step, a higher current is applied to the serpentine heater element 814 such that the temperature of the element 814 increases and some heat is lost to the flowing liquid. By again measuring the voltage across the element 814 while the higher current is being applied, the new resistance of the element 814 is determined and the increased temperature is again calculated by the CMOS circuitry 86. Using the new temperature of the serpentine heater element 814 and the known temperature of sample liquid calculated in the first step, the flow speed of the liquid is determined. From the known channel cross sectional geometry and the flow speed, the flow rate of the liquid in the channel is calculated.
Capillary Meniscus Marching Velocity Sensor
• The speed of the leading meniscus at the sample flow front can be determined by the time delay between triggering the various liquid sensors 174.
Mixers
• The LOC variants mix the sample and reagents using diffusion. Other embodiments of the LOC device mix the sample and the reagents using different mixing arrangements. A selection of mixer designs is described below:
• A CMOS-controlled mechanical bend-actuator mixer is schematically illustrated in FIG. 235. Reagent flowing through the reagent channel 818 combines with sample fluid flowing through the sample channel 820 in a mixing channel 822. The bend-actuator mixer 816 is a beam 824 within the mixing channel 822 with a resistive heater element 826 for bending via differential thermal expansion. The first variant thermal bend actuated valve 302, shown in FIGS. 68 and 69, uses a similar beam configured for bending though differential thermal expansion. The beam 824 is cantilevered in the mixing channel 822 and activated for cyclic bending in the combined streams. Mixing occurs when the flow experiences shear forces generated by the cyclic actuations of the bend actuator mixer 816.
• FIG. 236 schematically illustrates a CMOS-controlled thermal impulse mixer 832. Reagents from the reagent channel 818 and sample from the sample channel 820 combine in the mixing channel 822. The thermal impulse mixer 832 has a bubble generator 828 for rapidly heating and vaporizing some of the liquid to generate a vapour bubble 830. Expansion and subsequent collapse of the vapour bubble 830 rapidly displaces the adjacent liquids. Mixing occurs when the flow experiences shear forces generated by sequential vapour bubbles 830 generated by electric pulses applied to the bubble generator 828.
Additional Details on the Fluorescence Detection System
• FIGS. 58 and 59 show the hybridization-responsive FRET probes 236. These are often referred to as molecular beacons and are stem-and-loop probes, generated from a single strand of nucleic acid, that fluoresce upon hybridization to complementary nucleic acids. FIG. 58 shows a single FRET probe 236 prior to hybridization with a target nucleic acid sequence 238. The probe has a loop 240, stem 242, a fluorophore 246 at the 5′ end, and a quencher 248 at the 3′ end. The loop 240 consists of a sequence complementary to the target nucleic acid sequence 238. Complementary sequences on either side of the probe sequence anneal together to form the stem 242.
• In the absence of a complementary target sequence, the probe remains closed as shown in FIG. 58. The stem 242 keeps the fluorophore-quencher pair in close proximity to each other, such that significant resonant energy transfer can occur between them, substantially eliminating the ability of the fluorophore to fluoresce when illuminated with the excitation light 244.
• FIG. 59 shows the FRET probe 236 in an open or hybridized configuration. Upon hybridization to a complementary target nucleic acid sequence 238, the stem-and-loop structure is disrupted, the fluorophore and quencher are spatially separated, thus restoring the ability of the fluorophore 246 to fluoresce. The fluorescence emission 250 is optically detected as an indication that the probe has hybridized.
• The probes hybridize with very high specificity with complementary targets, since the stem helix of the probe is designed to be more stable than a probe-target helix with a single nucleotide that is not complementary. Since double-stranded DNA is relatively rigid, it is sterically impossible for the probe-target helix and the stem helix to coexist.
Primer-Linked Probes
• Primer-linked, stem-and-loop probes and primer-linked, linear probes, otherwise known as scorpion probes, are an alternative to molecular beacons and can be used for real-time and quantitative nucleic acid amplification in the LOC device. Real-time amplification could be performed directly in the hybridization chambers of the LOC device. The benefit of using primer-linked probes is that the probe element is physically linked to the primer, thus only requiring a single hybridization event to occur during the nucleic acid amplification rather than separate hybridizations of the primers and probes being required. This ensures that the reaction is effectively instantaneous and results in stronger signals, shorter reaction times and better discrimination than when using separate primers and probes. The probes (along with polymerase and the amplification mix) would be deposited into the hybridization chambers 180 during fabrication and there would be no need for a separate amplification section on the LOC device. Alternatively, the amplification section is left unused or used for other reactions.
Primer-Linked Linear Probe
• FIGS. 178 and 179 show a primer-linked linear probe 692 during the initial round of nucleic acid amplification and in its hybridized configuration during subsequent rounds of nucleic acid amplification, respectively. Referring to FIG. 178, the primer-linked linear probe 692 has a double-stranded stem segment 242. One of the strands incorporates the primer linked probe sequence 696 which is homologous to a region on the target nucleic acid 696 and is labelled on its 5′ end with fluorophore 246, and linked on its 3′ end to an oligonucleotide primer 700 via an amplification blocker 694. The other strand of the stem 242 is labelled at its 3 end with a quencher moiety 248. After an initial round of nucleic acid amplification has completed, the probe can loop around and hybridize to the extended strand with the, now complementary, sequence 698. During the initial round of nucleic acid amplification, the oligonucleotide primer 700 anneals to the target DNA 238 (FIG. 178) and is then extended, forming a DNA strand containing both the probe sequence and the amplification product. The amplification blocker 694 prevents the polymerase from reading through and copying the probe region 696. Upon subsequent denaturation, the extended oligonucleotide primer 700/template hybrid is dissociated and so is the double stranded stem 242 of the primer-linked linear probe, thus releasing the quencher 248. Once the temperature decreases for the annealing and extension steps, the primer linked probe sequence 696 of the primer-linked linear probe curls around and hybridizes to the amplified complementary sequence 698 on the extended strand and fluorescence is detected indicating the presence of the target DNA. Non-extended primer-linked linear probes retain their double-stranded stem and fluorescence remains quenched. This detection method is particularly well suited for fast detection systems as it relies on a single-molecule process.
Primer-Linked Stem-and-Loop Probes
• FIGS. 180A to 180F show the operation of a primer-linked stem-and-loop probe 704. Referring to FIG. 180A, the primer-linked stem-and-loop probe 704 has a stem 242 of complementary double-stranded DNA and a loop 240 which incorporates the probe sequence. One of the stem strands 708 is labelled at its 5′ end with fluorophore 246. The other strand 710 is labelled with a 3′-end quencher 248 and carries both the amplification blocker 694 and oligonucleotide primer 700. During the initial denaturation phase (see FIG. 180B), the strands of the target nucleic acid 238 separate, as does the stem 242 of the primer-linked, stem-and-loop probe 704. When the temperature cools for the annealing phase (see FIG. 180C), the oligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the target nucleic acid sequence 238. During extension (see FIG. 180D) the complement 706 to the target nucleic acid sequence 238 is synthesized forming a DNA strand containing both the probe sequence 704 and the amplified product. The amplification blocker 694 prevents the polymerase from reading through and copying the probe region 704. When the probe next anneals, following denaturation, the probe sequence of the loop segment 240 of the primer-linked stem-and-loop probe (see FIG. 180F) anneals to the complementary sequence 706 on the extended strand. This configuration leaves the fluorophore 246 relatively remote from the quencher 248, resulting in a significant increase in fluorescence emission.
Control Probes
• The hybridization chamber array 110 includes some hybridization chambers 180 with positive and negative control probes used for assay quality control. FIGS. 229 and 230 schematically illustrate negative control probes without a fluorophore 796, and FIGS. 231 and 232 are sketches of positive control probes without a quencher 798. The positive and negative control probes have a stem-and-loop structure like the FRET probes described above. However, a fluorescence signal 250 will always be emitted from positive control probes 798 and no fluorescence signal 250 is ever emitted from negative control probes 796, regardless of whether the probes hybridize into an open configuration or remain closed.
• Referring to FIGS. 229 and 230, the negative control probe 796 has no fluorophore (and may or may not have a quencher 248). Hence, whether the target nucleic acid sequence 238 hybridizes with the probe (see FIG. 230), or the probe remains in its stem-and-loop configuration (see FIG. 229), the response to the excitation light 244 is negligible. Alternatively, the negative control probe 796 could be designed so that it always remains quenched. For example, by synthesizing the loop 240 to have a probe sequence that will not hybridize to any nucleic acid sequence within the sample under investigation, the stem 242 of the probe molecule will re-hybridize to itself and the fluorophore and quencher will remain in close proximity and no appreciable fluorescence signal will be emitted. This negative control signal would correspond to low level emissions from hybridization chambers 180 in which the probes has not hybridized but the quencher does not quench all emissions from the reporter.
• Conversely, the positive control probe 798 is constructed without a quencher as illustrated in FIGS. 231 and 232. Nothing quenches the fluorescence emission 250 from the fluorophore 246 in response to the excitation light 244 regardless of whether the positive control probe 798 hybridizes with the target nucleic acid sequence 238.
• FIG. 52 shows a possible distribution of the positive and negative control probes (378 and 380 respectively) throughout the hybridization chamber array 110. The control probes 378 and 380 are placed in hybridization chambers 180 positioned in a line across the hybridization chamber array 110. However, the arrangement of the control probes within the array is arbitrary (as is the configuration of the hybridization chamber array 110).
Protein Detection Variants
• Some embodiments of the LOC device use a homogeneous protein detection assay to detect specific proteins within a crude cell lysate. Numerous homogeneous protein detection assays have been developed for use in these embodiments of the LOC device. Commonly, these assays utilize antibodies or aptamers to capture the target protein.
• In one type of assay, an aptamer 141 which binds to a particular protein 142 is labelled with two different fluorophores or luminophores 143 and 144 which can function as a donor and an acceptor in a fluorescence resonance energy transfer (FRET) or electrochemiluminescence resonance energy transfer (ERET) reaction (see FIGS. 256A and 256B). Both donor 143 and acceptor 144 are linked to the same aptamer 141, and the change in separation is caused by a change in conformation upon binding to the target protein 142. For example, an aptamer 141 in the absence of the target forms a conformation where the donor and acceptor are in close proximity (see FIG. 256A); upon binding to the target, the new conformation results in a larger separation between the donor and acceptor (see FIG. 256B). When the acceptor is a quencher and the donor is a luminophore, the effect of binding to the target is an increase in light emission 250 or 862 (see FIG. 256B).
• A second type of assay uses two antibodies 145 or two aptamers 141 that must independently bind to different, non-overlapping epitopes or regions of the target protein 142 (see FIGS. 257A, 257B, 258A and 258B). These antibodies 145 or aptamers 141 are labelled with different fluorophores or luminophores 143 and 144 which can function as a donor and an acceptor in a fluorescence resonance energy transfer (FRET) or electrochemiluminescence resonance energy transfer (ERET) reaction. The fluorophores or luminophores 143 and 144 form part of a pair of short complementary oligonucleotides 147 attached to the antibodies or aptamers via long, flexible linkers 149 (see FIGS. 257A and 258A). Once the antibodies 145 or aptamers 141 bind to the target protein 142, the complementary oligonucleotides 147 find each other and hybridize to one another (see FIGS. 257B and 258B). This brings the donors and acceptors 143 and 144 in close proximity to one another resulting in efficient FRET 250 or ERET 862 that is used as a signal for target protein detection.
• To ensure there is no, or very little, background signal as a result of the oligonucleotides 147 attached to the two antibodies 145 or aptamers 141 hybridizing to one another in the absence of their binding to the protein 142, it is necessary to carefully choose the length and sequence of the complementary oligonucleotides 147 so that the dissociation constant (k_d) for the duplex is relatively high (˜5 μM). Thus when free antibodies or aptamers labelled with these oligonucleotides are mixed at nanomolar concentrations, well below that of their k_d, the likelihood of duplex formation and a FRET 250 or ERET 862 signal being generated is negligible. However, when both antibodies 145 or both aptamers 141 bind to the target protein 142, the local concentration of the oligonucleotides 147 will be much higher than their k_d resulting in almost complete hybridization and generation of a detectable FRET 250 or ERET 862 signal.
• The choice of fluorophores and luminophores is an important consideration when designing a homogeneous protein detection assay. Crude cell lysates are often turbid and may contain substances which autofluoresce. In such cases, the use of molecules with long-lasting fluorescence or electrochemiluminescence and donor-acceptor pairs 143 and 144 which are optimized to give maximal FRET 250 or ERET 862 is desired. One such pair is europium chelate and Cy5, which has previously been shown to significantly improve signal-to-background ratio in such a system when compared with other donor-acceptor pairs, by allowing the signal to be read after interfering background fluorescence, electrochemiluminescence or scattered light has decayed. Europium chelate and AlexaFluor 647 or terbium chelate and Fluorescein FRET or ERET pairs also work well. The sensitivity and specificity of this approach is similar to that of enzyme-linked immunosorbent assays (ELISAs), but no sample manipulation is required.
• In some embodiments of the LOC device, one of the antibodies 145 or one of the aptamers 141 is attached to the base of the proteomic assay chamber 124 (see for example FIGS. 268 and 272) and the protein lysate is combined with the other antibody 145 or aptamer 141 during lysis within the chemical lysis section 130 to facilitate binding to the first antibody 145 or aptamer 141 prior to entering the proteomic assay chamber 124. This increases the subsequent speed with which a detectable signal is generated as only one conjugation or hybridization event is required within the proteomic assay chamber.
Fluorophore Design
• Fluorophores with long fluorescence lifetimes are required in order to allow enough time for the excitation light to decay to an intensity below that of the fluorescence emission at which time the photosensor 44 is enabled, thereby providing a sufficient signal to noise ratio. Also, longer fluorescence lifetime translates into larger integrated fluorescence photon count.
• The fluorophores 246 (see FIG. 59) have a fluorescence lifetime greater than 100 nanoseconds, often greater than 200 nanoseconds, more commonly greater than 300 nanoseconds and in most cases greater than 400 nanoseconds.
• The metal-ligand complexes based on the transition metals or lanthanides have long lifetimes (from hundreds of nanoseconds to milliseconds), adequate quantum yields, and high thermal, chemical and photochemical stability, which are all favourable properties with respect to the fluorescence detection system requirements.
• A particularly well-studied metal-ligand complex based on the transition metal ion Ruthenium (Ru (II)) is tris(2,2′-bipyridine) ruthenium (II) ([Ru(bpy)₃]²⁺) which has a lifetime of approximately 1 μs. This complex is available commercially from Biosearch Technologies under the brand name Pulsar 650.

• TABLE 1
  Photophysical properties of Pulsar 650 (Ruthenium chelate)

  Parameter	Symbol	Value	Unit
  Absorption Wavelength	λabs	460	nm
  Emission Wavelength	λ	em	650	nm
  Extinction Coefficient	E	14800	M−1cm−1
  Fluorescence Lifetime	τf	1.0	μs
  Quantum Yield	H	1 (deoxygenated)	N/A

• Terbium chelate, a lanthanide metal-ligand complex has been successfully demonstrated as a fluorescent reporter in a FRET probe system, and also has a long lifetime of 1600 μs.

• TABLE 2
  Photophysical properties of terbium chelate

  Parameter	Symbol	Value	Unit
  Absorption Wavelength	λabs	330-350	nm
  Emission Wavelength	λem	548	nm
  Extinction Coefficient	E	13800	M−1cm−1
  		(λabs and ligand depen-
  		dent, can be up to
  		30000 @ λe = 340 nm)
  Fluorescence Lifetime	τf	1600	μs
  		(hybridized probe)
  Quantum Yield	H	1	N/A
  		(ligand dependent)

• The fluorescence detection system used by the LOC device 301 does not utilize filters to remove unwanted background fluorescence. It is therefore advantageous if the quencher 248 has no native emission in order to increase the signal-to-noise ratio. With no native emission, there is no contribution to background fluorescence from the quencher 248. High quenching efficiency is also important so that fluorescence is prevented until a hybridization event occurs. The Black Hole Quenchers (BHQ), available from Biosearch Technologies, Inc. of Novato California, have no native emission and high quenching efficiency, and are suitable quenchers for the system. BHQ-1 has an absorption maximum at 534 nm, and a quenching range of 480-580 nm, making it a suitable quencher for the Tb-chelate fluorophore. BHQ-2 has an absorption maximum at 579 nm, and a quenching range of 560-670 nm, making it a suitable quencher for Pulsar 650.
• Iowa Black Quenchers (Iowa Black FQ and RQ), available from Integrated DNA Technologies of Coralville, Iowa, are suitable alternative quenchers with little or no background emission. Iowa Black FQ has a quenching range from 420-620 nm, with an absorption maximum at 531 nm and would therefore be a suitable quencher for the Tb-chelate fluorophore. Iowa Black RQ has an absorption maximum at 656 nm, and a quenching range of 500-700 nm, making it an ideal quencher for Pulsar 650.
• In the embodiments described here, the quencher 248 is a functional moiety which is initially attached to the probe, but other embodiments are possible in which the quencher is a separate molecule free in solution.
Excitation Source
• In the fluorescence detection based embodiments described herein, a LED is chosen as the excitation source instead of a laser diode, high power lamp or laser due to the low power consumption, low cost and small size. Referring to FIG. 181, the LED 26 is positioned directly above the hybridization chamber array 110 on an external surface of the LOC device 301. On the opposing side of the hybridization chamber array 110, is the photosensor 44, made up of an array of photodiodes 184 (see FIGS. 53, 54 and 77) for detection of fluorescence signals from each of the chambers.
• FIGS. 182, 183 and 184 schematically illustrate other embodiments for exposing the probes to excitation light. In the LOC device 30 shown in FIG. 182, the excitation light 244 generated by the excitation LED 26 is directed onto the hybridization chamber array 110 by the lens 254. The excitation LED 26 is pulsed and the fluorescence emissions are detected by the photosensor 44.
• In the LOC device 30 shown in FIG. 183, the excitation light 244 generated by the excitation LED 26 is directed onto the hybridization chamber array 110 by the lens 254, a first optical prism 712 and second optical prism 714. The excitation LED 26 is pulsed and the fluorescence emissions are detected by the photosensor 44.
• Similarly, the LOC device 30 shown in FIG. 184, the excitation light 244 generated by the excitation LED 26 is directed onto the hybridization chamber array 110 by the lens 254, a first minor 716 and second minor 718. Again, the excitation LED 26 is pulsed and the fluorescence emissions are detected by the photosensor 44.
• The excitation wavelength of the LED 26 is dependent on the choice of fluorescent dye. The Philips LXK2-PR14-R00 is a suitable excitation source for the Pulsar 650 dye. The SET UVTOP335TO39BL LED is a suitable excitation source for the Tb-chelate label.

• TABLE 3
  Philips LXK2-PR14-R00 LED specifications

  	Parameter	Symbol	Value	Unit
  	Wavelength	λex	460	nm
  	Emission Frequency	νem	6.52(10)14	Hz
  	Output Power	pl	0.515 (min) @ 1A	W
  	Radiation pattern		Lambertian profile	N/A

• TABLE 4
  SET UVTOP334TO39BL LED Specifications

  Parameter	Symbol	Value	Unit
  Wavelength	λe	340	nm
  Emission Frequency	νe	8.82(10)14	Hz
  Power	pl	0.000240 (min) @ 20 mA	W
  Pulse Forward Current	I	200	mA
  Radiation pattern		Lambertian	N/A

Ultra Violet Excitation Light
• Silicon absorbs little light in the UV spectrum. Accordingly, it is advantageous to use UV excitation light. A UV LED excitation source can be used but the broad spectrum of the LED 26 reduces the effectiveness of this method. To address this, a filtered UV LED can be used. Optionally, a UV laser can be the excitation source unless the relatively high cost of the laser is impractical for the particular test module market.
Led Driver
• The LED driver 29 drives the LED 26 at a constant current for the required duration. A lower power USB 2.0-certifiable device can draw at most 1 unit load (100 mA), with a minimum operating voltage of 4.4 V. A standard power conditioning circuit is used for this purpose.
Photodiode
• FIG. 54 shows the photodiode 184 integrated into the CMOS circuitry 86 of the LOC device 301. The photodiode 184 is fabricated as part of the CMOS circuitry 86 without additional masks or steps. This is one significant advantage of a CMOS photodiode over a CCD, an alternate sensing technology which could be integrated on the same chip using non-standard processing steps, or fabricated on an adjacent chip. On-chip detection is low cost and reduces the size of the assay system. The shorter optical path length reduces noise from the surrounding environment for efficient collection of the fluorescence signal and eliminates the need for a conventional optical assembly of lenses and filters.
• Quantum efficiency of the photodiode 184 is the fraction of photons impinging on its active area 185 that are effectively converted to photo-electrons. For standard silicon processes, the quantum efficiency is in the range of 0.3 to 0.5 for visible light, depending on process parameters such as the amount and absorption properties of the cover layers.
• The detection threshold of the photodiode 184 determines the smallest intensity of the fluorescence signal that can be detected. The detection threshold also determines the size of the photodiode 184 and hence the number of hybridization chambers 180 in the hybridization and detection section 52 (see FIG. 52). The size and number of chambers are technical parameters that are limited by the dimensions of the LOC device (in the case of the LOC device 301, the dimensions are 1760 μm×5824 μm) and the real estate available after other functional modules such as the pathogen dialysis section 70 and amplification section(s) 112 are incorporated.
• For standard silicon processes, the photodiode 184 detects a minimum of 5 photons. However, to ensure reliable detection, the minimum can be set to 10 photons. Therefore with the quantum efficiency range being 0.3 to 0.5 (as discussed above), the fluorescence emission from the probes should be a minimum of 17 photons but 30 photons would incorporate a suitable margin of error for reliable detection.
Calibration Chambers
• The non-uniformity of the electrical characteristic of the photodiode 184, autofluorescence, and residual excitation photon flux that has not yet completely decayed, introduce background noise and offset into the output signal. This background is removed from each output signal using one or more calibration signals. Calibration signals are generated by exposing one or more calibration photodiodes 184 in the array to respective calibration sources. A low calibration source is used for determining a negative result in which a target has not reacted with a probe. A high calibration source is indicative of a positive result from a probe-target complex. In the embodiment described here, the low calibration light source is provided by calibration chambers 382 in the hybridization chamber array 110 which:
• do not contain any probes;
• contain probes that have no fluorescent reporter; or,
• contain probes with a reporter and quencher configured such that quenching is always expected to occur.
• The output signal from such calibration chambers 382 closely approximates the noise and offset in the output signal from all the hybridization chambers in the LOC device. Subtracting the calibration signal from the output signals generated by the other hybridization chambers substantially removes the background and leaves the signal generated by the fluorescence emission (if any). Signals arising from ambient light in the region of the chamber array are also subtracted.
• It will be appreciated that the negative control probes described above with reference to FIGS. 229 to 232 can be used in calibration chambers. However, as shown in FIGS. 202 and 203, which are enlarged views of insets DG and DH of LOC variant X 728 shown in FIG. 195, another option is to fluidically isolate the calibration chambers 382 from the amplicon. The background noise and offset can be determined by leaving the fluidically isolated chambers empty, or containing reporterless probes, or indeed any of the ‘normal’ probes with both reporter and quencher as hybridization is precluded by fluidic isolation.
• The calibration chambers 382 can provide a high calibration source to generate a high signal in the corresponding photodiodes. The high signal corresponds to all probes in a chamber having hybridized. Spotting probes with reporters and no quenchers, or just reporters will consistently provide a signal approximating that of a hybridization chamber in which a predominant number of the probes have hybridized. It will also be appreciated that calibration chambers 382 can be used instead of control probes, or in addition to control probes.
• The number and arrangement of the calibration chambers 382 throughout the hybridization chamber array is arbitrary. However, the calibration is more accurate if photodiodes 184 are calibrated by a calibration chamber 382 that is relatively proximate. Referring to FIG. 56, the hybridization chamber array 110 has one calibration chamber 382 for every eight hybridization chambers 180. That is, a calibration chamber 382 is positioned in the middle of every three by three square of hybridization chambers 180. In this configuration, the hybridization chambers 180 are calibrated by a calibration chamber 382 that is immediately adjacent.
• FIG. 228 shows a differential imager circuit 788 used to substract the signal from the photodiode 184 corresponding to the calibration chamber 382 as a result of excitation light, from the fluorescence signal from the surrounding hybridization chambers 180. The differential imager circuit 788 samples the signal from the pixel 790 and a “dummy” pixel 792. In one embodiment, the “dummy” pixel 792 is shielded from light, so its output signal provides a dark reference. Alternatively, the “dummy” pixel 792 can be exposed to the excitation light along with the rest of the array. In the embodiment where the “dummy” pixel 792 is open to light, signals arising from ambient light in the region of the chamber array are also subtracted. The signals from the pixel 790 are small (i.e. close to dark signal), and without a reference to a dark level it is hard to differentiate between the background and a very small signal.
• During use, the “read_row” 794 and “read_row_d” 795 are activated and M4 797 and MD4 801 transistors are turned on. Switches 807 and 809 are closed such that the outputs from the pixel 790 and “dummy” pixel 792 are stored on pixel capacitor 803 and dummy pixel capacitor 805 respectively. After the pixel signals have been stored, switches 807 and 809 are deactivated. Then the “read_col” switch 811 and dummy “read_col” switch 813 are closed, and the switched capacitor amplifier 815 at the output amplifies the differential signal 817.
Suppression and Enablement of the Photodiode
• The photodiode 184 needs to be suppressed during excitation by the LED 26 and enabled during fluorescence. FIG. 83 is a circuit diagram for a single photodiode 184 and FIG. 84 is a timing diagram for the photodiode control signals. The circuit has photodiode 184 and six MOS transistors, M _shunt 394, M _tx 396, M _reset 398, M _sf 400, M _read 402 and M _bias 404. At the beginning of the excitation cycle, t1, the transistors M _shunt 394, and M _reset 398 are turned on by pulling the M_shunt gate 384 and the reset gate 388 high. During this period, the excitation photons generate carriers in the photodiode 184. These carriers have to be removed, as the amount of generated carriers can be sufficient to saturate the photodiode 184. During this cycle, M _shunt 394 directly removes the carriers generated in photodiode 184, while M M _reset 398 resets any carriers that have accumulated on node ‘NS’ 406 due to leakage in transistors or due to diffusion of excitation-produced carriers in the substrate. After excitation, a capture cycle commences at t4. During this cycle, the emitted response from the fluorophore is captured and integrated in the circuit on node ‘NS’ 406. This is achieved by pulling tx gate 386 high, which turns on the transistor M _tx 396 and transfers any accumulated carriers on the photodiode 184 to node ‘NS’ 406. The duration of the capture cycle can be as long as the fluorophore emits. The outputs from all photodiodes 184 in the hybridization chamber array 110 are captured simultaneously.
• There is a delay between the end of the capture cycle t5 and the start of the read cycle t6. This delay is due to the requirement to read each photodiode 184 in the hybridization chamber array 110 (see FIG. 52) separately following the capture cycle. The first photodiode 184 to be read will have the shortest delay before the read cycle, while the last photodiode 184 will have the longest delay before the read cycle. During the read cycle, transistor M _read 402 is turned on by pulling the read gate 393 high. The ‘NS’ node 406 voltage is buffered and read out using the source-follower transistor M _sf 400.
• There are additional, optional methods of enabling or suppressing the photodiode as discussed below:
1. Suppression Methods
• FIGS. 225, 226 and 227 show three possible configurations 778, 780, 782 for the M_shunt transistor 394. The M_shunt transistor 394 has a very high off ratio at maximum |V_Gs|=5 V which is enabled during excitation. As shown in FIG. 225, the M_shunt gate 384 is configured to be on the edge of the photodiode 184. Optionally, as shown in FIG. 226, the M_shunt gate 384 may be configured to surround the photodiode 184. A third option is to configure the M_shunt gate 384 inside the photodiode 184, as shown in FIG. 227. Under this third option there would be less photodiode active area 185.
• These three configurations 778, 780 and 782 reduce the average path length from all locations in the photodiode 184 to the M_shunt gate 384. In FIG. 225, the M_shunt gate 384 is on one side of the photodiode 184. This configuration is simplest to fabricate and impinges the least on the photodiode active area 185. However, any carriers lingering on the remote side of the photodiode 184 would take longer to propagate through to the M_shunt gate 384.
• In FIG. 226, the M_shunt gate 384 surrounds the photodiode 184. This further reduces the average path length for carriers in the photodiode 184 to the M_shunt gate 384. However, extending the M_shunt gate 384 about the periphery of the photodiode 184 imposes a greater reduction of the photodiode active area 185. The configuration 782 in FIG. 227 positions the M_shunt gate 384 within the active area 185. This provides the shortest average path length to the M_shunt gate 384 and hence the shortest transition time. However, the impingement on the active area 185 is greatest. It also poses a wider leakage path.
2. Enabling Methods
• a. A trigger photodiode drives the shunt transistor with a fixed delay.
  b. A trigger photodiode drives the shunt transistor with programmable delay.
  c. The shunt transistor is driven from the LED drive pulse with a fixed delay.
  d. The shunt transistor is driven as in 2c but with programmable delay.
• FIG. 89 is a schematic section view through a hybridization chamber 180 showing a photodiode 184 and trigger photodiode 187 embedded in the CMOS circuitry 86. A small area in the corner of the photodiode 184 is replaced with the trigger photodiode 187. A trigger photodiode 187 with a small area is sufficient as the intensity of the excitation light will be high in comparison with the fluorescence emission. The trigger photodiode 187 is sensitive to the excitation light 244. The trigger photodiode 187 registers that the excitation light 244 has extinguished and activates the photodiode 184 after a short time delay Δt 300 (see FIG. 2). This delay allows the fluorescence photodiode 184 to detect the fluorescence emission from the FRET probes 186 in the absence of the excitation light 244. This enables detection and improves the signal to noise ratio.
• Both photodiodes 184 and trigger photodiodes 187 are located in the CMOS circuitry 86 under each hybridization chamber 180. The array of photodiodes combines, along with appropriate electronics, to form the photosensor 44 (see FIG. 77). The photodiodes 184 are pn-junction fabricated during CMOS structure manufacturing without additional masks or steps. During MST fabrication, the dielectric layer (not shown) above the photodiodes 184 is optionally thinned using the standard MST photolithography techniques to allow more fluorescent light to illuminate the active area 185 of the photodiode 184. The photodiode 184 has a field of view such that the fluorescence signal from the probe-target hybrids within the hybridization chamber 180 is incident on the sensor face. The fluorescent light is converted into a photocurrent which can then be measured using CMOS circuitry 86.
• Alternatively, one or more hybridization chambers 180 can be dedicated to a trigger photodiode 187 only. These options can be used in these in combination with 2a and 2b above.
Delayed Detection of Fluorescence
• The following derivations elucidate the delayed detection of fluorescence using a long-lifetime fluorophore for the LED/fluorophore combinations described above. The fluorescence intensity is derived as a function of time after excitation by an ideal pulse of constant intensity I_e between time t₁ and t₂ as shown in FIG. 60.
• Let [S1](t) equal the density of excited states at time t, then during and after excitation, the number of excited states per unit time per unit volume is described by the following differential equation:
• $\begin{array}{cc}\frac{\left[S1\right]}{t}\left(t\right)+\frac{[S1]\left(t\right)}{{\tau }_F}=\frac{I_e\varepsilon c}{{\mathrm{hv}}_e}& \left(1\right)\end{array}$
• where c is the molar concentration of fluorophores, ε is the molar extinction coefficient, ν_e is the excitation frequency, and h=6.62606896(10)⁻³⁴ Js is the Planck constant.
  This differential equation has the general form:
• $\frac{y}{x}+p\left(x\right)y=q\left(x\right)$
• which has the solution:
• $\begin{array}{cc}y\left(x\right)=\frac{\int {}^{\int p\left(x\right)x}q\left(x\right)x+k}{{}^{\int p\left(x\right)x}}& \left(2\right)\end{array}$
• Using this now to solve equation (1),
• $\begin{array}{cc}\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{{\mathrm{hv}}_e}+k{}^{-t/{\tau }_f}& \left(3\right)\end{array}$
• Now at time t₁, [S1](t₁)=0, and from (3):
• $\begin{array}{cc}k=-\frac{I_e\varepsilon c{\tau }_f}{hv_e}{}^{{\mathrm{<figure-callout\; id="1"\; label="t"\; filenames="US20110312622A1-20111222-D00008.png,US20110312622A1-20111222-D00012.png"\; state="\{\{state\}\}">t</figure-callout>}}_1/{\tau }_f}& \left(4\right)\end{array}$
• Substituting (4) into (3):
• $\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{hv_e}-\frac{I_e\varepsilon c{\tau }_f}{hv_e}{}^{-\left(t-t_1\right)/{\tau }_f}$
• At time t₂:
• $\begin{array}{cc}\left[S1\right]\left(t_2\right)=\frac{I_e\varepsilon c{\tau }_f}{hv_e}-\frac{I_e\varepsilon c{\tau }_f}{hv_e}{}^{-\left(t_2-t_1\right)/{\tau }_f}& \left(5\right)\end{array}$
• For t≧t₂, the excited states decay exponentially and this is described by:
• 
  [S1](t)=[S1](t ₂)e ^{−(t−t 2 )/τ f}   (6)
• Substituting (5) into (6):
• $\begin{array}{cc}\left[S1\right]\left(t\right)=\frac{I_e\varepsilon c{\tau }_f}{hv_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t-t_2\right)/{\tau }_f}& \left(7\right)\end{array}$
• The fluorescence intensity is given by the following equation:
• $\begin{array}{cc}{I}_f\left(t\right)=-\frac{\left[S1\right]\left(t\right)}{x}hv_f\eta l& \left(8\right)\end{array}$
• where ν_f is the fluorescence frequency, η is the quantum yield and 1 is the optical path length.
• Now from (7):
• $\begin{array}{cc}\frac{\left[S1\right]\left(t\right)}{t}=-\frac{I_e\varepsilon c}{hv_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t-t_2\right)/{\tau }_f}& \left(9\right)\end{array}$
• Substituting (9) into (8):
• $\begin{array}{cc}{I}_f\left(t\right)=I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}\left[1-{}^{-\left(t_2-t_1\right)/{\tau }_f}\right]{}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{For}\frac{t_2-{\mathrm{<figure-callout\; id="1"\; label="t"\; filenames="US20110312622A1-20111222-D00008.png,US20110312622A1-20111222-D00012.png"\; state="\{\{state\}\}">t</figure-callout>}}_1}{{\tau }_f}\to \infty ,I_f\left(t\right)\to I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}& \left(10\right)\end{array}$
• Therefore, we can write the following approximate equation which describes the fluorescence intensity decay after a sufficiently long excitation pulse (t₂−t₁>>τ_f):
• $\begin{array}{cc}{I}_f\left(t\right)=I_e\varepsilon cl\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{for}t\ge t_2& \left(11\right)\end{array}$
• In the previous section, we concluded that for t₂−t₁>>τ_f,
• $I_f\left(t\right)=I_e\varepsilon \mathrm{cl}\eta \frac{v_f}{v_e}{}^{-\left(t-t_2\right)/{\tau }_f}\mathrm{for}t\ge {\mathrm{<figure-callout\; id="2"\; label="t"\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">t</figure-callout>}}_2.$
• From the above equation, we can derive the following:
• 
  

  

  (t)=

  

  εclηe ^{−(t−t 2 )/τ f}   (12)
• where
• ${\stackrel{⃛}{n}}_f\left(t\right)=\frac{I_f\left(t\right)}{{\mathrm{hv}}_f}$
• is the number of fluorescent photons per unit time per unit area and
• ${\stackrel{⃛}{n}}_e=\frac{I_e}{{\mathrm{hv}}_e}$
• is the number of excitation photons per unit time per unit area.
• Consequently,
• $\begin{array}{cc}{\ddot{n}}_f\left(t\right)={\int }_{{\mathrm{<figure-callout\; id="3"\; label="t"\; filenames="US20110312622A1-20111222-D00030.png,US20110312622A1-20111222-D00038.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_f\left(t\right)t& \left(13\right)\end{array}$
• where {umlaut over (n)}_f is the number of fluorescent photons per unit area and t₃ is the instant of time at which the photodiode is turned on. Substituting (12) into (13):
• $\begin{array}{cc}{\ddot{n}}_f={\int }_{{\mathrm{<figure-callout\; id="3"\; label="t"\; filenames="US20110312622A1-20111222-D00030.png,US20110312622A1-20111222-D00038.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_e\varepsilon \mathrm{cl}{\mathrm{\eta }}^{-\left(t-t_2\right)/{\tau }_f}t& \left(14\right)\end{array}$
• Now, the number of fluorescent photons that reach the photodiode per unit time per unit area,

  

  (t), is given by the following:
• 
  

  

  (t)=

  

  (t)φ₀  (15)
• where φ₀ is the light gathering efficiency of the optical system.
• Substituting (12) into (15) we find
• 
  

  

  (t)=φ₀

  

  εclηe ^{−(t−t 2 )/τ f}   (16)
• Similarly, the number of fluorescence photons that reach the photodiode per unit fluorescent area {umlaut over (n)}_s, will be as follows:
• ${\ddot{n}}_s={\int }_{{\mathrm{<figure-callout\; id="3"\; label="t"\; filenames="US20110312622A1-20111222-D00030.png,US20110312622A1-20111222-D00038.png"\; state="\{\{state\}\}">t</figure-callout>}}_3}^{\infty }{\stackrel{⃛}{n}}_s\left(t\right)t$
• and substituting in (16) and integrating:
• 
  {umlaut over (n)}_s=φ₀

  

  εclητ _f e ^{−(t 3 −t 2 )/τ f}
• 
  Therefore,
• 
  n _s=φ₀ {dot over (n)} _e εclητ _f e ^{−Δt/τ f}   (17)
• The optimal value of t₃ is when the rate of electrons generated in the photodiode 184 due to fluorescence photons becomes equal to the rate of electrons generated in the photodiode 184 by the excitation photons, as the flux of the excitation photons decays much faster than that of the fluorescence photons.
• The rate of sensor output electrons per unit fluorescent area due to fluorescence is:
• 
  

  

  (t)=φ_f

  

  (t)
• where φ_f is the quantum efficiency of the sensor at the fluorescence wavelength.
• Substituting in (17) we have:
• 
  

  

  (t)=φ_fφ₀

  

  εclηe ^{−(t−t 2 )/τ f}   (18)
• Similarly, the rate of sensor output electrons per unit fluorescent area due to the excitation photons is:
• 
  

  

  (t)=φ_e

  

  e ^{−(t−t 2 )/τ e}   (19)
• where φ_e is the quantum efficiency of the sensor at the excitation wavelength, and τ_e is the time-constant corresponding to the “off” characteristics of the excitation LED. After time t₂, the LED's decaying photon flux would increase the intensity of the fluorescence signal and extend its decay time, but we are assuming that this has a negligible effect on I_f(t), thus we are taking a conservative approach.
  Now, as mentioned earlier, the optimal value of t₃ is when:
• 
  

  

  (t ₃)=

  

  (t ₃)
• Therefore, from (18) and (19) we have:
• 
  φ_fφ₀

  

  εclηe ^{−(t 3 −t 2 )/τ f} =φ_e

  

  e ^{−(t 3 −t 2 )/τ e}
• and rearranging we find:
• $\begin{array}{cc}{t}_3-{\mathrm{<figure-callout\; id="2"\; label="t"\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">t</figure-callout>}}_2=\frac{\ln \left(\varepsilon \mathrm{cl}\eta \frac{{\phi }_f{\mathrm{<figure-callout\; id="0"\; label="\phi "\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0}{{\phi }_e}\right)}{\frac{1}{{\tau }_f}-\frac{1}{{\tau }_e}}& \left(20\right)\end{array}$
• From the previous two sections, we have the following two working equations:
• 
  n _s=φ₀ {dot over (n)} _e Fτ _f e ^{−Δt/τ f}   (21)
• $\begin{array}{cc}\Delta t=\frac{\ln \left(F\frac{{\phi }_f{\mathrm{<figure-callout\; id="0"\; label="\phi "\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">\phi </figure-callout>}}_0}{{\phi }_e}\right)}{\frac{1}{{\tau }_f}-\frac{1}{{\tau }_e}}& \left(22\right)\end{array}$
• where F=εclη and Δt=t₃−t₂. We also know that, in practice, t₂−t₁>>τ_f.
• The optimal time for fluorescence detection and the number of fluorescence photons detected using the Philips LXK2-PR14-R00 LED and Pulsar 650 dye are determined as follows. The optimum detection time is determined using equation (22):
• Recalling the concentration of amplicon, and assuming that all amplicons hybridize, then the concentration of fluorescent fluorophores is: c=2.89(10)⁻⁶ mol/L
• The height of the chamber is the optical path length l=8(10)⁻⁶m.
• We have taken the fluorescence area to be equal to our photodiode area, yet our actual fluorescence area is substantially larger than our photodiode area; consequently we can approximately assume φ₀=0.5 for the light gathering efficiency of our optical system. From the photodiode characteristics,
• $\frac{{\phi }_f}{{\phi }_e}=10$
• is a very conservative value for the ratio of the photodiode quantum efficiency at the fluorescence wavelength to its quantum efficiency at the excitation wavelength.
• With a typical LED decay lifetime of τ_e=0.5 ns and using Pulsar 650 specifications, Δt can be determined:
• $\begin{array}{cc}\begin{array}{c}F=\left[1.48{\left(10\right)}^6\right]\left[2.89{\left(10\right)}^{-6}\right]\left[8{\left(10\right)}^{-6}\right]\\ =3.42{\left(10\right)}^{-5}\end{array}\begin{array}{c}\Delta t=\frac{\ln \left(\left[3.42{\left(10\right)}^{-5}\right]\left(10\right)\left(0.5\right)\right)}{\frac{1}{1{\left(10\right)}^{-6}}-\frac{1}{0.5{\left(10\right)}^{-9}}}\\ =4.34{\left(10\right)}^{-9}s\end{array}& \left(1\right)\end{array}$
• The number of photons detected is determined using equation (21). First, the number of excitation photons emitted per unit time {dot over (n)}_e is determined by examining the illumination geometry.
• The Philips LXK2-PR14-R00 LED has a Lambertian radiation pattern, therefore:
• 
  

  

  =

  

  (θ)  (23)
• where

  

  is the number of photons emitted per unit time per unit solid angle at an angle of θ off the LED's forward axial direction, and

  

  is the valve of

  

  in the forward axial direction.
• The total number of photons emitted by the LED per unit time is:
• $\begin{array}{cc}\begin{array}{c}{\stackrel{.}{n}}_l={\int }_{\Omega }{\stackrel{\cdots }{n}}_l\Omega \\ ={\int }_{\Omega }{\stackrel{\cdots }{n}}_{l0}\cos \left(\theta \right)\Omega \end{array}& \left(24\right)\end{array}$
• Now,
• $\mathrm{\Delta \Omega }=2\pi \left[1-\cos \left(\theta +\Delta \theta \right)\right]-2\pi \left[1-\cos \left(\theta \right)\right]$ $\begin{array}{c}\mathrm{\Delta \Omega }=2\pi \left[\cos \left(\theta \right)-\cos \left(\theta +\Delta \theta \right)\right]\\ =4\pi \sin \left(\theta \right)\cos \left(\frac{\Delta \theta }{2}\right)\sin \left(\frac{\Delta \theta }{2}\right)+\\ 4\pi \cos \left(\theta \right){\mathrm{<figure-callout\; id="2"\; label="sin"\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">sin</figure-callout>}}^2\left(\frac{\Delta \theta }{2}\right)\end{array}$ $d\Omega =2\pi \sin \left(\theta \right)d\theta$
• Substituting this into (24):
• $\begin{array}{c}{\stackrel{.}{n}}_l={\int }_0^{\frac{\mathrm{<figure-callout\; id="2"\; label="\pi "\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">\pi </figure-callout>}}{2}}2\pi {\stackrel{\cdots }{n}}_{l0}\cos \left(\theta \right)\sin \left(\theta \right)\theta \\ =\pi {\stackrel{\cdots }{n}}_{l0}\end{array}$
• Rearranging, we have:
• $\begin{array}{cc}{\stackrel{\cdots }{n}}_{l0}=\frac{{\stackrel{.}{n}}_l}{\pi }& \left(26\right)\end{array}$
• The LED's output power is 0.515 W and ν_e=6.52(10)¹⁴ Hz, therefore:
• $\begin{array}{cc}\begin{array}{c}{\stackrel{.}{n}}_l=\frac{p_l}{hv_e}\\ =\frac{0.515}{\left[6.63{\left(10\right)}^{-34}\right]\left[6.52{\left(10\right)}^{14}\right]}\\ =1.19{\left(10\right)}^{18}\mathrm{photons}\text{/}s\end{array}& \left(27\right)\end{array}$
• Substituting this value into (26) we have:
• $\begin{array}{c}{\stackrel{\cdots }{n}}_{l0}=\frac{1.19{\left(10\right)}^{18}}{\pi }\\ =3.79{\left(10\right)}^{17}\mathrm{photons}\text{/}s\text{/}\mathrm{sr}\end{array}$
• Referring to FIG. 61, the optical centre 252 and the lens 254 of the LED 26 are schematically shown. The photodiodes are 16 μm×16 μm, and for the photodiode in the middle of the array, the solid angle (Ω) of the cone of light emitted from the LED 26 to the photodiode 184 is approximately:
• $\begin{array}{c}\Omega =\mathrm{area}\mathrm{of}\mathrm{sensor}/{\mathrm{<figure-callout\; id="2"\; label="r"\; filenames="US20110312622A1-20111222-D00012.png,US20110312622A1-20111222-D00013.png"\; state="\{\{state\}\}">r</figure-callout>}}^2\\ =\frac{\left[16{\left(10\right)}^{-6}\right]\left[16{\left(10\right)}^{-6}\right]}{{\left[2.825{\left(10\right)}^{-3}\right]}^2}\\ =3.21{\left(10\right)}^{-5}\mathrm{sr}\end{array}$
• It will be appreciated that the central photodiode 184 of the photodiode array 44 is used for the purpose of these calculations. A sensor located at the edge of the array would only receive 2% less photons upon a hybridization event for a Lambertian excitation source intensity distribution.
• The number of excitation photons emitted per unit time is:
• 
  {dot over (n)} _e=

  

  _lΩ  (28)
• 
  [3.79(10)¹⁷][3.21(10)⁻⁵]
• 
  =1.22(10)¹³ photons/s
• Now referring to equation (29):
• 
  n _s=φ₀ {dot over (n)} _e Fτ _f e ^{−Δt/τ f}
• 
  n _s=(0.5)[1.22(10)¹³][3.42(10)⁻⁵][1(10)⁻⁶ ]e ^{−4.34(10) −9 /1(10) −6}
• 
  =208 photons per sensor.
• Therefore, using the Philips LXK2-PR14-R00 LED and Pulsar 650 fluorophore, we can easily detect any hybridization events which results in this number of photons being emitted.
• The SET LED illumination geometry is shown in FIG. 62. At I_D=20 mA, the LED has a minimum optical power output of p₁=240 μW centred at λ_e=340 nm (the absorption wavelength of the terbium chelate). Driving the LED at I_D=200 mA would increase the output power linearly to p₁=2.4 mW. By placing the LED's optical centre 252, 17.5 mm away from the hybridization chamber array 110, we would approximately concentrate this output flux in a circular spot size which has a maximum diameter of 2 mm.
• The photon flux in the 2 mm-diameter spot at the hybridization away plane is given by equation 27.
• $\begin{array}{c}{\stackrel{.}{n}}_l=\frac{p_l}{hv_e}\\ =\frac{2.4{\left(10\right)}^{-3}}{\left[6.63{\left(10\right)}^{-34}\right]\left[8.82{\left(10\right)}^{14}\right]}\\ =4.10{\left(10\right)}^{15}\mathrm{photons}\text{/}s\end{array}$
• Using equation 28, we have:
• $\begin{array}{c}{\stackrel{.}{n}}_e={\stackrel{\cdots }{n}}_l\Omega \\ =4.10\left(10\right)15\frac{{\left[16{\left(10\right)}^{-6}\right]}^2}{{\pi \left[1{\left(10\right)}^{-3}\right]}^2}\\ =3.34{\left(10\right)}^{11}\mathrm{photons}\text{/}s\end{array}$
• Now, recalling equation 22 and using the Tb chelate properties listed previously,
• $\begin{array}{c}\Delta t=\frac{\ln \left[\left(6.94{\left(10\right)}^{-5}\right)\left(10\right)\left(0.5\right)\right]}{\frac{1}{1{\left(10\right)}^{-3}}-\frac{1}{0.5{\left(10\right)}^{-9}}}\\ =3.98{\left(10\right)}^{-9}s\end{array}$
• Now from equation 21:
• 
  n _s=(0.5)[3.34(10)¹¹][6.94(10)⁻⁵][1(10)⁻³ ]e ^{−3.98(10) −9 /1(10) −3}
• 
  =11,600 photons per sensor.
• The theoretical number of photons emitted by hybridization events using the SET LED and terbium chelate system are easily detectable and well over the minimum of 30 photons required for reliable detection by the photosensor as indicated above.
Maximum Spacing Between Probes and Photodiode
• The on-chip detection of hybridization avoids the needs for detection via confocal microscopy (see Background of the Invention). This departure from traditional detection techniques is a significant factor in the time and cost savings associated with this system. Traditional detection requires imaging optics which necessarily uses lenses or curved mirrors. By adopting non-imaging optics, the diagnostic system avoids the need for a complex and bulky optical train. Positioning the photodiode very close to the probes has the advantage of extremely high collection efficiency: when the thickness of the material between the probes and the photodiode is of the order of 1 micron, the angle of collection of emission light is up to 173°. This angle is calculated by considering light emitted from a probe at the centroid of the face of the hybridization chamber closest to the photodiode, which has a planar active surface area parallel to that chamber face. The cone of emission angles within which light is able to be absorbed by the photodiode is defined as having the emitting probe at its vertex and the corner of the sensor on the perimeter of its planar face. For a 16 micron×16 micron sensor, the vertex angle of this cone is 170°; in the limiting case where the photodiode is expanded so that its area matches that of the 29 micron×19.75 micron hybridization chamber, the vertex angle is 173°. A separation between the chamber face and the photodiode active surface of 1 micron or less is readily achievable.
• Employing a non-imaging optics scheme does require the photodiode 184 to be very close to the hybridization chamber in order to collect sufficient photons of fluorescence emission. The maximum spacing between the photodiode and probes is determined as follows with reference to FIG. 54.
• Utilizing a terbium chelate fluorophore and a SET UVTOP335TO39BL LED, we calculated 11600 photons reaching our 16 micron×16 micron photodiode 184 from the respective hybridization chamber 180. In performing this calculation we assumed that the light-collecting region of our hybridization chamber 180 has a base area which is the same as our photodiode active area 185, and half of the total number of the hybridization photons reaches the photodiode 184. That is, the light gathering efficiency of the optical system is φ₀=0.5.
• More accurately we can write φ₀=[(base area of the light-collecting region of the hybridization chamber)/(photodiode area)][Ω/4π], where Ω=solid angle subtended by the photodiode at a representative point on the base of the hybridization chamber. For a right square pyramid geometry:
• 
  Ω=4 arcsin(a ²/(4d ₀ ² +a ²)), where d₀=distance between the chamber and the photodiode, and a is the photodiode dimension.
• Each hybridization chamber releases 23200 photons. The selected photodiode has a detection threshold of 17 photons; therefore, the minimum optical efficiency required is:
• 
  φ₀=17/23200=7.33×10⁻⁴
• The base area of the light-collecting region of the hybridization chamber 180 is 29 micron×19.75 micron.
• Solving for d₀, we will get the maximum limiting distance between the bottom of our hybridization chamber and our photodiode 184 to be d₀=249 microns. In this limit, the collection cone angle as defined above is only 0.8°. It should be noted this analysis ignores the negligible effect of refraction.
LOC Device Fabrication Process Variant I
• In addition to standard digital and analog functional blocks, an array of photodiodes 184 (and possibly trigger photodiodes 187) is fabricated during CMOS processing for the hybridization chambers 180 (see FIGS. 54, 77 and 89).
• In ascending order, the four metal layers of the CMOS circuitry 86 are referred to as layers M1 197, M2 201, M3 203 and top metal layer 195 (see FIG. 37). The top metal layer 195 acts as the electrical interface between: (a) the CMOS circuitry 86 and an external test module reader 12 by defining the bond-pads 104 (see FIGS. 5 and 261B) along the edge of the LOC device 301 that connect the CMOS circuitry to an external test module reader 12 through the USB plug 14 (see FIG. 1), and (b) the CMOS circuitry 86 and MST layer 87 by transmitting signals between these to actuate or monitor MST transducers (e.g. heaters, active valves etc) (see FIG. 261A).
• Upon the top metal layer 195 sits the passivation layer 88 to protect the CMOS circuitry 86 from the external environment (see FIG. 261A). Processing of the top metal 195 and passivation 88 layers is outlined in the following section.
• In the second phase of LOC device 301 fabrication the microfluidic structures and associated transducers are fabricated above the CMOS circuitry 86 using a series of standard surface micromachining techniques.
• The top metal layer 195 and passivation layer 88, with which structures in the MST layer 87 interface, are processed as follows. The top metal layer 195 is deposited to a thickness of 1.0 micron. Photoresist is spun-on and patterned. Exposed regions of the top metal layer 195 are etched to a depth of 1.0 micron, stopping on the underlying dielectric layer 199. A passivation layer 88 is then deposited to a thickness of 1.0 micron. The passivation layer 88 is subjected to Chemical Mechanical Planarization (CMP) to remove all topography propagated through the passivation layer 88 from underlying top metal layer 195 features. Photoresist is spun-on and patterned. Exposed regions of the passivation layer 88 are etched to bare the top metal layer 195 (see FIG. 261A).
• At this stage the CMOS circuitry 86 top surface is planar except for the openings through the passivation layer 88 for bond-pads 104 and liquid sensors 174. A layer of sacrificial photoresist is now spun-on to a thickness of 1.0 micron and patterned to protectively cover the bond-pads 104 and liquid sensors 174. The sacrificial photoresist features are not removed until MST processing is near complete.
• The MST channel layer 100 is processed next to form features such as microchannels 158 (see FIG. 50) and hybridization chambers 180 (see FIG. 54). Silicon dioxide is deposited in a layer to a thickness slightly in excess of 8.0 microns, with the expectation that subsequent CMP will erode the layer thickness down to 8.0 microns. Photoresist is spun-on and patterned. Exposed regions of silicon dioxide are subjected to a timed etch to a depth that removes all silicon dioxide from the top surface of the sacrificial photoresist layer protecting the bond-pads 104 and liquid sensors 174. Sacrificial photoresist is spun-on to a layer thickness of 10.0 microns (measured from the base of the MST channel layer 100), filling all MST channel layer 100 topography. This sacrificial photoresist layer is patterned to eliminate excess sacrificial photoresist and then a CMP applied.
• At this stage the top surface is planar, with regions of MST channel layer 100 silicon dioxide and regions of sacrificial photoresist. Referring to FIG. 261A, a layer of silicon dioxide is deposited to form a roof layer 66. The roof layer 66 is less than 5 microns thick, with a preferred thickness which is between 0.5 micron and 2.5 microns.
• Electrical vias, such as heater vias 228 (see FIGS. 56 and 57) and MEMS vias 224 (see FIG. 41), are now fabricated to connect the CMOS circuitry 86 with the MST transducers. Photoresist is spun-on and patterned to expose regions of silicon dioxide with underlying top metal layer 195 features. A timed etch is performed through the roof 66, MST channel layer 100 and passivation 88 layers to a total thickness of approximately 10.0 microns. Etched vias are filled with a metal such as copper using electroless plating and then planarized against the roof layer 66 silicon dioxide with a CMP step.
• The heater layer is processed next to form MST transducers such as heater elements 154 (see FIG. 42), cap channel liquid sensor electrodes 218 and 220 (see FIG. 25), and boiling-initiated valves 106 and 108 (see FIG. 11). Heater layer material, such as TiAl, is deposited. The heater is less than 4 microns thick, with a preferred thickness between 0.25 microns and 2 microns. Photoresist is spun-on and patterned. Exposed regions of heater layer material are completely etched.
• At this stage the top surface is composed of either regions of roof layer 66 silicon dioxide or heater layer features. Openings in the roof layer 66 are now fabricated to form features such as windows into the hybridization chambers 180 and plasma etch holes 200 (see FIGS. 14 and 15). Photoresist is spun-on and patterned. Exposed regions of roof layer 66 silicon dioxide are completely etched. A plasma etch is performed to remove all photoresist, sacrificial or otherwise, leaving the final MST layer 87 (see FIG. 54). Etchant species access to the sacrificial photoresist layers occurs through all roof layer 66 openings, with plasma etch holes 200 arranged primarily to optimise the etch.
• In the third phase of LOC device 301 fabrication, the cap 46 is fabricated independently of the CMOS+MST device 48 before being bonded to the chip top surface. Referring to FIGS. 22 and 127, the cap 46 substrate is 310 microns thick and processed using standard bulk micromachining techniques. The substrate reservoir layer 78 is patterned and etched to a depth of 250 microns. The cap channel layer 80 is patterned and etched to a depth of 60 microns.
• Assembly of the cap 46 upon the CMOS+MST device 48 is performed by first bonding the cap lower seal layer 64 to the top of the roof layer 66 (see FIG. 22). The lower seal layer 64 is 10 microns thick and conforms to topography, sealing the CMOS+MST device 48 top surface where it is in contact with the roof layer 66 and heater layer. The lower seal layer 64 does not encroach upon the MST channels 90 in the MST channel layer 100. The cap substrate (reservoir and cap channel layers 78 and 80) is then attached, with the cap channel layer 80 bonded to the lower seal layer 64 (see FIG. 22). Next, reagents are spotted into reservoirs 54, 56, 58, 60 and 62 and FRET probes 186 are spotted into hybridization chambers 180.
• Finally, a 10 micron-thick cap upper seal layer 82 is bonded to the substrate reservoir layer 78 (see FIG. 22). The upper seal layer 82 has holes for vents 122, the sample inlet 68, the evaporator 190 and the waste reservoir 76. Its attachment completes the LOC device 301.
• In an alternative cap structure, the cap 46 has an interface layer 594, such as a die attach tape, combined with the previously described substrate reservoir layer 78 and cap channel layer 80 (see FIGS. 109 and 127). Comparing LOC variant VIII 518, which employs this alternative cap (see FIG. 127), to LOC variant VII 492 (see FIG. 94) which uses the cap described in the previous section, the difference in physical layers is the replacement of the lower seal layer 64 by the interface layer 594 and the omission of the upper seal layer 82.
• The interface layer 594 is 100 microns thick. Interface layer channels (such as the reservoir-side interface channel 596 and sample-side interface channel 598 displayed in FIG. 111) connect cap channels 94 to microchannels 158 in the MST channel layer 100 through openings in the roof layer 66 like those shown in FIGS. 116A, 116B and 117A. Interface layer channels can also connect multiple MST microchannels 158 as in the case of a valve interface cavity 616 (see FIG. 133).
• The interface layer 594 greatly increases the possible fluid channel configurations connecting the reservoirs 54, 56, 58, 60 and 62 to the CMOS+MST device 48 at the cost of increased fabrication complexity. This increased fabrication complexity is mitigated partially by dispensing with the upper seal layer 82 and harnessing surface tension to retain reagents in their reservoirs (indeed, as done in all LOC devices described herein). Reagent dehydration is minimised using the humidifier 196 and humidity sensor 232 (see FIGS. 4 and 6).
Alternative Systems Incorporating the LOC Device
• FIG. 223 is a sketch of an embodiment of the test module 10 configured to measure the glucose content of blood. The LED spectrometer 772 consists of a set of LEDs 26.1, 26.2, 26.3, each of which transmit a different wavelength through the optical window 136 into the hybridization chambers 180. The photodiode 184 detects the transmission of the different wavelengths through the unprocessed blood sample 770 and a spectrogram is generated. Note that LOC device 301 (see FIG. 5) would be suitable for use in this type of test module.
• Alternative excitation sources can be used which do not require portability or low cost. Alternative excitation sources include lasers and gas flash lamps (for example, xenon). FIG. 224 is a sketch of a configuration using a laser 774 directed onto the hybridization chamber array 110 via an optical train 776. The laser 774 is activated for short pulses and the photosensor 44 (comprising an array of photodiodes 184) acquires the fluorescence emissions.
• A combination of different types of excitation source can be used within the same system to excite fluorophores with different excitation spectra linked to multiple probes in the array of hybridization chambers. By sequentially activating the different excitation sources, fluorescence data for different probes can be obtained, which is, in effect, using each hybridization chamber to perform multiple assays.
Electrochemiluminescence as an Alternative Detection Method
• Electrochemiluminescence (ECL) involves the generation of species at electrode surfaces that then undergo electron-transfer reactions to form excited states that emit light. Electrochemiluminescence differs from normal chemiluminescence in that formation of the excited species relies on oxidation or reduction of the luminophore or a coreactant at an electrode. Coreactants, in this context, are additional reagents added to the ECL solution which enhance the efficiency of ECL emission. In normal chemiluminescence, the excited species form purely through mixing of suitable reagents. The emitting atom or complex is traditionally referred to as a luminophore. In brief, ECL relies on generating an excited state of the luminophore, at which point a photon will be emitted. As with any such process, it is possible for an alternate path to be taken from the excited state which does not lead to the desired light emission (i.e. quenching).
• Embodiments of the test module that use ECL instead of fluorescence detection do not require an excitation LED. Electrodes are fabricated within the hybridization chambers to provide the electrical pulse for ECL generation and the photons are detected using the photosensor 44. The duration and voltage of the electrical pulse are controlled; in some embodiments, control over the current is used as an alternative to controlling the voltage.
Luminophore and Quencher
• The ruthenium complex, [Ru(bpy)₃]²⁺, described previously for use as a fluorescent reporter in the probes, can also be used as a luminophore in an ECL reaction in the hybridization chambers, with TPrA (tri-n-propylamine (CH₃CH₂—CH₂)₃N) as the coreactant. Coreactant ECL has the benefit that luminophores are not consumed after photon emission and the reagents are available for the process to repeat. Furthermore, the [Ru(bpy)₃]²⁺/TPrA ECL system provides good signal levels at physiologically relevant conditions of pH in aqueous solutions. Alternative coreactants which can produce equivalent or better results than TPrA with ruthenium complexes are N-butyldiethanolamine and 2-(dibutylamino)ethanol.
• FIG. 237 illustrates the reactions occurring during an ECL process in which [Ru(bpy)₃]²⁺ is the luminophore 864 and TPrA is the coreactant 866. ECL emission 862 in the [Ru(bpy)₃]²⁺/TPrA ECL system follows the oxidation of both Ru(bpy)₃ ²⁺ and TPrA at the anode 860. The reactions are as follows:
• 
  Ru(bpy)₃ ²⁺ −e ⁻→Ru(bpy)₃ ³⁺  (1)
• 
  TPrA−e ⁻→[TPrA^.]⁺→TPrA^.+H⁺  (2)
• 
  Ru(bpy)₃ ³⁺+TPrA^.→Ru(bpy)₃ ^.2++products  (3)
• 
  Ru(bpy)₃ ^.2+→Ru(bpy)₃ ²⁺ +hν  (4)
• The wavelength of the emitted light 862 is around 620 nm and the anode potential is around 1.1V with respect to a Ag/AgCl reference electrode. For the [Ru(bpy)₃]²⁺/TPrA ECL system, either the Black Hole Quencher, BHQ 2, or Iowa Black RQ described previously, would be a suitable quencher. In the embodiments described here, the quencher is a functional moiety which is initially attached to the probe, but other embodiments are possible in which the quencher is a separate molecule free in solution.
Hybridization Probes for ECL Detection
• FIGS. 281 and 282 show the hybridization-responsive ECL probes 237. These are often referred to as molecular beacons and are stem-and-loop probes, generated from a single strand of nucleic acid, that luminesce upon hybridization to complementary nucleic acids. FIG. 281 shows a single ECL probe 237 prior to hybridization with a target nucleic acid sequence 238. The probe has a loop 240, stem 242, a luminophore 864 at the 5′ end, and a quencher 248 at the 3′ end. The loop 240 consists of a sequence complementary to the target nucleic acid sequence 238. Complementary sequences on either side of the probe sequence anneal together to form the stem 242.
• In the absence of a complementary target sequence, the probe remains closed as shown in FIG. 281. The stem 242 keeps the luminophore-quencher pair in close proximity to each other, such that significant resonant energy transfer can occur between them, substantially eliminating the ability of the luminophore to emit light after electrochemical excitation.
• FIG. 282 shows the ECL probe 237 in an open or hybridized configuration. Upon hybridization to a complementary target nucleic acid sequence 238, the stem-and-loop structure is disrupted, the luminophore 864 and quencher 248 are spatially separated, thus restoring the ability of the luminophore 864 to emit light. The ECL emission 862 is optically detected as an indication that the probe has hybridized.
• The probes hybridize with very high specificity with complementary targets, since the stem helix of the probe is designed to be more stable than a probe-target helix with a single nucleotide that is not complementary. Since double-stranded DNA is relatively rigid, it is sterically impossible for the probe-target helix and the stem helix to coexist.
Primer-Linked ECL Probes
• Primer-linked stem-and-loop probes and primer-linked linear probes, otherwise known as scorpion probes, are an alternative to molecular beacons and can be used for real-time and quantitative nucleic acid amplification in the LOC device. Real-time amplification is performed directly in the hybridization chambers of the LOC device. The benefit of using primer-linked probes is that the probe element is physically linked to the primer, thus only requiring a single hybridization event to occur during the nucleic acid amplification rather than separate hybridizations of the primers and probes being required. This ensures that the reaction is effectively instantaneous and results in stronger signals, shorter reaction times and better discrimination than when using separate primers and probes. The probes (along with polymerase and the amplification mix) would be deposited into the hybridization chambers 180 during fabrication and there would be no need for an amplification section on the LOC device. Alternatively, the amplification section is left unused or used for other reactions.
Primer-Linked Linear ECL Probes
• FIGS. 283 and 284 show a primer-linked linear ECL probe 693 during the initial round of nucleic acid amplification and in its hybridized configuration during subsequent rounds of nucleic acid amplification, respectively. Referring to FIG. 283, the primer-linked linear ECL probe 693 has a double-stranded stem segment 242. One of the strands incorporates the primer linked probe sequence 696 which is homologous to a region on the target nucleic acid 696 and is labelled on its 5′ end with luminophore 864, and linked on its 3′ end to an oligonucleotide primer 700 via an amplification blocker 694. The other strand of the stem 242 is labelled at its 3 end with a quencher molecule 248. After the initial round of nucleic acid amplification has completed, the probe can loop around and hybridize to the extended strand with the, now, complementary sequence 698. During the initial round of nucleic acid amplification, the oligonucleotide primer 700 anneals to the target DNA 238 (see FIG. 283) and is then extended, forming a DNA strand containing both the probe sequence and the amplification product. The amplification blocker 694 prevents the polymerase from reading through and copying the probe region 696. Upon subsequent denaturation, the extended oligonucleotide primer 700/template hybrid is dissociated and so is the double stranded stem 242 of the primer-linked linear probe, thus releasing the quencher 248. Once the temperature decreases for the annealing and extension steps, the primer linked probe sequence 696 of the primer-linked linear ECL probe curls around and hybridizes to the amplified complementary sequence 698 on the extended strand and light emission is detected indicating the presence of the target DNA. Non-extended primer-linked linear ECL probes retain their double-stranded stem and light emission remains quenched. This detection method is particularly well suited for fast detection systems as it relies on a single-molecule process.
Primer-Linked Stem-and-Loop ECL Probes
• FIGS. 285A to 285F show the operation of a primer-linked stem-and-loop ECL probe 705. Referring to FIG. 285A, the primer-linked stem-and-loop ECL probe 705 has a stem 242 of complementary double-stranded DNA and a loop 240 which incorporates the probe sequence. One of the stem strands 708 is labelled at its 5′ end with luminophore 864. The other strand 710 is labelled with a 3′-end quencher 248 and carries both the amplification blocker 694 and oligonucleotide primer 700. During the initial denaturation phase (see FIG. 285B), the strands of the target nucleic acid 238 separate, as does the stem 242 of the primer-linked stem-and-loop ECL probe 705. When the temperature cools for the annealing phase (see FIG. 285C), the oligonucleotide primer 700 on the primer-linked stem-and-loop ECL probe 705 hybridizes to the target nucleic acid sequence 238. During extension (see FIG. 285D), the complement 706 to the target nucleic acid sequence 238 is synthesized forming a DNA strand containing both the probe sequence 705 and the amplified product. The amplification blocker 694 prevents the polymerase from reading through and copying the probe region 705. When the probe next anneals, following denaturation (see FIG. 285E), the probe sequence of the loop segment 240 of the primer-linked stem-and-loop probe (see FIG. 285F) anneals to the complementary sequence 706 on the extended strand. This configuration leaves the luminophore 864 relatively remote from the quencher 248, resulting in a significant increase in light emission.
ECL Control Probes
• The hybridization chamber array 110 includes some hybridization chambers 180 with positive and negative ECL control probes used for assay quality control. FIGS. 286 and 287 schematically illustrate negative control ECL probes 786 without a luminophore, and FIGS. 288 and 289 are sketches of positive control ECL probes 787 without a quencher. The positive and negative control ECL probes have a stem-and-loop structure like the ECL probes described above. However, an ECL signal 862 (see FIG. 282) will always be emitted from positive control ECL probes 787 and no ECL signal 862 is ever emitted from negative control ECL probes 786, regardless of whether the probes hybridize into an open configuration or remain closed.
• Referring to FIGS. 286 and 287, the negative control ECL probe 786 has no luminophore (and may or may not have a quencher 248). Hence, whether the target nucleic acid sequence 238 hybridizes with the probe as shown in FIG. 287, or the probe remains in its stem 242 and loop 240 configuration as shown in FIG. 286, the ECL signal is negligible. Alternatively, the negative control ECL probe could be designed so that it always remains quenched. For example, by having an artificial probe (loop) sequence 240 that will not hybridize to any nucleic acid sequence within the sample under investigation, the stem 242 of the probe molecule will re-hybridize to itself and the luminophore and quencher will remain in close proximity and no appreciable ECL signal will be detected. This negative control would account for any low level emission that may occur if the quenching is not complete.
• Conversely, the positive control ECL probe 787 is constructed without a quencher as illustrated in FIGS. 288 and 289. Nothing quenches the ECL emission 862 from the luminophore 864 regardless of whether the positive control probe 787 hybridizes with the target nucleic acid sequence 238.
• FIGS. 275 and 276 show another possibility for constructing a positive control chamber. In this case, the calibration chambers 382 which are sealed from the amplicon (or any flow containing target molecules) can be filled with the ECL luminophore solution such that a positive signal is always detected at the electrode
• Similarly, the control chambers can be negative control chambers because the lack of inlets prevents any targets from reaching the probes such that an ECL signal is never detected.
• FIG. 52 shows a possible distribution of the positive and negative control probes (378 and 380 respectively) throughout the hybridization chamber array 110. For ECL, positive and negative control ECL probes 786 and 787 would replace control fluorescent probes 378 and 380, respectively. The control probes are placed in hybridization chambers 180 along a line extending diagonally across the hybridization chamber array 110. However, the arrangement of the control probes within the array is arbitrary (as is the configuration of the hybridization chamber array 110).
Calibration Chambers for ECL Detection
• The non-uniformity of the electrical characteristic of the photodiode 184, response to any ambient light present at the sensor array, and light originating at other locations in the array, introduce background noise and offset into the output signal. This background is removed from each output signal by calibration chambers 382 in the hybridization chamber array 110 which either do not contain any probes, contain probes that have no ECL luminophore, or contain probes with a luminophore and quencher configured such that quenching is always expected to occur. The number and arrangement of the calibration chambers 382 throughout the hybridization chamber array is arbitrary. However, the calibration is more accurate if photodiodes 184 are calibrated by a calibration chamber 382 that is relatively proximate. Referring to FIG. 304, the hybridization chamber array 110 has one calibration chamber 382 for every eight hybridization chambers 180. That is, a calibration chamber 382 is positioned in the middle of every three by three square of hybridization chambers 180. In this configuration, the hybridization chambers 180 are calibrated by a calibration chamber 382 that is immediately adjacent.
• FIG. 228 shows a differential imager circuit 788 used to substract the signal from the photodiode 184 corresponding to the calibration chamber 382 as a result of the applied electrical pulse, from the ECL signal from the surrounding hybridization chambers 180. The differential imager circuit 788 samples the signal from the pixel 790 and a “dummy” pixel 792. Signals arising from ambient light in the region of the chamber array are also subtracted. The signals from the pixel 790 are small (i.e. close to dark signal), and without a reference to a dark level it is hard to differentiate between the background and a very small signal.
• During use, the “read_row” 794 and “read_row_d” 795 are activated and M4 797 and MD4 801 transistors are turned on. Switches 807 and 809 are closed such that the outputs from the pixel 790 and “dummy” pixel 792 are stored on pixel capacitor 803 and dummy pixel capacitor 805 respectively. After the pixel signals have been stored, switches 807 and 809 are deactivated. Then the “read_col” switch 811 and dummy “read_col” switch 813 are closed, and the switched capacitor amplifier 815 at the output amplifies the differential signal 817.
ECL Levels and Signal Efficiency
• The normal metric of efficiency in ECL is the number of photons obtained per “Faradaic” electron, i.e. per electron which participates in the electrochemistry. The ECL efficiency is denoted φ_ECL:
• $\begin{array}{cc}{\phi }_{\mathrm{ECL}}=\frac{{\int }_0^tI\tau }{\left(\frac{N_A}{F}\right){\int }_0^ti\tau }& \left(5\right)\end{array}$
• where I is the intensity in photons per second, i is the current in amperes, F is Faraday's constant, and N_A is Avogadro's constant.
Efficiency of Coreactant ECL
• Annihilation ECL in deoxygenated, aprotic solutions (e.g. nitrogen-flushed acetonitrile solutions) is simple enough to allow efficiency measurements, and the consensus value of φ_ECL, is around 5%. Coreactant systems, however, have been generally declared to be beyond meaningful direct measurements of efficiency. Instead, emission intensity is related by scaling to easily-prepared standard solutions such as Ru(bpy)₃ ²⁺, measured in the same format. The literature (see for example J. K. Leland and M. J. Powell, J. Electrochem. Soc., 137, 3127 (1990), and R. Pyati and M. M. Richter, Annu. Rep. Prog. Chem. C, 103, 12-78 (2007)) indicates that (without enhancers such as surfactants), the efficiency of Ru(bpy)₃ ²⁺ ECL with TPrA coreactants peaks at levels comparable to the 5% seen for annihilation ECL in acetonitrile (e.g. 2% efficiency; see I. Rubinstein & A. J. Bard, J. Am. Chem. Soc., 103 512-516 (1981)).
ECL Potentials
• The voltage at the working electrode for the Ru(bpy)₃ ²⁺/TPrA system is approximately +1.1 V (generally measured in the literature with respect to a reference Ag/AgCl electrode). Voltages this high shorten electrode lifetimes but this is not an issue for single-use devices such as the LOC device used in the present diagnostic system.
• The ideal voltage between the anode and cathode depends on the combination of solution components and electrode materials. Selecting the correct voltage can require compromising between the highest signal levels, reagent and electrode stability, and the activation of undesired side reactions such as electrolysis of the water in the chamber. In tests on buffered aqueous Ru(bpy)₃]²⁺/coreactant solution and platinum electrodes, the ECL emission is maximized at 2.1-2.2 V (depending on the coreactant choice). Emission intensities drop to <75% of the peak values for voltages below 1.9 V and above 2.6 V, and to <50% of the peak values for voltages below 1.7 V and above 2.8 V. A preferred anode-cathode voltage difference for ECL operation in such systems is therefore 1.7-2.8 V, with the range 1.9-2.6 V being particularly preferred. This allows maximization of the emission intensity as a function of voltage, while avoiding voltages at which significant gas evolution at the electrodes is observed.
ECL Emission Wavelength
• The wavelength of the emitted light 862 from ECL has an intensity peak at around 620 nm (measured in air or vacuum), and the emission spans a relatively broad wavelength range. Significant emission occurs at wavelengths from around 550 nm to 700 nm. Furthermore, the peak emission wavelength can vary by ˜10% due to changes in the chemical environment around the active species. The LOC device embodiments described here, which incorporate no wavelength-specific filters, have two advantages for capturing signals with such a broad and variable spectrum. The first advantage is sensitivity: any wavelength filter reduces light transmission, even within its pass band, so efficiency is improved by not including a filter. The second advantage is flexibility: adjustment of filter pass bands is not required after minor reagent changes, and the signals are less dependent on minor differences in non-target components of the input sample.
Solution Volume Participating in ECL
• ECL relies on the availability of luminophore (and coreactant) in solution. However, as illustrated in FIG. 239, the excited species 868 are generated only in the solution 872 near the electrodes 860 and 870. The parameter boundary layer depth in the models presented here, is the depth of the layer of solution 872 around the electrode 860 in which the excited species 868 are generated.
• This is a simplification, since solution dynamics can drive the available concentration upward or downward:
• • Increased availability: diffusion and electrophoretic effects will allow exchange with more of the solution.
  • Decreased availability: reagents can adsorb onto the electrodes and may become unavailable to the ECL process.
• For a boundary layer depth value of 0.5 μm, the following observations are made:
• ECL is observed in experiments where conjugation to magnetic beads with diameters up to 4.5 μm is used to attract the luminophore 864 to the anode 860.
• Ru(bpy)₃ ²⁺/TPrA ECL emission 862 as a function of electrode spacing, for interdigitated electrode arrays, was found to be maximised at a 0.8 μm electrode spacing. The requirement for a coreactant 866 in aqueous solutions 872 can be lifted when electrode spacings are ˜2 μm. This indicates that the excited species 868 diffuse multiple microns, which implies diffusive exchange on a similar scale for the species in the ground state.
Steady State and Pulsed Operation
• During pulsed activation of the electrodes 860 and 870, the intensity of the ECL emission 862 (see FIG. 282) is generally higher than the intensity of the emission 862 from steady-state activation of the electrodes. Accordingly, the activation signal to the electrodes 860 and 870 is pulse-width modulated (PWM) by the CMOS circuitry 86 (see FIG. 244).
Reagent Recycling and Species Lifetime
• The Ru complex is not consumed in the Ru(bpy)₃ ²⁺/TPrA ECL system, so the intensity of emission 862 does not reduce with successive reaction cycles. The lifetime of the rate-limiting step is approximately 0.2 milliseconds giving a total reaction recycling time of approximately 1 millisecond.
Electrophoretic Effects and Other Constraints
• Given the complexity of the solutions in the hybridization chamber, a large number of phenomena take place when the ECL voltage is turned on. Electrophoresis of macromolecules, ohmic conduction, and capacitive effects from small ion migration occur simultaneously.
• Electrophoresis of the oligonucleotides (probes and amplicon) can complicate the detection of probe-target hybrids, as DNA is highly negatively charged and attracted to the anode 860. The time scale for this motion is typically short (in the order of milliseconds). Electrophoretic effects are strong even though the voltages are moderate (˜1 V), because the separation between the anode 860 and cathode 870 is small.
• Electrophoresis enhances the ECL emission 862 in some embodiments of the LOC device and degrades the emission in others. This is addressed by increasing or decreasing the electrode spacing to get the associated increases or decreases in electrophoretic effect. Interdigitation of the anode 860 and the cathode 870 above the photodiode 184 represents the extreme case of minimizing this separation. Such an arrangement produces ECL, even in the absence of a coreactant 866 at carbon electrodes 860 and 870.
Ohmic Heating (DC Current)
• The current required to maintain an ECL voltage of ˜2.2 V, is determined as follows with reference to the ECL cell 874 schematically illustrated in FIG. 240.
• The DC current through the chamber is determined by two resistances: the interface resistance R_i between the electrodes 860 and 870 and the bulk of the solution, and the solution resistance R_s which is derived from the bulk solution resistivity and conduction path geometry. For solutions with ionic strengths relevant to the conditions in LOC devices, the chamber resistance is dominated by interfacial resistances at the electrodes 860 and 870, and R_s can be neglected.
• The effect of the interfacial resistance is estimated by scaling measurements of macroscopic current flow through similar solutions for the electrode geometries in the LOC devices.
• Macroscopic measurements of current density through a similar solution, at platinum electrodes, were taken. Consistent with the worst-case (high current) approach being taken, overall ionic strength and ECL reactant concentrations in the test solution were higher than those used in the LOC devices. The anode area was smaller than the cathode area, and was surrounded by a cathode with comparable area in a ring geometry. For an anode consisting of a circle 2 mm in diameter, the current measured was 1.1 mA, giving a current density of 350 A/m².
• In the heating model, the electrode area is for the square ring geometry schematically illustrated in FIG. 240. The anode is a ring with width 1 μm and thickness 1 μm. The surface area is 196 square microns, and therefore the calculated current I=69 nA.
• The heating (power=V²/R) was modelled for the worst case in which all the heat goes into raising the temperature of the water in the chamber. This leads to heating of chamber contents at 5.8° C./s, at a voltage difference of 2.2 V, if no allowance for heat removal by the bulk of the LOC device is made.
• Heating of the chambers by ˜20° C. can cause denaturation of most hybridization probes. For highly specific probes intended for mutation detection, it is preferable to further restrict heating to 4° C. or less. With this level of temperature stability, single base mismatch-sensitive hybridization, using appropriately designed sequences, becomes feasible. This allows the detection of mutations and allelic differences at the level of single nucleotide polymorphisms. Hence the DC current is applied to the electrodes 860 and 870 for 0.69 s, to limit the heating to 4° C.
• A current of ˜69 nA passing through the chamber is far more than can be accommodated as Faradaic current by the ECL species at micromolar concentrations. Therefore, low-duty-cycle pulsing of the electrodes 860 and 870 to further reduce heating (to 1° C. or less) while maintaining sufficient ECL emission 862, does not introduce complications associated with reagent depletion. In other embodiments, the current is reduced to 0.1 nA which removes the need for pulsed activation of the electrodes. Even at currents as low as 0.1 nA, the ECL emission 862 is luminophore-limited.
Chamber and Electrode Geometry Maximizing Optical Coupling Between ECL Luminescence and Photosensor
• The immediate chemical precursors of ECL luminescence are generated within nanometres of the working electrode. Referring again to FIG. 239, light emission (the excited species 868) generally occurs within microns or less of that location. Hence the volume immediately adjacent to the working electrode (anode 860) is visible to the corresponding photodiode 184 of the photosensor 44. Accordingly, the electrodes 860 and 870 are directly adjacent the active surface area 185 of the corresponding photodiode 184 in the photosensor 44. Furthermore, the anode 860 is shaped to increase the length of its lateral periphery ‘seen’ by the photodiode 184. This aims to maximize the volume of excited species 868 that can be detected by the underlying photodiode 184.
• FIG. 238 schematically illustrates three embodiments of the anode 860. A comb structure anode 878 has the advantage that the parallel fingers 880 can be interdigitated with the fingers of a cathode 870. The interdigitated configuration is shown in FIG. 245, and in a partial view of a LOC layout in FIGS. 272 and 276. The interdigitated configuration provides a uniform dielectric gap 876 (see FIG. 239) that is relatively narrow (1 to 2 microns) and the interdigitated comb structure is relatively simple for the lithographic fabrication process. As discussed above, a relatively narrow dielectric gap 876 between the electrodes 860 and 870 obviates the need for a coreactant in some solutions 872, as the excited species 868 will diffuse between anode and cathode. The removal of the requirement for a coreactant removes the potential chemical impact of the coreactant on the various assay chemistries and provides a wider range of possible assay options.
• Referring again to FIG. 238, some embodiments of the anode 860 have a serpentine configuration 882. To achieve high periphery length while maintaining tolerance against fabrication errors, it is convenient to form wide, rectangular meanders 884.
• The anode may have a more complex configuration 886 if necessary or desirable. For example, it may have a crenulated section 888, a branched structure 890, or a combination of the two. Partial views of LOC designs incorporating a branched structure 890 are shown in FIGS. 303 and 304. The more complicated configurations such as 886 provide a long length of lateral periphery, and are best suited to solution chemistries where a coreactant is employed since patterning a closely-spaced opposing cathode is more difficult.
Electrode Thickness
• Generally, ECL cells involve a planar working electrode which is viewed externally. Also, traditional microfabrication techniques for metal layers tend to lead to planar structures with metal thicknesses of approximately 1 micron. As has been indicated earlier, and shown schematically in FIGS. 238, 241 and 242, increasing the length of lateral periphery enhances the coupling between the ECL emission and the photodiode 184.
• A second strategy to further increase the efficiency of collection of emitted light 862 (see FIG. 282) by the photodiode 184 is to increase the thickness of the anode 860. This is shown schematically in FIG. 239. The part of the participating volume 892 adjacent to the walls of the working electrode is the region most efficiently coupled to the photodiode 184. Therefore, for a given width of working electrode 860, the overall collection efficiency of the emitted light 862 can be improved by increasing the thickness of the electrodes. Further, since high current carrying capacity is not required, the width of the working electrode 860 is reduced as far as is practical. The thickness of the electrodes 860 and 870 can not increase without restrictions. Noting that the feature and separation sizes of the electrodes are likely to be of the order of 1 micron, and that liquid filling makes gaps which are wider than they are deep unfavourable, the optimum practical thickness for the electrodes is 0.25 micron to 2 microns.
Electrode Spacing
• The spacing between the electrodes 860 and 870 is important for the quality of signals in LOC devices, particularly in embodiments where the electrodes are interdigitated. In embodiments where the anode 860 is a branched structure such as shown in FIG. 238 and FIG. 242, the spacing between adjacent elements can also be important. ECL emission efficiency, and the collection efficiency of the emitted light, should both be maximised.
• Generation of ECL emission tends to favour electrode spacings on the order of one micron or less. Small spacings are particularly attractive when performing ECL in the absence of a coreactant. The fact that the spacing can be comparable to the wavelength of the emitted light 862 is of limited importance. Therefore, in many embodiments where the emitted light 862 (see FIG. 282) is measured at a location which does not require that the light have passed between the electrodes 860 and 870, making the electrode spacing as small as practical is often the goal. In embodiments where the emitted light 862 must pass between the electrodes 860 and 870, however, it becomes necessary to move beyond considering just the ECL emission process, and consider the wave properties of light.
• The wavelength of the emitted light 862 from ECL of Ru(bpy)₃ ²⁺ is around 620 nm, and therefore 460 nm (0.46 microns) in water. In embodiments where the photodiode 184 and the ECL excited species 868 are on different sides of the electrode structure, and the electrode structure is metallic, the emitted light 862 must pass through a gap between elements of the metallic structures. If this gap is comparable to the wavelength of the light, diffraction generally reduces the intensity of propagating light which reaches the photodiode 184. In cases where the emitted light 862 is incident on the gap at large angles, however, evanescent mode coupling can be harnessed to improve the strength of collected signals. Two measures are taken in the LOC devices to enhance the efficiency of coupling between the photodiode 184 and the emitted light 862.
• First, the separation between metallic elements is not reduced below approximately the wavelength of the emitted light in water, i.e. approximately 0.4 microns. When combined with other observations regarding small separations between interdigitated electrodes, this indicates an optimal range for the electrode spacing of 0.4 to 2 microns.
• Second, the distance from the gap between elements to the photodiode 184 is minimised. In the LOC device embodiments described here, this indicates that the total thickness of layers between the electrodes 860 and 870 and the photodiode 184 be one micron or less. In embodiments where multiple layers are present between the electrodes and the photodiode, arranging their thicknesses to be quarter-wave or three-quarter wave layers has the further benefit of suppressing reflection of the emitted light 862.
Electrode Models
• FIG. 239 is a schematic partial cross-section of the electrodes 860 and 870 in the hybridization chamber. The volume around the lateral periphery of the anode 860 occupied by the excited species 868, is sometimes referred to as the participating volume 892. The occluded region 894 above the anode 860 is ignored because its optical coupling to the photodiode 184 is negligible.
• A technique for determining whether a particular electrode configuration provides a foundation for the level of ECL emission 862 for the underlying photodiode 184 is set out below with reference to FIGS. 240, 241 and 242.
• FIG. 240 is a ring geometry in which the anode 860 is around the edge of photodiode 184. In FIG. 241, the anode 860 is positioned within the periphery of the photodiode 184. FIG. 242 shows a more complex configuration in which the anode 860 has a series of parallel fingers 880 to increase the length of its lateral edges.
• For all of the above configurations, the model calculations are as follows. For a participating volume 892 of solution V_ECL, the total effective number of emitters N_em is:
• 
  N _em =N _lum·τ_p/τ_ECL =V _ECL C _L N _A·τ_p/τ_ECL  (6)
• where the participating number of luminophores N_lum=V_ECLC_LN_A, τ_ECL is the lifetime of the ECL process, C_L is the luminophore concentration, T_p is the pulse duration, and N_A is Avogadro's number.
• The number of isotropically emitted photons N_phot is:
• 
  N _phot=φ_ECL N _em  (7)
• where φ_ECL is the ECL efficiency, defined as the average number of photons emitted by the ECL reaction of a single luminophore.
• The signal count of electrons, S, from the photodiode is then
• 
  S=N _phot. φ_oφ_q,  (8)
• where φ_o is the optical coupling efficiency (the number of photons absorbed by the photodiode 184) and φ_q is the photodiode quantum efficiency. The signal is therefore:
• $\begin{array}{cc}S=V_{\mathrm{ECL}}C_LN_A\frac{{\tau }_p}{{\tau }_{\mathrm{ECL}}}{\phi }_{\mathrm{ECL}}{\phi }_o{\phi }_q& \left(9\right)\end{array}$
• For FIGS. 240 and 241 electrode configurations, φ_o is:
• 
  φ_o=(25% photons which are directed towards the photodiode 184)×(10% of photons which are not reflected)
• i.e., φ_o=2.5% for configurations shown in FIGS. 240 and 241
• For the electrode configuration of FIG. 242, 50% of photons are emitted in a direction pointing towards the photodiode 184, but the absorption efficiency as a function of angle is unchanged, so
• 
  φ_o=(50% photons which are directed towards the photodiode)×(10% of photons which are not reflected)
• i.e., φ_o=5% for the configuration of FIG. 242.
• The participating volume 892 depends on the electrode configuration, and details are presented in the corresponding sections.
• The input parameters for the calculations are listed in the following:

• TABLE 5
  Input Parameters

  Parameter	Value	Comment

  Luminophore concen-	2.89	μM	Probe concentration
  tration CL			calculated previously
  ECL recycling period	1	ms	Combined lifetimes of
  (lifetime) τECL			reaction steps for
  			luminophore.
  Boundary layer	0.5	μm	Effective volume (including
  depth D			diffusion and electro-
  			phoresis) of solution
  			participating in ECL
  Duration of current	0.69	s	Chosen to limit ohmic
  application τp			heating to 4° C. (as
  			described previously)
  Chamber X dimension	28	μm
  Chamber Y dimension	28	μm
  Chamber height Z	8	μm
  Photodiode X dimension	16	μm
  Photodiode Y dimension	16	μm
  Electrode thickness (i.e.,	1	μm
  exposed edge height)
  Electrode layer minimum	1	μm	Process critical
  width and gap			dimension
  Electrode interfacial	350	A/m2	For ohmic heating
  current density
  Solution volume	0.5	Ω · m	For ohmic heating
  resistivity
  Voltage difference	2.2	V
  applied (working −
  counter electrode)

Ring Geometry Around Periphery of Photodiode
• Referring to FIG. 240, the anode 860 is a ring around the edge of the photodiode 184. In this configuration, the participating volume 892 is:
• 
  V _ECL=4×[(layer beside the electrode wall)+(quarter-cylinder above the electrode wall)]
• Calculation Results:
• Photons generated from a 0.5 μm boundary layer: 3.1×10⁵
  Electron counts in photodiode: 2.3×10³
• This signal is readily detectable by the underlying photodiode 184 of the LOC device photosensor 44.
Additional Fingers to Increase Edge Length
• Referring to FIG. 242, parallel fingers 880 are added across the anode 860. Only horizontal edges shown in figure contribute to the participating volume 892, to avoid double-counting the perpendicular edges. The participating volume 892 is then:
• 
  V _ECL=(8×2)×[(layer beside the electrode wall)+(quarter-cylinder above the electrode wall)]
• Calculation Results for FIG. 242 Configuration:
• Photons generated from a 0.5 μm boundary layer: 1.1×10⁶
  Electron counts in photodiode 184: 8.0×10³
• This signal is easily detectable in the photodiode 184.
Complete Overlay
• This configuration shown in FIG. 243 and FIG. 244 is included as a limiting case of maximum surface area coupling. In practice, 90% or better coupling between the electrode surface area and the active surface area 185 of the photodiode 184 achieves a nearly optimal result, and even coupling of 50% of the photodiode active surface area 185 to the electrode surface area provides most of the benefit of the complete overlay configuration. Complete overlay can be achieved in two embodiments: first, as indicated schematically in FIG. 243, by employing a transparent anode 860, in a plane parallel with that of the photodiode 184 and with an area matched to that of the photodiode, and arranging the anode in immediate proximity to the photodiode 184, such that emitted light 862 passes through the anode and onto the photodiode. In a second embodiment shown schematically in FIG. 244, the anode 860 is again parallel to and registered with the photodiode area, but the solution 872 fills a void between the anode 860 and the photodiode 184. For signal modelling of a complete overlay configuration, the anode is assumed to be a complete layer above the photodiode 184, with half of the photons directed toward the photodiode 184 (absorption efficiency still 10%).
• Photons generated from a 0.5 μm boundary layer: 7.7×10⁵
  Electron counts in photodiode: 1.2×10⁴
• It is possible to improve the signal and assay beyond the above models by using surfactants and probe immobilization at the anode.
Maximum Spacing Between ECL Probes and Photodiode
• The on-chip detection of hybridization avoids the needs for detection via confocal microscopy (see Background of the Invention). This departure from traditional detection techniques is a significant factor in the time and cost savings associated with this system. Traditional detection requires imaging optics which necessarily uses lenses or curved mirrors. By adopting non-imaging optics, the diagnostic system avoids the need for a complex and bulky optical train. Positioning the photodiode very close to the probes has the advantage of extremely high collection efficiency: when the thickness of the material between the probes and the photodiode is on the order of 1 micron, the angle of collection of emission light is up to 174°. This angle is calculated by considering light emitted from a probe at the centroid of the face of the hybridization chamber closest to the photodiode, which has a planar active surface parallel to that chamber face. The cone of emission angles within which light is able to be absorbed by the photodiode is defined as having the emitting probe at its vertex and the corner of the sensor on the perimeter of its planar face. For a 16 micron×16 micron sensor, the vertex angle of this cone is 170°; in the limiting case where the photodiode is expanded so that its area matches that of the 28 micron×26.5 micron hybridization chamber, the vertex angle is 174°. A separation between the chamber face and the photodiode active surface of 1 micron or less is readily achievable.
• Employing a non-imaging optics scheme does require the photodiode 184 to be very close to the hybridization chamber in order to collect sufficient photons of fluorescence emission. The maximum spacing between the photodiode and probes is determined as follows.
• Utilizing a ruthenium chelate luminophore and the electrode configuration of FIG. 242, we calculated 27,000 photons being absorbed by our 16 micron×16 micron sensor from the respective hybridization chamber, to generate 8000 electrons assuming a sensor quantum efficiency of 30%. In performing this calculation we assumed that the light-collecting region of our hybridization chamber has a base area which is the same as our sensor area, one quarter of the total number of the hybridization photons is angled so as to reach the sensor, and a conservative 10% estimate for the proportion of photons which do not scatter away from the sensor-dielectric interface. That is, the light gathering efficiency of the optical system is φ₀=0.025.
• More accurately we can write φ₀=[(base area of the light-collecting region of the hybridization chamber)/(photodetector area)][Ω/4π][10% absorbed], where Ω=solid angle subtended by the photodetector at a representative point on the base of the hybridization chamber. For a right square pyramid geometry:
• 
  Ω=4 arcsin(a ²/(4d ₀ ² +a ²)), where d₀=distance between the chamber and the photodiode, and a is the photodiode dimension.
• Each hybridization chamber releases 1.1×10⁶ photons. The selected photodetector has a detection threshold of 17 photons, and for values of d₀ greater than ten times the sensor size (i.e., essentially normal incidence) the proportion of photons not reflected at the sensor surface can be increased from 10% to 90%. Therefore, the minimum optical efficiency required is:
• 
  φ₀=17/(1.1×10⁶×0.9)=1.72×10⁻⁵
• The base area of the light-emitting region of the hybridization chamber 180 is 29 micron×19.75 micron.
• Solving for d₀, we will get the maximum limiting distance between the bottom of our hybridization chamber and our photodetector to be d₀=1600 microns. In this limit, the collection cone angle as defined above is only 0.8°. It should be noted this analysis ignores the negligible effect of refraction.
LOC Device Fabrication Process Variant II
• The fabrication process for the ECL LOC devices is such that the electrodes for ECL are constructed inside the hybridization chambers using the heater material. Consequently, the heaters 154 or 182 are also constructed inside the heated microchannels 158 (see FIG. 262A) and within the walls of the hybridization chambers 180 rather than on top of the roof layer 66, as was the case in the previously described fabrication process (see FIG. 54).
• The previously described fabrication process for the CMOS circuitry 86 applies to ECL LOC devices with the exception that the CMOS top metal layer 195 (see FIG. 37) also provides current to the electrodes for ECL generation.
• As discussed in the previous fabrication process, MST processing builds up the MST layer on top of the CMOS circuitry 86 using a series of standard deposition, patterning and etching steps. Patterning, sealing and bonding of the cap 46 is also in accordance with the previously described fabrication process.
• The fabrication process will now be briefly described with reference to FIGS. 239, 262A and 262B. The photodiodes 184 are fabricated for each of the yet to be fabricated hybridization chambers 180 (see FIG. 239). Next the CMOS top metal layer 195 is deposited as a laminate of TiN/AlCu/TiN (see FIG. 262A). The TiN layers are each 0.1 microns while the AlCu layer is 0.8 microns. The final TiN layer acts as an antireflective coating (ARC) which reduces unwanted reflections during subsequent exposure steps. It also has suitable composition to form electrical connections with the heater material.
• A layer of photoresist is deposited and patterned. The exposed areas of the top metal layer 195 are etched to a depth of 1.0 micron.
• Next the passivation layer 88 is deposited as a 1.0 micron stack of oxide/nitride/oxide. The top surface is planarized using chemical mechanical polishing (CMP) to remove any topography caused by deposition over the etched CMOS top metal layer 195 (see FIG. 262A).
• Photoresist is spun onto the passivation layer 88 and patterned with the appropriate mask. The vias to the CMOS circuitry 86 are then formed by etching through the exposed areas of the passivation layer.
• Next, the heater material is deposited to a thickness of 1.0 micron (See FIG. 262A). The heater material is photoresist coated and patterned to define the heater elements 154, cap channel liquid sensor TiAl electrodes 218 and 220, boiling-initiated valves 108 and boiling-initiated valve contacts 153.
• Photoresist is then spun over the wafer and patterned to expose the pad oxide (the CMOS top metal layer 195) which is then etched to open the bond-pads 104 (see FIG. 262B) and MST channel liquid sensors 174.
• Next, a sacrificial layer is deposited to a thickness of 2 μm and patterned to protect heater material, the bond-pads 104 and the MST channel liquid sensors 174 during subsequent steps.
• Silicon dioxide is deposited to a thickness of 8.0 microns plus an allowance for subsequent planarization. The silicon dioxide is coated with photoresist and patterned to define the features within the MST channel layer 100 such as the heated microchannels 158 and the hybridization chambers 180 (see FIGS. 239 and 262A). The etch depth into the silicon dioxide is 8.0 microns.
• A 10 micron layer of sacrificial photoresist material is deposited to act as a scaffold for the roof layer 66 and is then planarized. Once planarized, the silicon dioxide roof layer 66 is deposited on the scaffold (see FIGS. 239 and 262A). The roof layer 66 is less than 5 microns thick, with a preferred thickness which is between 0.5 micron and 2.5 microns.
• The roof layer 66 is coated with photoresist and patterned such that the roof layer 66 can be etched to form the holes where the meniscus forms in the boiling-initiated valves. Also, the grid of small ashing holes 200 is etched through the roof layer 66 (see FIG. 262A).
• Dicing tape is then attached and the wafer is partially sawn to the top of the dicing streets. The wafer is then cleaned and ashed to remove the sacrificial scaffold and to release the individual LOC devices. The die are mounted onto a PCB wafer and the cap 46 is applied to each LOC device. The cap 46 is as described previously. Briefly, 100 micron thick double sided tape is patterned and bonded to the roof of each LOC device. This forms the interface layer 594 to connect the cap channel layer 80 to the downtake openings in the roof layer 66 (see for example FIG. 127). The reservoir layer 78 is then patterned to a depth of 200 microns to form the reservoirs 54, 56, 58.1, 58.2, 60.1, 60.2, 62.1 and 62.2. The cap channel layer 80 is then patterned to a depth of 60 microns to form the cap channels 94. The cap channel layer 80 of the cap 46 is then bonded to the interface layer 594 and the LOC device is then spotted with reagents and probes as described previously (see FIG. 127).
• It is also possible to use the above fabrication process to construct LOC devices that use fluorescence detection. LOC variant IX 726 described earlier has been redesigned using this fabrication process as shown in FIGS. 187 to 189. Note that the heater elements 154 are now positioned within the heated microchannels 158 and hybridization chambers 180.
LOC Variants
• The LOC device 301 described and illustrated above in full is just one of many possible LOC device designs. Variations of the LOC device that use different combinations of the various functional sections described above will now be described and/or shown as schematic flow-charts, from sample inlet to detection, to illustrate some of the combinations possible. The flow-charts have been divided, where appropriate, into sample input and preparation stage 288, extraction stage 290, incubation stage 291, amplification stage 292, pre-hybridization stage 293 and detection stage 294. For all the LOC variants that are briefly described or shown only in schematic form, the accompanying full layouts are not shown for reasons of clarity and succinctness. Also in the interests of clarity, smaller functional units such as liquid sensors and temperature sensors are not shown but it will be appreciated that these have been incorporated into the appropriate locations in each of the following LOC device designs.
LOC Variant I
• Referring to FIG. 79, LOC variant 1346 is a pathogenic DNA detection LOC device like the LOC device 301 described above with the exception that the first variant of the thermal bend actuated valves 302 are used instead of the boiling-initiated valves 126, 106 and 108. LOC variant I also omits the humidity sensor 232 but retains the humidifier 196.
LOC Variant II
• FIG. 80 shows LOC variant II 348. This LOC device extracts 290, incubates 291, amplifies 292, and detects 294 human DNA by using the leukocyte dialysis section 328 shown in FIG. 76. The second variant of thermal bend actuated valve 308 is used instead of boiling-initiated valves 126, 106 and 108 at the output to the chemical lysis 130, incubation 114 and amplification 112 sections.
LOC Variant V
• LOC variant V 362 shown in FIG. 81 extracts 290, incubates 291, amplifies 292 and detects 294 human DNA and so uses the leukocyte dialysis section 328. The reagent reservoirs 54, 56, 58, 60 and 62 use the third variant of thermal bend actuated valve 312 at their respective outlets.
LOC Variant VI
• LOC variant VI 364 shown in FIG. 82 is a complex design for extraction 290, incubation 291, nucleic acid amplification 292 and detection 294 of both human and pathogen target sequences, as well as human and pathogen protein detection. By using the leukocyte dialysis section 328 and pathogen dialysis section 70 in combination, three output streams are generated; leukocytes, pathogens and erythrocytes. Each stream is processed separately for greater sensitivity and parallel processing. The pathogen process stream has pathogen lysis reagent reservoir 56.2, pathogen target restriction enzyme, ligase and linker reservoir 58.2, pathogen amplification mix reservoir 60.2 and pathogen polymerase reservoir 62.2. LOC variant VI 364 utilises the second variant of thermal bend actuated valves 308 at the output of the chemical lysis sections 130.1 to 130.3, incubation sections 114.1 and 114.2 and amplification sections 112.1 and 112.2.
• The leukocyte and the pathogen process streams also separately direct a stream of lysed cells directly to the proteomic assay chamber arrays 124.1 and 124.2, respectively, for protein detection. The remaining lysed leukocyte and pathogen cells are amplified in the amplification sections 112.1 and 112.2 prior to hybridization and target nucleic acid sequence detection in the hybridization chamber arrays 110.1 and 110.2, respectively.
• In contrast, the erythrocyte stream is lysed in the chemical lysis section 130.3 by reagent from lysis reservoir 56.3 and fed directly into the proteomic assay chamber array 124.3, without nucleic acid amplification, for protein detection by the photosensor 44.
LOC Variant VII
• FIGS. 94 to 108 show LOC variant VII 492. Features and structures that correspond to equivalent features or structures shown in previous figures are indicated by the same reference numeral.
• As diagrammatically shown in FIG. 108, this variant extracts 290, incubates 291, amplifies 292 and detects 294 human DNA using parallel nucleic acid amplification. Four separate amplification sections 112.1 to 112.4 are included to increase assay sensitivity and improve signal-to-noise ratio of the detected fluorescence. The design also uses the leukocyte dialysis section 328, lysis reagent 56, chemical lysis section 130, restriction enzymes, ligase and linkers 58 and incubation section 114 as previously described. However, this LOC variant uses several of the fault tolerant valve arrays 309, 313, 462, 464, 466 and 468 instead of single active valves at the output of the chemical lysis section 130, incubation section 114 and amplification sections 112.1 to 112.4. The valve arrays are 2×2 arrays of the second type of thermal bend actuated valve 308.
• Separate amplicons from each of the amplification sections 112.1 to 112.4 feed into separate hybridization sections 110.1 to 110.4 where fluorescence is detected by the photosensor 44.
• FIG. 94 is a perspective of LOC variant VII 492 showing the vent holes 122, the sample inlet 68 and the evaporator 190 in the upper seal layer 82. Waste reservoirs 76 are unsealed such that excess waste can be transferred to a porous element 49 (see FIG. 1) within the test module. A series of bond-pads 104 extend along one edge and the humidity sensor 232 is also exposed for sensing the humidity of the microenvironment within the test module.
• FIG. 95 is an exploded perspective of the cap 46 in isolation. Removing the upper seal 82 reveals the reagent reservoirs (54, 56, 58, 188, 60.1-60.4 and 62.1-62.4) beneath the vent holes 122. LOC variant VII 492 has four amplification sections 112 to 112.4 and hence four amplification mix reservoirs 60.1 to 60.4 and four polymerase reservoirs 62.1 to 62.4, which provide the reagents for respective amplification sections 112.1 to 112.4.
• FIG. 96 is a perspective of the underside of the cap 46 showing the configuration of the cap channels 94 and the lower portions of the reagent reservoirs (60.1, 60.2, . . . 62.1, 62.2 . . . etc). FIG. 97 superimposes the features of the cap 46 on the features of the CMOS+MST device 48. FIG. 98 shows the features of the CMOS+MST device 48 in isolation. FIG. 99 shows the cap channels 94, reservoirs and valve array components in isolation. The waste channel 72 leads to the underside of the waste reservoirs 76 while the target channel 74 leads to the chemical lysis section 130 downstream of the lysis reagent reservoir 56. FIGS. 100, 101, 102, 103, 104, 105 and 106 are enlargements of Insets BA to BG respectively.
• The blood sample enters through the sample inlet 68. Capillary action draws the sample through the cap channel 94 to the anticoagulant surface tension valve 118 (see FIG. 100). Anticoagulant from the reservoir 54 combines with the blood sample which continues onto the leukocyte dialysis section 328 (see FIG. 99). As best shown in FIG. 105, the target channel 74 and the waste cell channel 72 are connected by a series of dialysis MST channels 204 through the MST layer 87. The target channel 74 connects to the dialysis MST channels 204 through respective arrays of 7.5 micron holes 165. The dialysis MST channels 204 connect to the waste channel 72 via dialysis uptakes 168. The dialysis MST channel 204 at the upstream end 502 of the dialysis section has no downtake. Capillary initiation features 166 ensure that the sample flow does not pin at the array of 7.5 micron diameter apertures 165 but rather flows through to the dialysis MST channels 204.
• Referring back to FIGS. 97, 99 and 100, the sample flow, now with a much higher concentration of the target cells, flows to the lysis reagent surface tension valve 128. Lysis reagent from the reservoir 56 combines with the sample flow and enters into the chemical lysis section 130. The flow stops at the 2×2 fault tolerant array 309 of thermal bend actuated valves of the second type 308. The CMOS circuitry 86 is programmed with a dwell time for diffusive mixing of the lysis reagent such that enough of target cells are lysed. After sufficient time (less than 0.5 sec), the valves in the fault tolerant valve array 309 are activated (i.e. opened) and the flow continues into the downstream section of the mixing section 131.
• With the genetic material released, the restriction enzymes, ligase and linkers from reservoir 58 are added via surface tension valve 132. The sample flow continues through the remainder of the mixing section 131 to the downtake 134 and into the heated microchannels of the incubation section 114 (see FIG. 99). The sample flow fills the incubation section 114 until it reaches the 2×2 fault tolerant valve array 313. After sufficient incubation time, the fault tolerant valve array 313 activates.
• The sample flows into the common supply channel 504 for the amplification sections. The common supply channel 504 feeds into the inlets 506, 508, 510 and 512 of the four separate amplification sections 112.1 to 112.4 (see FIG. 101). Each of the amplification mix reservoirs 60.1 to 60.4 has a surface tension valve 138 connecting to each of the amplification section inlets 506, 508, 510 and 512 respectively. Similarly, each of the polymerase reservoirs 62.1 to 62.4 have a surface tension valve 140 connecting to each of the amplification section inlets 506, 508, 510 and 512 respectively (see FIG. 99). The surface tension valves 138 and 140 retain the reagents in their respective reservoirs by pinning a meniscus. As the sample flows down each inlet, the meniscus is removed and the reagents combine with the sample flow; firstly the amplification mix of primers, dNTPs and buffer, then the polymerase.
• Each of the four separate amplification sections 112.1 to 112.4 fills with the sample, amplification mix and polymerase. The respective flow through each of the amplification sections stops at the respective amplification exit fault tolerant valve arrays 462, 464, 466 and 468 (see FIG. 102). The CMOS-controlled thermal cycling amplifies the genetic material. Referring specifically to FIGS. 97, 103 and 107, the amplification exit valve arrays 462, 464, 466 and 468 activate such that the four amplicons flow into separate hybridization chamber arrays 110.1 to 110.4 via separate inlets 494, 496, 498, 500. Each of the separate hybridization chamber arrays has its own end-point liquid sensor 178. Feedback from the respective end-point liquid sensors 178 triggers the hybridization heaters 182 and the LED driver 29 in the CMOS circuitry signals the excitation LED 26 to illuminate. Hybridization with the FRET probes 182 in any of the individual hybridization chambers 180 is detected by the photodiodes 184 underlying those chambers (see FIG. 54).
LOC Variant VIII
• FIGS. 109 to 113 and 114 to 140 show LOC variant VIII 518. Features and structures that correspond to equivalent features or structures shown in the LOC device 301 are indicated by the same reference numeral. Features that do not correspond to previously described features are indicated with new reference numerals.
• As diagrammatically shown in FIG. 140, this variant of the LOC 518 extracts 290, incubates 291, amplifies 292 and detects 294 human DNA using twelve separate amplification sections (112.1 to 112.12). LOC variant VIII 518 uses multiple amplification sections to increase assay sensitivity and improve signal to noise ratio of the detected fluorescence.
• Referring to FIGS. 109, 110 and 111, the blood sample enters the sample inlet 68 and capillary action draws it along the cap channel 94 to the anticoagulant surface tension valve 118. The cap 46 is fabricated with an alternative layer to the lower seal 64. In this design, an interface layer 594 is positioned between the cap channel layer 80 and the MST channel layer 100 of the CMOS+MST device 48. The interface layer 594 allows a more complex fluidic interconnection between the reagent reservoirs and the MST layer 87 without increasing the size of the silicon substrate 84. FIG. 111 superimposes the reservoirs, the top channels and the interface channels to illustrate the more sophisticated plumbing achieved with the interface layer 594.
• As best shown in FIG. 139, the interface layer 594 requires the anticoagulant surface tension valve 118 to have two interface channels 596 and 598. A reservoir-side interface channel 596 connects the reservoir outlet with the downtakes 92 and a sample-side interface channel 598 connects the uptakes 96 with the cap channel 94. Anticoagulant from the reservoir 54 flows through the MST channels 90 via the reservoir-side interface channel 596 to pin a meniscus at the uptakes 96. The sample flow along the cap channel 94 dips into the sample-side interface channel 598 to remove the meniscus so that the anticoagulant combines with the blood sample as it continues on to the leukocyte dialysis section 328.
• Referring to FIGS. 114 and 139, the leukocyte dialysis section 328 incorporates a bypass channel 600 for filling the flow channel structures without trapped air bubbles. The blood sample flows through the cap channel 94 to the upstream end of the interface target channel 602. The interface target channel 602 is in fluid communication with dialysis MST channels 204 via apertures in the form of 7.5 micron diameter holes 165. Each of the dialysis MST channels 204 lead from the 7.5 micron diameter holes 165 to respective dialysis uptakes 168. The dialysis uptakes 168 are open to the interface waste channel 604. However the uptakes are configured to pin a meniscus rather than allow capillary driven flow to continue.
• Conversely, the bypass channel 600 at the very upstream end of the leukocyte dialysis section 328, has a CIF (capillary initiation feature) 202 to promote capillary driven flow from the bypass channel 600 into the interface waste channel 604 (see FIGS. 114 and 139). The bypass channel also has a wide meander to lengthen the flow-path from the interface target channel 602 to the interface waste channel 604. The longer flow-path delays the sample flow such that it fills the interface waste channel 604 after the meniscus forms at the most upstream dialysis MST channel 204. The sample flow starts at the upstream end and unpins the meniscus at each of the dialysis uptake holes 168 as the flow moves downstream along the interface waste channel 604. This ensures all the dialysis MST channels fill with sample flow as the dialysis section fills.
• Without the bypass channel 600, or dialysis uptakes 168 configured to pin a meniscus, some dialysis MST channels 204 may not fill. Similarly, an air bubble may form in the interface waste channel 604. In either case, flow through the dialysis section can be substantially throttled.
• Referring back to FIGS. 110 and 111, the interface waste channel 604 feeds into the waste channel 72 which flows to the waste reservoir 76. The interface target channel 602 feeds into the target channel 74. The sample flow with the target cells is drawn along the target channel 74 to the lysis surface tension valve 128.
• As with the anticoagulant surface tension valve 118 described above, the lysis surface tension valve 128 has a lysis reservoir-side interface channel 606 and a lysis sample-side interface channel 608 (see FIG. 111). Lysis reagent flows from the reservoir 56 to the lysis reservoir-side interface channel 606 via a cap channel 94. The reagent flows into the downtakes 92, through the MST channels 90 to the uptakes 96 where the reagents pin a meniscus (see FIG. 110). Sample flow from the target channel 74 fills the lysis sample-side interface channel 608. The sample flow removes the menisci at the uptakes 96 and the lysis reagent combines with the sample as it flows into the chemical lysis section 130.
• In the chemical lysis section 130, the lysis reagent diffusively mixes through the flow to lyse the target cells and release the genetic material therein. The sample flow stops at the mixing section exit valve 206. As best shown in FIGS. 114 and 139, the mixing section exit valve is a boiling-initiated valve 206. The lysed sample flows into a mixing section exit downtake 612 via an interface duct 610. The sample continues along the MST channel 90 to the boiling-initiated valve 206 where it stops when a meniscus pins at the valve uptake 151 in the roof layer 66 (see in particular FIGS. 116A and 117A). The liquid sensor 174 upstream of the valve provides feedback that the sample flow is about to reach the valve uptake 151. If the CMOS circuitry 86 is programmed with a delay to ensure the target cells are completely lysed, the liquid sensor feedback initiates the delay period.
• After any delay period, the annular heater 152 is activated via the heater contacts 156 (see FIGS. 117A and 117B). The sample liquid at the valve uptake 151 boils and the meniscus is unpinned. The sample flows into the boiling-initiated valve interface cavity 616 (see FIG. 118) and out of the valve downtake 150 (see FIG. 117B). The downstream liquid sensor 174 registers that the flow has resumed along the MST channel 90.
• The lysed sample flow continues to the uptakes 96 of the restriction enzyme, ligase and linker surface tension valve 132 (see FIG. 114). Referring to FIGS. 116A, 117A, 118, 119 and 120, restriction enzymes, ligase and linker primers in the reservoir 58 flow into the cap channel 94 leading to the restriction enzyme, ligase and linker valve interface channel 614. The restriction enzyme, ligase and linker valve interface channel 614 opens to three uptakes 96 where the enzymes and linker primers are retained by a meniscus. The lysed sample flow in the MST channel 90 passes the uptakes 96 and removes the menisci such that the restriction enzymes, ligase and linker primers mix with the sample flow.
• Referring to FIG. 114, the sample, restriction enzymes, ligase and linker primers flow through a MST channel mixing section 131 for diffusion mixing prior to entering the heated microchannels of the incubation section 114. The incubation section 114 is composed of a serpentine microchannel 210 (see FIG. 115) heated by respective heaters 154 (see FIG. 117A) supported on the roof layer 66 above. The heaters 154 extend between respective pairs of heater contacts 156 connected to the CMOS circuitry 86.
• Referring to FIG. 121, the sample flow is stopped at the incubator exit valve 207. The incubator exit valve 207 is a boiling-initiated valve similar to the mixing section exit valve 206. The liquid sensor 174 immediately upstream of the incubator exit valve 207 indicates when the sample flow is about to stop at the valve uptake 151 (see FIGS. 121, 123 and 124B). The CMOS circuitry 86 initiates an incubation time delay (if required) in response to the liquid sensor.
• After sufficient incubation, the annular heater 152 boils liquid at the valve uptake 151 to unpin the meniscus. Flow resumes into the boiling-initiated valve interface cavity 616 (see FIG. 125) and out of the valve downtake 150 (see FIG. 123). From the valve downtake 150, the sample flows along the MST incubation exit channel 630 (see FIG. 121) to the polymerase surface tension valve 140 (see FIGS. 110 and 111). Polymerase from the reservoir 62 combines with the sample flow as it travels the serpentine path of the amplification input channel 632.
• Referring back to FIG. 121, the amplification input channel 632 directs the sample flow past the twelve amplification mix surface tension valves 138. Amplification mix in each of the amplification mix reservoirs 60.1 to 60.12 (see FIG. 111) flows through respective cap channels 94 (see FIGS. 126 and 127) and respective amplification interface ducts 618-629 (see FIG. 125) to pin menisci at the amplification mix surface tension valves 138 (see FIG. 131). The sample flow opens each of the surface tension valves in turn, and the amplification mix from the respective amplification mix reservoirs 60.1 to 60.12 (see FIG. 111) entrains with the sample flow into the respective heated microchannel 158 of each of the twelve amplification sections 112.1 to 112.12.
• Referring to FIG. 128, each of the twelve amplification sections 112.1 to 112.12 has one of the amplification outlet valves 108 respectively. The sample flow stops at the valve uptakes 151 of each amplification outlet valve 108. After thermal cycling, the valve heater 152 boils liquid at the valve uptake 151 (best shown in FIG. 132B) and sample flows to the valve interface cavity 616 (see FIG. 133) and out through the valve downtake 150.
• Downstream of the amplification outlet valves 108 are the separate arrays 110.1 to 110.12 of hybridization chambers 180 for each of the twelve separate amplicons (see FIGS. 128 and 136). The sample is drawn along the flow-path 176 through each of the separate arrays 110.1 to 110.12 and into the individual hybridization chambers 180 via respective diffusion barrier inlets 175. Referring to FIG. 136, when the sample flow reaches the end-point liquid sensor 178, the hybridization heaters 182 are energized for optimum probe-target hybridization.
• Referring to FIG. 113 and FIGS. 136 to 138, the humidity sensor 232 and hybridization and detection section 52 are surrounded by a strip of titanium nitride deposited on the roof layer 66. The strip of TiN provides a LED chip support surface 634 for the excitation LED 26 (see FIG. 3). The excitation LED is sealed to the LED chip support surface 634 and the air pressure in the hybridization chambers 180 is equalized with the atmosphere through the vent holes 122 and the vent channel 636 in the MST layer 87 (see FIGS. 136 and 138).
• Referring to FIG. 140, the hybridization chamber arrays 110.1 to 110.12 for each of the twelve amplicons have a single photosensor 44 in the underlying CMOS circuitry 86 (see FIG. 12). Probe-target hybrids in any of the hybridization chambers 180 emit a fluorescence signal that is detected by the corresponding photodiode 184. Each of the hybridization chamber arrays 110.1 to 110.12 has at least one calibration chamber 382 which is isolated from the sample flow such that no amplicon enters the calibration chamber 382. The calibration chambers 382 are used to calibrate the photodiode outputs to adjust for system noise, in the sense of readout error, as described elsewhere in this specification.
LOC Variant IX
• FIG. 187 schematically illustrates the operation of LOC variant IX 726 shown in FIG. 188. LOC variant IX 726 extracts 290, incubates 291, amplifies 292 and detects 294 pathogenic DNA, and uses the pathogen dialysis section 70. As best shown in FIG. 189, the bypass channel 600 structure is used to prevent trapped air bubbles. LOC variant IX 726 also uses the alternative cap fabrication process as shown in FIGS. 109 to 111 in relation to LOC variant VIII 518.
LOC Variant X
• FIGS. 190 to 206 show LOC variant X 728. Referring to the operation schematic shown in FIG. 190, this LOC device extracts 290, incubates 291, amplifies 292 and detects 294 both human and pathogen nucleic acids, as well as human and pathogen proteins. It does so by combining the leukocyte and pathogen dialysis sections (328 and 70 respectively) to produce leukocyte, pathogen and erythrocyte output streams that are lysed and separately directed to the proteomic assay chamber arrays 124.1-124.3 for protein detection. In the case of the leukocytes and pathogens, part of the output stream is also directed to the incubation 114.1-114.2 and amplification 112.1-112.2 sections for nucleic acid amplification and then onto hybridization chambers 110.1 and 110.2 for nucleic acid detection. Each output is processed separately for higher sensitivity and parallel analysis.
• To start, a biological sample (such as whole blood) is added to the sample inlet 68, as best seen in FIG. 196. The sample flows through the cap channel 94 to the anticoagulant reservoir surface tension valve 118. Anticoagulant (or other reagents) from the reservoir 54 mixes with the sample as it flows on to the upstream end of the leukocyte output dialysis section 328.
• The anticoagulant surface tension valve 118 has two channels 596 and 598 in the interface layer 594 (see FIGS. 191 and 196). The reservoir-side interface channel 596 connects the reservoir outlet with the downtakes 92, while the sample-side interface channel 598 connects the uptakes 96 with the cap channel 94. The valve is filled by anticoagulant from the reservoir 54 flowing through the reservoir-side interface channel 596 into the MST channels 90 to pin a meniscus at the uptakes 96. The sample flowing along the cap channel 94 is forced into the sample-side interface channel 598 so that the sample and anticoagulant meniscus interact to release the anticoagulant into the sample as it continues back on through the cap channel 94 and on towards the upstream end of leukocyte output dialysis section 328.
• The sample enters the upstream end of the leukocyte output dialysis section 328 through the large constituents interface channel 730. The large constituents interface channel opens upon arrays of 7.5 micron filter holes 165, each a filter for a lateral dialysis MST channel 204 that connects to an uptake hole 168 that in turn opens within the small constituents interface channel 732 (see FIG. 200). The first lateral dialysis MST channel of the dialysis section is a bypass channel 600 that allows all the other lateral dialysis MST channels 204 to fill without trapping bubbles (see FIG. 205). All lateral dialysis MST channels 204 except the bypass channel have uptake 168 that pin the meniscus, and only release once there is interaction with flow in the small constituents interface channel 732. Flow in the small constituents interface channel 732 begins upstream of the uptakes 168, at the bypass channel 600 comprising a capillary initiation feature 202 that is an aperture with geometry configured to promote meniscus instability such that the meniscus remains unpinned and capillary driven flow is uninterrupted. Sample flow from the capillary initiation feature progresses downstream to sequentially unpin the menisci at the uptakes 168 (see FIG. 205).
• The downstream end of the leukocyte output dialysis section 328 is shown in FIG. 200. The large constituents interface channel 730 feeds into the large constituents cap channel 736 and the small constituents interface channel 732 feeds into the small constituents cap channel 734. The large constituents cap channel 736 directs the leukocytes (and any other large components) past the lysis surface tension valve 128.1, where lysis reagent from reservoir 56.1 is added to the leukocyte lysis section 130.1 (see FIG. 194). The leukocyte lysis section 130.1 outlet has a filter downtake 738 (see FIG. 196) that prevents large components occluding the MST channel or boiling-initiated valve 206 before complete lysis. After sufficient time for lysis, the boiling-initiated valve 206 opens the lysis section 130.1 outlet and the sample flow is split into two streams, one stream flows past the restriction enzyme, ligase and linker reservoir 58.1 surface tension valve 132.1, while the other stream is drawn along a lysed leukocyte bypass channel 742 directly to the proteomic assay chamber array 124.1 within the hybridization and detection section (see FIG. 196).
• In the case of the lysed leukocyte bypass channel 742 flow, the sample fills the proteomic assay chamber array 124.1 (see FIG. 198) containing probes for hybridization with target human proteins. Probe-target hybrids are detected with the photosensor 44 (see FIG. 190).
• In the case of the flow past the restriction enzyme, ligase and linker reservoir 58.1, the sample enters the leukocyte incubation section 114.1 while continuously mixing with restriction enzymes, ligase and linker primers from reservoir 58.1 (see FIG. 196). After restriction enzyme digestion and linker ligation, the incubator outlet valve 207 (also a boiling-initiated valve) releases flow to continue past the PCR mix reservoir 60.1 surface tension valve 138.1 and then the polymerase reservoir 62.1 surface tension valve 140.1 (see FIGS. 195 and 197), the respective reservoir reagents mixing with the sample as it flows to the leukocyte DNA amplification section 112.1.
• Thermal cycling is performed in the amplification section 112.1, before the boiling-initiated valve 108 opens to send the amplicon to the hybridization chamber array 110.1 containing probes for human DNA targets (see FIG. 198). Probe-target hybrids are detected with photosensor the 44 (see FIG. 190).
• Returning to the downstream end of the leukocyte output dialysis section (see FIG. 200), the small constituents cap channel 734 directs erythrocytes and pathogens to the upstream side of the pathogen output dialysis section 70 (see FIGS. 194, 196 and 206).
• The pathogen output dialysis section 70 operates in the same manner as leukocyte output dialysis section 328 with the exception that the filter downtakes have 3 microns holes 164 rather than 7.5 micron holes. Erythrocytes remain in the large constituents interface channel 730 while pathogens are filtered into the small constituents interface channel 732.
• At the downstream end of the pathogen output dialysis section 70, as FIG. 201 illustrates, the erythrocyte and pathogen flows exit into the large constituents cap channel 736 and small constituents cap channel 734, respectively.
• Note that the designations of dialysis section flows is based on a relative indication of size as ‘large constituents’ and ‘small constituents’ holds only with respect to the dialysis section considered. While it is therefore consistent that the small constituents output of the leukocyte output dialysis section 328 feeds into the large constituents interface channel 730 of the pathogen output dialysis section 70, no relationship of component size between the two dialysis section streams is implied.
• The erythrocyte flow in the large constituents cap channel 736 is mixed with lysis reagent as it is directed past the lysis reagent reservoir surface tension valve 128.3 to fill the erythrocyte lysis chamber 130.3 (see FIGS. 194 and 195). The erythrocyte lysis chamber 130.3 outlet incorporates a filter downtake 738 to prevent large components from occluding the MST channel or boiling-initiated valve 206 before complete lysis as explained above with reference to the leukocyte lysis chamber 130.1 outlet. Upon complete lysis, the boiling-initiated valve 206 releases the flow into the proteomic assay chamber array 124.3 (see FIG. 198) containing probes for conjugation or hybridization with target human proteins. Probe-target complexes are detected with the photosensor 44 (see FIG. 190).
• The small constituents cap channel 734 pathogen flow is mixed with lysis reagent as it is directed past the lysis reagent reservoir 56.2 surface tension valve 128.2 to fill the lysis chamber 130.2 (see FIGS. 194 and 195). After lysis is complete, the boiling-initiated valve 206 opens the lysis chamber 130.2 outlet and the sample flow is split into two streams, one stream flows past the restriction enzyme, ligase and linker reservoir 58.2 surface tension valve 132.2, while the other stream is drawn along a lysed pathogen bypass channel 744 directly to the proteomic assay chamber array 124.2 within the hybridization and detection section 294 (see FIGS. 194, 195, 197 and 198).
• In the case of the lysed pathogen bypass channel 744 flow, the sample fills the proteomic assay chamber array 124.2 (FIG. 198) containing probes for conjugation or hybridization with target pathogen proteins. Probe-target complexes are detected with the photosensor 44 (FIG. 190).
• In the case of the flow past restriction enzyme, ligase and linker reservoir 58.2, the sample enters the pathogen incubation section 114.2 while continuously mixing with restriction enzymes, ligase and linker primers from reservoir 58.2 (see FIG. 197). After restriction enzyme digestion and linker ligation, the incubator outlet valve 207 (also a boiling-initiated valve) releases flow to continue past the PCR mix reservoir 60.2 surface tension valve 138.2 and then the polymerase reservoir 62.2 surface tension valve 140.2 (see FIGS. 195 and 197), the respective reservoir reagents mixing with the sample as it flows onto the pathogen DNA amplification section 112.2 (see FIG. 197).
• Thermal cycling is performed in the amplification section 112.2, before the boiling-initiated valve 108 opens to send the amplicon to the hybridization chamber array 110.2 containing probes for pathogen DNA targets (see FIG. 198). Once the required hybridization chambers have filled, hybridization heaters 182 are activated after a time delay which is referenced to the flow rate sensor 740 (see FIG. 204) in the pathogen incubation section 114.2. Probe-target hybrids are detected with the photosensor 44 (see FIG. 190). Within the hybridization chamber array 180 and proteomic assay chamber array 124 areas, calibration chambers 382 (see FIGS. 202 and 203) are inserted for photosensor 44 output noise reduction.
LOC Variant XI
• FIGS. 207 to 214 show LOC variant XI 746. This LOC device extracts 290, incubates 291, amplifies 292 and detects 294 pathogenic DNA. The sample input and preparation phase 288, and the extraction phase 290 are the same as those of the LOC device 301 (see FIG. 4). In the incubation stage 291, four parallel incubation sections 114.1 to 114.4 are used. Referring to FIGS. 209, 210 and 212, a shared restriction enzyme, ligase and linker reservoir 58 adds enzymes to the sample flow via surface tension valve 132 into the common incubator feed channel 748. The common incubator feed channel 748 fills all the incubation sections 114.1 to 114.4 through first, second, third and fourth incubator inlets 750, 752, 754 and 756 respectively.
• Referring to FIG. 213, incubation sections 114.1 to 114.4 flow to the four parallel amplification sections 112.1 to 112.4, respectively. After sufficient incubation time, boiling-initiated valves 207 at each incubator section outlet open. Each amplification section 112.1 to 112.4 has respective amplification mix reservoirs 60.1 to 60.4 and polymerase reservoirs 62.1 to 62.4 (see FIGS. 207 to 210). The polymerase is added immediately before nucleic acid amplification to optimise the amplification process.
• Referring to FIG. 214, each of the amplification sections 112.1 to 112.4 have boiling-initiated valves 108 at their respective outlets. After thermal cycling, the boiling-initiated valves 108 open such that amplicon from each amplification sections 112.1-112.4 flow into respective hybridization chamber arrays 110.1 to 110.4. The hybridization chamber arrays 110.1 to 110.4 are surrounded by a strip of titanium nitride to provide a LED chip support surface 634 for the excitation LED 26 (see FIG. 3). The excitation LED is sealed to the LED chip support surface 634. As shown in FIGS. 195 and 199, the air pressure in the hybridization chambers 180 is equalized with the atmosphere through the vent holes 122 and the vent channel 636 in the MST layer 87 (see FIG. 191).
• As shown in FIG. 212, the pathogen dialysis section 70 has a bypass channel 600 to prevent air entrapment. LOC variant XI 746 also has a flow rate sensor 740 and liquid sensor 174 (see FIG. 209) for the timed operation of the heater elements in each of the functional sections (i.e. hybridization arrays, incubation and amplification section etc).
LOC Variant XII
• LOC variant XII 758 is shown in FIGS. 215 to 222. This LOC device extracts 290, incubates 291, amplifies 292 and detects 294 pathogenic DNA, and uses a pre-hybridization purification step 293 to increase hybridization efficiency. The sample (such as whole blood) is added to the sample inlet 68 (see FIG. 217) and capillary action draws the sample to the surface tension valve 118 where anticoagulant is added from reservoir 54. The sample continues in the cap channel 94 to the pathogen dialysis section 70. The dialysis section 70 has a bypass channel 600 to prevent trapped air bubbles (see FIG. 217).
• After dialysis in the pathogen dialysis section 70, the erythrocyte and leukocyte stream is directed to a waste reservoir 76 while the pathogens continue in the sample flow to surface tension valve 128 where a lysis reagent is added from reservoir 56. The sample fills the chemical lysis chamber 130 where it is retained by boiling-initiated valve 206 until the lysis reagent has diffused through the sample to release most, if not all, of the pathogenic DNA. When the boiling-initiated valve 206 opens, the sample flows to surface tension valve 132 where restriction enzymes, ligase and linker primers are added from reservoir 58. The sample fills the incubation section 114 and is heated while restriction digestion and linker ligation of the pathogenic DNA occurs (see FIG. 217).
• After restriction digestion and linker ligation, the boiling-initiated valve 207 opens for the sample to flow into the amplification section 112. Amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 through surface tension valve 140 as the sample flows into the amplification section 112. The pathogenic DNA is amplified by thermal cycling before the boiling-initiated valve 108 opens for the amplicon to flow to the small constituents dialysis section 682 where large components are removed (see FIG. 217).
• As best shown in FIGS. 220 and 221, the small constituents dialysis section 682 has a large constituents channel 760 between two small constituents channels 762 formed in the bottom channel layer 100 (see FIG. 216). The large constituents channel 760 is connected to the small constituents channels 762 by stoma in the form of a series of inverse tapered openings 764 (smaller at the large constituents channel end). In most practical applications, the stoma will be between 1 to 8 microns wide and 1 to 8 microns high. As the amplicon flows into the large constituents channel 760, small constituents (smaller than the inverse tapered openings 764) start to diffuse into the small constituents channels 762. The concentration of small constituents in the large constituents channel 760 reduces as the flow progresses to the downstream end of the small constituents dialysis section 682. An additional advantage of microfabricated stoma is that the number of stoma per unit length along the channel is very high, so that the separation is more efficient. For efficient separation of constituents with the sizes of interest, the spacing between adjacent stoma is between 1 micron and 10 microns; in the embodiment shown in FIG. 221, the spacing between adjacent stoma is 8 microns.
• FIG. 222 shows the downstream end of the small constituents dialysis section 682. The large constituents channel 760 diverts into a wide meander ending at a blind end 766 which provides a waste reservoir. The two small constituents channels 762 lead to opposite sides of the hybridization chamber array 110 where they both follow serpentine paths through the array to respective blind ends 768. The small constituents amplicon fills all the individual hybridization chambers 180 prior to timed initiation of the hybridization heaters and subsequent probe-target hybrid detection (as described previously).
• The small constituents dialysis section 682 removes cell debris which may still remain in the sample flow following cell lysis. Cell debris may interfere with hybridization efficiency.
LOC Variant XIII
• FIG. 141 shows the operation of a LOC variant XIII 640 for pathogen detection. It uses a pathogen dialysis section 70, a chemical lysis section 130 and a thermal lysis section 638. This LOC variant XIII 640 does not have a restriction enzyme, ligase and linker primer reservoir (see for example reservoir 58 in FIG. 4). Single flow stream (as opposed to parallel flow streams) nucleic acid amplification functionality (see amplification mix reservoir 60, polymerase reservoir 62 and amplification section 112) is retained.
• Boiling-initiated valves 108 (other types of active valve could also be used) are at the exit to the chemical 130 and thermal lysis 638 sections, and the amplification section 112. Surface tension valves 118, 128, 138 and 140 are at the outlets to the reagent reservoirs 54, 56, 60 and 62.
LOC Variant XIV
• FIG. 142 is a schematic of LOC variant XIV 641 for pathogen detection. The LOC device uses a single pathogen dialysis section 70 and both chemical 130 and thermal 638 lysis sections. LOC variant XIV 641 has parallel nucleic acid amplification functionality (amplification sections 112.1, 112.2 . . . 112.X operating in parallel), with respective hybridization chamber arrays 110.1, 110.2, . . . 110.X and on-chip photosensor 44 (each hybridization chamber array having separate photodiode arrays). Each nucleic acid amplification stream has separate amplification mix reservoirs (60.1, 60.2 . . . 60.X) and separate polymerase reservoirs (62.1, 62.2 . . . 62.X). LOC variant XIV 641 provides pathogen detection with the benefits of parallel nucleic acid amplification. Boiling-initiated valves 108 are used at the exit to the lysis 130 and amplification 112.1-112.X sections and surface tension valves 118, 128, 138.1-138.X and 140.1-140.X are at the outlets to the reagent reservoirs 54, 56, 60.1-60.X and 62.1-62.X, respectively.
LOC Variant XV
• FIG. 143 shows LOC variant XV 642. LOC variant XV 642 uses the pathogen dialysis section 70 (for pathogen detection) and both chemical 130 and thermal 638 lysis sections. LOC variant XV 642 has tandem nucleic acid amplification functionality where two amplification sections 112.1 and 112.2 are arranged in series. Immediately upstream of each amplification section 112.1 and 112.2 are respective amplification mix reservoirs 60.1 and 60.2 and polymerase reservoirs 62.1 and 62.2. The amplicon from the tandem amplification fills the hybridization chamber array 110 for detection with the photosensor 44. Boiling-initiated valves 108 are used at the exit to the lysis sections 130 and 638 and amplification sections 112.1-112.2. Surface tension valves 118, 128, 138.1-138.2 and 140.1-140.2 are at the outlets to the reagent reservoirs 54, 56, 60.1-60.2 and 62.1-62.2.
LOC Variant XVI
• FIG. 144 is a schematic illustration of a LOC variant XVI 643 for pathogen detection with the pathogen dialysis section 70, thermal lysis section 638, nucleic acid amplification functionality (amplification section 112 and reagent reservoirs 60 and 62) and hybridization (hybridization chamber array 110) with on-chip detection (photosensor 44).
LOC Variant XVII
• FIG. 145 is a schematic of LOC variant XVII 644 for pathogen detection. The LOC device uses a single pathogen dialysis section 70 and a thermal lysis section 638. LOC variant XVII has parallel nucleic acid amplification functionality (amplification sections 112.1, 112.2 . . . 112.X operating in parallel), with respective hybridization chamber arrays 110.1, 110.2, . . . 110.X and on-chip photo sensor 44 (each hybridization chamber array having separate photodiode arrays). Each nucleic acid amplification stream has separate amplification mix reservoirs (60.1, 60.2 . . . 60.X) and separate polymerase reservoirs (62.1, 62.2 . . . 62.X). LOC variant XVII 644 provides pathogen detection with the benefits of parallel nucleic acid amplification. Boiling-initiated valves 108 are used at the exit to the lysis 130 and amplification 112.1-112.X sections and surface tension valves 118, 138.1-138.X and 140.1-140.X are at the outlets to the reagent reservoirs 54, 60.1-60.X and 62.1-62.X, respectively.
LOC Variant XVIII
• FIG. 146 shows LOC variant XVIII 645. This variant uses the pathogen dialysis section 70 (for pathogen detection) and the thermal lysis section 638. LOC variant XVIII 645 has tandem nucleic acid amplification functionality where two amplification sections 112.1 and 112.2 are arranged in series. Immediately upstream of each amplification sections 112.1 and 112.2 are respective amplification mix reservoirs 60.1 and 60.2 and polymerase reservoirs 62.1 and 62.2. The amplicon from the tandem amplification fills the hybridization chamber array 110 for detection with the photosensor 44. Boiling-initiated valves 108 are used at the exit to the thermal lysis section 638 and amplification sections 112.1-112.2. Surface tension valves 118, 138.1-138.2 and 140.1-140.2 are at the outlets to the reagent reservoirs 54, 60.1-60.2 and 62.1-62.2, respectively.
LOC Variant XIX
• The LOC variant XIX 646 shown in FIG. 147 has pathogen dialysis section 70 and thermal lysis section 638 of the sample prior to nucleic acid amplification (amplification section 112), but also adds a pre-hybridization purification step 293 between the amplification 292 and detection phases 294. The pre-hybridization, small constituents dialysis section 682 removes cell debris in the sample flow resulting from cell lysis. Most nucleic acid amplification protocols are sufficiently tolerant of cell debris in the sample. However, hybridization can be affected by cell debris and so pre-hybridization dialysis via the small constituents dialysis section 682 is used to substantially reduce the concentration of debris in the amplicon immediately prior to filling the hybridization chamber array 110.
LOC Variant XX
• The LOC variant XX 647 shown in FIG. 148 is configured for pathogen detection with pathogen dialysis section 70, chemical lysis section 130 and a single amplification section 112.
LOC Variant XXI
• FIG. 149 is a schematic of LOC variant XXI 648 for pathogen detection. The LOC device uses a single pathogen dialysis section 70 and a chemical lysis section 130. LOC variant XXI 648 has parallel nucleic acid amplification functionality (amplification sections 112.1, 112 2 . . . 112.X operating in parallel), with respective hybridization chamber arrays 110.1, 110.2, . . . 110.X and on-chip photo sensor 44 (each hybridization chamber array having separate photodiode arrays). Each nucleic acid amplification stream has separate amplification mix reservoirs (60.1, 60.2 . . . 60.X) and separate polymerase reservoirs (62.1, 62.2 . . . 62.X). LOC variant XXI 648 provides pathogen detection with the benefits of parallel nucleic acid amplification. Boiling-initiated valves 108 are used at the exit to the chemical lysis section 130 and amplification sections 112.1-112.X and surface tension valves 118, 128, 138.1-138.X and 140.1-140.X are at the outlets to the reagent reservoirs 54, 56, 60.1-60.X and 62.1-62.X, respectively.
LOC Variant XXII
• FIG. 150 shows LOC variant XXII 649. LOC variant XXII 649 uses the pathogen dialysis section 70 (for pathogen detection) and chemical lysis section 130. LOC variant XXII 649 has tandem nucleic acid amplification functionality where two amplification sections 112.1 and 112.2 are arranged in series. Immediately upstream of each amplification section 112.1 and 112.2 are respective amplification mix reservoirs 60.1 and 60.2 and polymerase reservoirs 62.1 and 62.2. The amplicon from the tandem amplification fills the hybridization chamber array 110 for detection with the photosensor 44. Boiling-initiated valves 108 are used at the exit to the chemical lysis section 130 and amplification sections 112.1-112.2. Surface tension valves 118, 128, 138.1-138.2 and 140.1-140.2 are at the outlets to the reagent reservoirs 54, 56, 60.1-60.2 and 62.1-62.2, respectively.
LOC Variant XXIII
• The LOC variant XXIII 650 of FIG. 151 is for genetic analysis and uses a leukocyte dialysis section 328 to substantially reduce the erythrocyte concentration in the sample. In the chemical lysis section 130, lysis reagent from reservoir 56 releases the genetic material in the leukocytes. Downstream of the chemical lysis section 130, the sample combines with restriction enzymes, ligase and linker primers from reservoir 58 and fills the incubation section 114. After incubation, the boiling-initiated valve 108 immediately downstream of the incubation section 114 opens for the sample to flow into the amplification section 112 and ultimately the hybridization chamber array 110.
LOC Variant XXIV
• LOC variant XXIV 651 is a genetic analysis LOC device (see FIG. 152) with a leukocyte dialysis section 328, chemical lysis section 130 and restriction enzyme, ligase and linker incubation section 114. LOC variant XXIV 651 uses parallel amplification sections 112.1, 112.2, . . . 112.X and respective hybridization chamber arrays 110.1, 110.2, . . . 110.X.
LOC Variant XXV
• LOC variant XXV 652 (see FIG. 153) is an embodiment of a genetic analysis LOC device. LOC variant XXV 652 uses a leukocyte dialysis section 328, chemical lysis section 130 and restriction enzyme, ligase and linker incubation section 114. The sample is then fed to tandem amplification sections 112.1 and 112.2 before detection in a single hybridization chamber array 110.
LOC Variant XXVI
• LOC variant XXVI 653 shown in FIG. 154 is for genetic analysis and uses a leukocyte dialysis section 328 to substantially reduce the erythrocyte concentration in the sample. In the chemical lysis section 130, lysis reagent from reservoir 56 releases the genetic material in the leukocytes. After chemical lysis, the boiling-initiated valve 108 immediately downstream of the chemical lysis section 114 opens for the sample to flow into the amplification section 112 and ultimately the hybridization chamber array 110.
LOC Variant XXVII
• LOC variant XXVII 654 shown in FIG. 155 is a genetic analysis LOC device with a leukocyte dialysis section 328 and chemical lysis section 130. LOC variant XXVII 654 then uses parallel amplification sections 112.1, 112.2, . . . 112.X and respective hybridization chamber arrays 110.1, 110.2, . . . 110.X.
LOC Variant XXVIII
• LOC variant XXVIII 655 shown in FIG. 156 is an embodiment of a genetic analysis LOC device. LOC variant XXVIII 655 uses a leukocyte dialysis section 328 and chemical lysis section 130. The sample is then fed to tandem amplification sections 112.1 and 112.2 before detection in a single hybridization chamber array 110.
LOC Variant XXIX
• LOC variant XXIX 659 shown in FIG. 157 is a LOC device for pathogen detection and genetic analysis. The sample input and preparation section 288 has been simplified so as not to include any dialysis. Removing dialysis can increase the sensitivity of detection for pathogens that may interact unfavourably with dialysis functionality. The whole blood sample is added to the sample inlet 68 and anticoagulant from reservoir 54 is added via the surface tension valve 118. The extraction phase 290 lyses the pathogens and leukocytes with lysis reagent from reservoir 56 in the chemical lysis section 130. The incubation stage 291 involves incubation of the sample with restriction enzymes, ligase and linker primers from reservoir 58 and incubation in the incubation section 114 prior to entering the amplification stage 292 where amplification mix from reservoir 60 and the polymerase from reservoir 62 are added and the sample is amplified in the amplification section 112. Detection phase 294 occurs in the hybridization chamber arrays 110.
LOC Variant XXX
• LOC variant XXX 660 shown in FIG. 158 is a LOC device for pathogen detection and genetic analysis. The sample input and preparation section 288 has been simplified so as not to include any dialysis. Removing dialysis can increase the sensitivity of detection for pathogens that may interact unfavourably with dialysis functionality. The whole blood sample is added to the sample inlet 68 and anticoagulant from reservoir 54 is added via the surface tension valve 118. The extraction phase 290 lyses the pathogens and leukocytes with lysis reagent from reservoir 56 in the chemical lysis section 130. Following incubation with the restriction enzymes, ligase and linkers from the reservoir 58 in the incubation section 114, the sample is amplified in parallel amplification sections 112.1, 112.2, . . . 112.X and detected in respective hybridization chamber arrays 110.1, 110.2 . . . 110.X.
LOC Variant XXXI
• LOC variant XXXI 661 in FIG. 159 is a LOC device for pathogen detection and genetic analysis. The sample input and preparation section 288 has been simplified so as not to include any dialysis. Removing dialysis can increase the sensitivity of detection for pathogens that may interact unfavourably with dialysis functionality. The whole blood sample is added to the sample inlet 68 and anticoagulant from reservoir 54 is added via the surface tension valve 118. The extraction phase 290 lyses the pathogens and leukocytes with lysis reagent from reservoir 56 in the chemical lysis section 130. Following incubation with the restriction enzymes, ligase and linkers in the incubation section 114, the sample is amplified in tandem amplification sections 112.1 and 112.2, and detected in a single hybridization chamber array 110.
LOC Variant XXXII
• LOC variant XXXII 662 in FIG. 160 is configured for pathogen detection, genetic analysis and proteomic analysis. A blood sample enters through the sample inlet 68 and combines with anticoagulant from reservoir 54 via the surface tension valve 118. The sample then flows through a leukocyte dialysis section 328 with the leukocyte output flowing on to the extraction phase 290 and the pathogens and erythrocytes within the ‘waste’ output flow into a pathogen dialysis section 70. The pathogen output feeds into the extraction phase 290, and the erythrocytes in the waste output also flows into the extraction phase as a separate stream. In the extraction phase 290, the leukocytes, pathogens and erythrocytes are separately lysed in respective chemical lysis sections 130.1, 130.2 and 130.3.
• As the leukocyte sample flow enters the incubation phase 291, it splits into two streams; one stream is incubated in the incubation section 114.1 and subsequently thermally cycled in the amplification section 112.1, while the other stream passes directly to the detection phase 294. The amplified leukocyte stream flows into the hybridization chamber array 110.1 whereas the non-amplified leukocyte stream enters the proteomic assay chamber array 124.1 for protein detection where the photosensor 44 detects any probe-target complexes.
• Similarly, the pathogen stream splits into two as it enters the incubation phase 291. Again, one stream is diverted directly to the detection phase 294 while the other stream is firstly digested in the incubation section 114.2, and then amplified in the amplification section 112.2. The amplified pathogen stream flows into the hybridization chamber array 110.2 while the non-amplified pathogen stream flows into a second proteomic assay chamber array 124.2. The photosensor 44 detects any probe-target hybrids in hybridization chamber array 110.3 and any probe-target complexes in the proteomic assay chamber array 124.2 respectively.
• Once the erythrocyte stream has been chemically lysed in the lysis section 130.3, it bypasses the incubation 291 and amplification 292 phases completely and fills the proteomic assay chamber array 124.3 for detection by the photosensor 44.
LOC Variant XXXIII
• LOC variant XXXIII 663 is schematically illustrated in FIG. 161. LOC variant XXXIII 663 is configured for pathogen detection, genetic analysis and proteomic analysis. A blood sample is combined with anticoagulant from reservoir 54 via the surface tension valve 118 and then flows through a leukocyte dialysis section 328 with the leukocyte output flowing on to the extraction phase 290 and the pathogens and erythrocytes within the waste output flow into a pathogen dialysis section 70. The pathogen output feeds into the extraction phase 290, and the erythrocytes in the waste output of that dialysis section also flows into the extraction phase as a separate stream. In the extraction phase 290, the leukocytes, pathogens and erythrocytes are separately lysed in respective chemical lysis sections 130.1, 130.2 and 130.3.
• As the leukocyte sample flow enters the incubation phase 291, it splits into two streams; one stream is incubated in the incubation section 114.1 and subsequently thermally cycled in parallel amplification sections 112.11 to 112.1X, while the other stream passes directly to the detection phase 294. The amplified leukocyte stream flows into separate hybridization chamber arrays 110.11 to 110.1X where the photosensor 44 detects any probe-target hybrids. The non-amplified leukocyte stream flows into a proteomic assay chamber array 124.1, for protein analysis and detection by the photosensor 44.
• Similarly, the pathogen stream splits into two as it enters the incubation phase 291. Again, one stream is diverted directly to the detection phase 294 while the other stream is firstly digested in the incubation section 114.2, and then amplified in the parallel amplification sections 112.21 to 112.2Y. The amplified pathogen stream flows into the hybridization chamber arrays 110.21 to 110.2Y while the non-amplified pathogen stream flows into a proteomic assay chamber array 124.2. The photosensor 44 detects any probe-target hybrids in hybridization chamber arrays 110.21 to 110.2Y and proteomic assay chamber array 124.2.
• Once the erythrocyte stream has been chemically lysed in the lysis section 130.3, it bypasses the incubation 291 and amplification 292 phases completely and fills the proteomic assay chamber array 124.3 for detection by the photosensor 44.
LOC Variant XXXIV
• LOC variant XXXIV 664 shown in FIG. 162 is for pathogen detection, genetic analysis and proteomic analysis. A blood sample is combined with anticoagulant from reservoir 54 via the surface tension valve 118 and then flows through a leukocyte dialysis section 328 with the leukocyte output flowing on to the extraction phase 290 and the pathogens and erythrocytes within the waste output flow into a pathogen dialysis section 70. The pathogen output feeds into the extraction phase 290, and the erythrocytes in the waste output of that dialysis section also flows into the extraction phase as a separate stream. In the extraction phase 290, the leukocytes, pathogens and erythrocytes are separately lysed in respective chemical lysis sections 130.1, 130.2 and 130.3 and the leukocyte and pathogen streams are split into two, one of which proceeds to the amplification phase 292 and the other goes directly to the proteomic assay chamber arrays 124.1 and 124.2, respectively, for protein detection by the photosensor 44. The lysed erythrocyte stream proceeds directly to the proteomic assay chamber array 124.3 for protein detection by the photosensor 44.
• The separate leukocyte stream and pathogen stream are amplified by respective tandem nucleic acid amplification configurations. The targets within the leukocyte stream are amplified in amplification sections 112.1 and 112.2, while the targets in the pathogen stream are amplified in amplification sections 112.3 and 112.4. Each amplified stream flows to a respective hybridization chamber array 110.1 and 110.2 for detection by the photosensor 44.
LOC Variant XXXV
• LOC variant XXXV 665 shown in FIG. 163 has a simple design for genetic analysis of a sample without any dialysis or lysis prior to nucleic acid amplification. This LOC device is used for samples in which the target nucleic acid sequences are not within cells so that an extraction phase is not required. Accordingly, the sample is not likely to be blood so reservoir 54 releases a diluent or other reagent via surface tension valve 118 during the sample input and preparation phase 288. The sample combines with amplification mix from reservoir 60 and polymerase from reservoir 62 via the surface tension valves 138 and 140 respectively. Thermal cycling in the amplification section 112 amplifies the targets and the boiling-initiated valve 108 opens to allow the amplicon to fill the hybridization chamber array 110. Any probe-target hybrids are detected with photosensor 44.
LOC Variant XXXVI
• LOC variant XXXVI 666 shown in FIG. 164 is a genetic analysis LOC device that does not extract the target nucleic acid sequences prior to nucleic acid amplification. LOC variant XXXVI 666 utilises a small constituents dialysis section 682 to purify the sample after nucleic acid amplification in the amplification section 112. The small constituents dialysis section 682 removes cells, particles and debris greater than a certain size and diverts them to a waste reservoir 766. The smaller sample constituents (such as dissolved molecules and amplified nucleic acids) flow into the hybridization chamber array 110 containing sequence specific probes in each chamber. Photosensor 44 detects light emitted from the probes which is modified by the degree of hybridization as determined by the probe design. As with all LOC devices, a humidifier 196 and humidity sensor 232 are used to control evaporation and condensation in the LOC device 666, particularly the hybridization chamber array 110.
LOC Variant XXXVII
• LOC variant XXXVII 667 is schematically illustrated in FIG. 165 and is a genetic analysis LOC device. Genetic material is extracted from sample cells prior to nucleic acid amplification by chemical lysis in the chemical lysis chamber 130. After nucleic acid amplification in the amplification section 112, the sample is purified with the small constituents dialysis section 682 before flowing into the hybridization chamber array 110 for detection of probe-target hybrids with the photosensor 44.
LOC Variant XXXVIII
• LOC variant XXXVIII 668 is schematically illustrated in FIG. 166. This LOC variant is configured for genetic analysis without dialysis, an extraction phase 290 or an incubation phase 291 prior to nucleic acid amplification 292. LOC variant XXXVIII 668 amplifies the target sequences in the sample using tandem amplification sections 112.1 and 112.2 configured in series. After thermal cycling in the amplification section 112.2, the boiling-initiated valve 108 opens to allow the amplicon to fill the hybridization chamber array 110. Any probe-target hybrids are detected with the photosensor 44.
LOC Variant XXXIX
• LOC variant XXXIX 669 shown in FIG. 167 does not lyse sample cells or use dialysis prior to nucleic acid amplification. This LOC device 669 has tandem amplification sections 112.1 and 112.2 arranged in series to amplify the nucleic acids. A small constituents dialysis section 682 purifies the sample after nucleic acid amplification in the amplification section 112.2. The small constituents dialysis section 682 removes cells, particles and debris greater than a certain size and diverts them to a waste reservoir 766. The smaller sample constituents (such as dissolved molecules and amplified nucleic acids) flow into the hybridization chamber array 110 containing sequence specific probes in each chamber. The photosensor 44 detects light emitted from the probes which is modified by the degree of hybridization as determined by the probe design. As with all LOC devices, a humidifier 196 and humidity sensor 232 are used to control evaporation and condensation in the LOC variant XXXIX 669, particularly the hybridization chamber array 110.
LOC Variant XL
• LOC variant XL 670 is schematically illustrated in FIG. 168. This LOC device lyses the cells (lysis reagent reservoir 56 and chemical lysis section 130) to extract genetic material from sample cells prior to nucleic acid amplification in the amplification section 112. After amplification in tandem amplification sections 112.1 and 112.2, the sample is purified with the small constituents dialysis section 682 (where large constituents are removed to waste reservoir 766) before flowing into the hybridization chamber array 110 for detection of hybrids with the photosensor 44.
LOC Variant XLI
• LOC variant XLI 671 shown in FIG. 169 is for the analysis of a sample in which the smaller, generally soluble, constituents are of interest. The sample is added to the sample inlet 68. Solid or powdered sample is combined with a suitable liquid in the sample inlet 68 for capillary flow to the surface tension valve 118. Reagents in reservoir 54 mix with the sample via surface tension valve 118 and flow continues to the small constituents dialysis section 682. Sample constituents below a particular size threshold, such as salts, metabolites, DNA and proteins, remain in the sample. Larger constituents such as cells, pathogens and debris, are diverted to the waste reservoir 766. The purified sample continues into other functional units (as described in relation to previous LOC devices) for further processing such as incubation, nucleic acid amplification and hybridization 684.
LOC Variant XLII
• LOC variant XLII 672 in FIG. 170 is for the analysis of a sample in which the larger, generally insoluble constituents are of interest. The sample is added to the sample inlet 68. Solid or powdered sample is combined with a suitable liquid in the sample inlet 68 for capillary flow to the surface tension valve 118. Reagents in reservoir 54 mix with the sample via surface tension valve 118 and flow continues to the large constituents dialysis section 686. Sample constituents above a particular size threshold, such as cells, pathogens and particulates remain in the sample. Smaller constituents such as salts, metabolites, DNA and proteins, are diverted to the waste reservoir 768. The purified sample continues into other functional sections for further processing such as incubation 291, nucleic acid amplification 292 and hybridization and detection 294.
LOC Variant XLIII
• LOC variant XLIII 673 shown in FIG. 171 detects pathogens in a sample by enrichment of the pathogen particles according to size in a two-stage dialysis process. A sample is added to the sample inlet 68 and capillary action draws it to the surface tension valve 118. Reagents in the reservoir 54 mix with the sample via surface tension valve 118 and flow continues to the large constituents dialysis section 686. Sample constituents above a particular size threshold (e.g. 1 μm) such as cells, pathogens and particulates remain in the sample. Smaller constituents such as salts, metabolites and proteins, are diverted to the first waste reservoir 768. The sample containing the large constituents then flows into the small constituents dialysis section 682. Here, smaller sample constituents such as pathogens and nucleic acids are retained. Larger constituents such as leukocytes and other large cells divert to the second waste reservoir 766. The purified sample, enriched for pathogen particles, continues into the amplification phase 292 and finally into the hybridization chamber array 110 where the probe-nucleic acid hybrid is detected by the photosensor 44.
LOC Variant XLIV
• FIG. 172 shows LOC variant XLIV 674 which detects pathogens via nucleic acid amplification. The sample is added to the sample inlet 68 and capillary action draws it to the surface tension valve 118. Reagents in reservoir 54 mix with the sample via surface tension valve 118 and flow continues to the amplification phase 292. Reagent reservoir 54 may also contain a chemical lysis reagent if desired. Amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 is added via surface tension valve 140. Thermal cycling in the amplification section 112 amplifies genetic material from any lysed cells. Chemical and/or thermal lysis can occur in the amplification section 112 prior to commencement of thermal cycling. When sufficient amplicon has been generated, the boiling-initiated valve 108 opens for capillary driven flow into the small constituents dialysis section 682. Small sample constituents such as dissolved molecules and amplified nucleic acids are retained in the sample. Large constituents such as cell membrane debris and any non-lysed pathogens flow to the waste reservoir 766. The small constituents in the purified sample continue to the hybridization chamber array 110 for hybrid detection by the photosensor 44.
LOC Variant XLV
• Referring to FIG. 173, LOC variant XLV 675 detects pathogens using a large constituents dialysis section 686, incubation section 114, amplification section 112, hybridization chamber array 110 and photosensor 44. The large constituents dialysis section 686 is designed to retain constituents above a certain threshold size (including pathogens). Constituents smaller than that threshold are diverted to the waste reservoir 768. The purified sample proceeds to the incubation phase 291 where it combines with restriction enzymes, ligase and linker primers from reservoir 58 via surface tension valve 132 and restriction digestion and linker ligation proceeds in the incubation section 114. After digestion, the boiling-initiated valve 108 opens and flow continues to the amplification section 112 where amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 is added via surface tension valve 140. When sufficient amplicon has been generated, the boiling-initiated valve 108 opens for capillary driven flow into the hybridization chamber array 110. Sequence specific probes in each chamber hybridize with any target sequences in the sample and the hybrids are detected with the photosensor 44.
LOC Variant XLVI
• FIG. 174 schematically illustrates LOC variant XLVI 676 which detects pathogens using a large constituents dialysis section 686, incubation section 114, amplification section 112, hybridization chamber array 110 and photosensor 44. The large constituents dialysis section 686 is designed to retain constituents above a certain threshold size (including pathogens). Constituents smaller than that threshold are diverted to the waste reservoir 768. The purified sample continues to the incubation phase 291 where it combines with restriction enzymes, ligase and linker primers from reservoir 58 via surface tension valve 132 and restriction digestion and linker ligation proceeds in the incubation section 114. After digestion, the boiling-initiated valve 108 opens and the sample enters the amplification section 112 where amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 is added via surface tension valve 140. After nucleic acid amplification, the amplicons are subjected to a pre-hybridization purification phase 293. A small constituents dialysis section 682 removes the large constituents from the amplicon flowing from the amplification section 112. The large constituents such as cells, pathogens, particles and debris are diverted to the second waste reservoir 766 while smaller constituents remaining in the sample, e.g. amplicons, fill the hybridization chamber array 110. Hybrids are detected with the photosensor 44.
LOC Variant XLVII
• FIG. 175 schematically illustrates LOC variant XLVII 677 which detects pathogens using a large constituents dialysis section 686, followed by nucleic acid amplification and hybridization and detection. The large constituents dialysis section 686 is designed to retain constituents above a certain threshold size (including pathogens). Constituents smaller than that threshold are diverted to the waste reservoir 768. Amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 is added via surface tension valve 140 prior to nucleic acid amplification in the amplification section 112. When sufficient amplicon has been generated, the boiling-initiated valve 108 opens for capillary driven flow into the hybridization chamber array 110. Sequence specific probes in each chamber hybridize with any target sequences in the sample and the hybrids are detected with the photosensor 44.
LOC Variant XLVIII
• FIG. 176 schematically illustrates LOC variant XLVIII 678 which is for the detection of pathogens using the restriction enzyme, ligase and linker incubation section 114, the amplification section 112, a small constituents dialysis section 682, hybridization chamber array 110 and photosensor 44. The sample is added to the sample inlet 68 and capillary action draws it to the surface tension valve 118. Reagents in reservoir 54, which can include a chemical lysis reagent, mix with the sample via surface tension valve 118 and flow continues to the incubation phase 291. Here the sample combines with restriction enzymes, ligase and linkers from reservoir 58 via a surface tension valve 132 and restriction digestion and linker ligation proceeds in the incubation section 114 until the boiling-initiated valve 108 at the outlet to the incubation section 114 opens and capillary drive flow resumes into the amplification phase 292. Amplification mix from reservoir 60 is added via surface tension valve 138 and polymerase from reservoir 62 is added via surface tension valve 140. Thermal cycling in the amplification section 112 amplifies genetic material from any lysed cells. When sufficient amplicon has been generated, the boiling-initiated valve 108 opens for capillary driven flow into the small constituents dialysis section 682. Small sample constituents such as dissolved molecules and amplified nucleic acids are retained in the sample. Large constituents such as cell membrane debris and any non-lysed pathogens flow to the waste reservoir 766. The small constituents in the purified sample continue to the hybridization chamber array 110 for hybrid detection by the photosensor 44.
LOC Variant XLIX
• FIGS. 177A and 177B schematically illustrate LOC variant XLIX 679 which analyses DNA from the nuclei of cells, and separately but simultaneously analyses the mitochondrial DNA from the cells. Sample cells (for example whole blood) are added to the sample inlet 68 and a reagent (such as anticoagulant) is added from reservoir 54 through surface tension valve 118. The sample flows into a first large constituents dialysis section 686 where small constituents such as salts, proteins and other molecules are removed to the waste reservoir 768.
• A first lysis reagent in reservoir 56.1 is added to the sample via surface tension valve 128.1. The first lysis reagent targets only the outer cell membranes and the mitochondrial membranes. It does not disrupt the membranes of the nuclei (released when the outer cell membranes break). The sample remains in the first chemical lysis section 130.1 until diffusive mixing of the lysis reagent disrupts all cells. When fully lysed, the boiling-initiated valve 108 at the outlet of the first chemical lysis section 130.1 opens for the capillary-driven flow through the LOC variant XLIX 679 to continue. Non-ionic detergents, such as Nonidet P40 (0.05-5%) and Triton X-100 (0.1-1%), or plant glycosides, such as saponin (0.01-0.1%) and Octy Glycoside, are examples of suitable lysis reagents that selectively lyse the outer cell membrane and mitochondrial membrane but not the nuclear membrane.
• The sample flows into a second large constituents dialysis section 686 where the small suspended constituents (including mitochondrial DNA) are removed from the sample containing the nuclei. A second lysis reagent from reservoir 56.2 is added to the sample via surface tension valve 128.2. The second lysis reagent diffusively mixes through the sample within a second chemical lysis chamber 130.2 to release the nuclear DNA. An example of a suitable lysis reagent is 1% sodium dodecyl sulfate or a higher concentration of the lysis reagent used in the first lysis reagent reservoir 56.1. When mixed and lysed, the boiling-initiated valve 108 at the outlet of the second chemical lysis section 130.2 opens and the sample flows to the first incubation section 114.1. The person skilled in the art will recognise that the lysis reagents and concentrations given are indicative only and any suitable lysis reagent can be used at a suitable concentration and for an appropriate amount of time in order to achieve this objective.
• The first restriction enzyme, ligase and linker reservoir 58.1 feeds into the sample flow via the surface tension valve 132.1. Simultaneously, the mitochondrial DNA in the small constituents output from the large constituents dialysis section 686 flows into the second incubation section 114.2. Enzymes from the second restriction enzyme, ligase and linker reservoir 58.2 are added via surface tension valve 132.2. After sufficient time for restriction digestion and linker ligation, the boiling-initiated valves 108 at the outlets of each incubation section 114.1 and 114.2, respectively, open and the separate nuclear and mitochondrial streams feed into respective sets of parallel amplification sections for nucleic acid amplification. The nuclear DNA fills parallel amplification sections 112.11, 112.12, . . . 112.1X while the mitochondrial DNA fills parallel amplification sections 112.21, 112.22, . . . 112.2Y. Each amplification section adds amplification mix from corresponding amplification mix reservoirs 60.11, 60.12, . . . 60.1X, and 60.21, 60.22, . . . 60.2Y through surface tension valves 138.11, 138.12, . . . 138.1X and 138.21, 138.22 . . . 138.2Y Likewise, polymerase for each of the amplification sections is added from corresponding polymerase reservoirs 62.11, 62.12, . . . 62.1× and 62.21, 62.22, . . . 62.2Y via surface tension valves 140.11, 140.12, . . . 140.1X, and 140.21, 140.22, . . . 140.2Y.
• Parallel nucleic acid amplification allows multiple amplification assays to be performed simultaneously. The nuclear DNA and the mitochondrial DNA are processed and amplified separately for more sensitive nucleic acid amplification. Furthermore, the amplicon from each of the amplification sections is detected separately using hybridization chamber arrays 110.11, 110.12, . . . 110.1X and 110.21, 110.22, . . . 110.2Y containing probes specifically designed for detection of the amplicon by the photosensor 44. This allows multiple targets to be detected without performing a multiplex amplification and therefore increases the assay sensitivity and signal to noise ratio.
• LOC Device with ECL Detection
• FIGS. 263 to 279 show a LOC variant 729 with electrochemiluminescence (ECL) detection. This LOC device prepares 288, extracts 290, incubates 291, amplifies 292 and detects 294 both human and pathogen nucleic acids, as well as human and pathogen protein detection. ECL is used in the hybridization chamber arrays and proteomic assay chamber arrays for target detection.
• As best shown in FIG. 269, a biological sample (for example, whole blood) is added to the sample inlet 68. The sample flows through the cap channel 94 to the anticoagulant surface tension valve 118. The cap 46 is fabricated with an interface layer 594 positioned between the cap channel layer 80 and the MST channel layer 100 of the CMOS+MST device 48 (see FIG. 264). The interface layer 594 allows a more complex fluidic interconnection between the reagent reservoirs and the MST layer 87 without increasing the size of the silicon substrate 84.
• FIG. 265 shows the MST layer 87 visible on the top surface of the CMOS+MST device 48. FIG. 266 shows the cap channel layer 80 on the underside of the cap 46. FIG. 267 superimposes the reservoirs, the cap channels 94 and the interface channels to illustrate the more sophisticated plumbing achieved with a cap 46 incorporating an interface layer 594.
• As best shown in FIG. 269, the interface layer 594 requires the anticoagulant surface tension valve 118 to have two interface channels 596 and 598. A reservoir-side interface channel 596 connects the reservoir outlet with the downtakes 92 and a sample-side interface channel 598 connects the uptakes 96 with the cap channel 94.
• Anticoagulant from the reservoir 54 flows through the MST channels 90 via the reservoir-side interface channel 596 to pin a meniscus at the uptakes 96. The sample flow along the cap channel 94 dips into the sample-side interface channel 598 to remove the meniscus so that the anticoagulant combines with the blood sample as it continues onto the leukocyte dialysis section 328.
• The leukocyte dialysis section 328 incorporates a bypass channel 600 for filling the flow channel structures without trapped air bubbles (see FIGS. 269 and 278). The blood sample flows through cap channel 94 to the upstream end of the large constituents interface channel 730. The large constituents interface channel 730 is in fluid communication with the dialysis MST channels 204 via apertures in the form of 7.5 micron diameter holes 165 (see FIG. 278).
• Referring to FIG. 278, each of the dialysis MST channels 204 lead from the 7.5 micron diameter holes 165 to respective dialysis uptakes 168. The dialysis uptake holes 168 are open to the small constituents interface channel 732. However the uptakes are configured to pin a meniscus rather than allow capillary driven flow to continue. The uptake belonging to the bypass channel 600 has a capillary initiation feature 202 configured to initiate capillary driven flow into the small constituents interface channel 732. This ensures the flow begins at the upstream end of the small constituents interface channel 732 and sequentially unpins the menisci at the dialysis uptakes 168 as the flow progresses downstream.
• FIG. 273 shows the downstream end of the leukocyte dialysis section 328. The large constituents interface channel 730 feeds into the large constituents cap channel 736 and the small constituents interface channel 732 feeds the small constituents cap channel 734. As best shown in FIG. 267, the large constituents cap channel 736 feeds the leukocytes (and any other large constituents) into the chemical lysis section 130.1 via the lysis surface tension valve 128.1 where lysis reagent from reservoir 56.1 is added. The chemical lysis section 130.1 has a 3 micron filter downtake 738 at the outlet (see FIG. 269). The filter downtake ensures that no large constituents reach the lysis chamber exit boiling-initiated valve 206. After sufficient time, the boiling-initiated valve 206 opens the chemical lysis section 130.1 outlet and the sample flow is split into two streams. As best shown in FIG. 269, one stream flows to the surface tension valve 132.1 for the first restriction enzyme, ligase and linker reservoir 58.1 and the other stream is drawn along a lysed leukocyte bypass channel 742 directly to the proteomic assay chamber array 124.1 in the hybridization and detection section 294. Here the sample fills the proteomic assay chamber array 124.1 (see FIG. 271) containing probes for hybridization with target human proteins. Probe-target hybrids are detected with a photosensor 44 (see FIG. 263). The other stream flows into the leukocyte incubation section 114.1 together with restriction enzymes, ligase and linker primers from reservoir 58.1.
• Referring to FIG. 270, after restriction enzyme digestion and linker ligation, the incubator outlet valve 207 (also a boiling-initiated valve) opens and flow continues into the leukocyte DNA amplification section 112.1. The amplification mix and polymerase in reservoirs 60.1 and 62.1 are added via surface tension valves 138.1 and 140.1 respectively. Referring to FIG. 271, after thermal cycling, the boiling-initiated valve 108 opens for the amplicon to enter the hybridization chamber array 110.1 containing probes for human DNA targets. Probe-target hybrids are detected with the photosensor 44.
• The erythrocytes and pathogens from the leukocyte dialysis section 328 are fed to the pathogen dialysis section 70 via the cap channel 734 (see FIGS. 269 and 279). This operates in the same manner as the leukocyte dialysis section 328 with the exception that the filter downtakes have 3 micron holes 164 instead of the 7.5 micron holes 165 used for leukocyte dialysis. The erythrocytes remain in the large constituents interface channel 730 while the pathogens diffuse to the small constituents interface channel 732.
• FIG. 274 shows the downstream end of the pathogen dialysis section 70. The erythrocytes flow into the large constituents cap channel 736 and the pathogens fill the small constituents cap channel 734. It will be appreciated that ‘large constituents’ and ‘small constituents’ are used in a relative sense as the large constituents output of the pathogen dialysis section is part of the small constituents output of the leukocyte dialysis section. The constituents in the large constituents cap 736 or interface channels are simply larger than the constituents in the small constituents cap 734 or interface channels within that particular dialysis section. As best shown in FIGS. 267 and 268, the erythrocytes in the large constituents cap channel 736 are directed to the surface tension valve 128.3 for the lysis reagent reservoir 56.3. The lysis reagent combines with the erythrocytes as the sample fluid fills the chemical lysis section 130.3. Boiling-initiated valve 206 at the outlet of the third chemical lysis section 130.3 retains the pathogens until lysis is complete. When the boiling-initiated valve 206 opens, the erythrocyte DNA flows directly into the proteomic assay chamber array 124.3 for protein analysis and detection by the photosensor 44 (see FIG. 271).
• The pathogens in the small constituents cap channel 734 are directed to the surface tension valve 128.2 of the second lysis reagent reservoir 56.2. The lysis reagent combines with the pathogens as the sample fluid fills the second chemical lysis section 130.2. After sufficient time, the boiling-initiated valve 206 opens the chemical lysis section 130.2 outlet and the sample flow is split into two streams. As best shown in FIGS. 268 and 270, one stream flows to the surface tension valve 132.2 for the second restriction enzyme, ligase and linker reservoir 58.2 and the other stream is drawn along a bypass channel 744 directly to the hybridization and detection section 294. Here the sample fills the proteomic assay chamber array 124.2 (see FIG. 271) containing probes for hybridization with target pathogen proteins or other biomolecules. Probe-target hybrids are detected with the photosensor 44 (see FIG. 263).
• The other stream flows into the pathogen incubation section 114.2 together with restriction enzymes, ligase and linker primers from reservoir 58.2. After restriction digestion and linker ligation, the incubator exit valve 207 (also a boiling-initiated valve) opens and flow continues into the pathogenic DNA amplification section 112.2 (see FIG. 270). As the chamber fills, the amplification mix and polymerase in reservoirs 60.2 and 62.2 are added via surface tension valves 138.2 and 140.2 respectively. After thermal cycling, the boiling-initiated valve 108 opens for the amplicon to flow into the second hybridization chamber array 110.2 containing probes for pathogenic DNA targets. Probe-target hybrids are detected with the photosensor 44 (see FIG. 271).
• Referring to FIG. 272, the hybridization chamber arrays 110.1 and 110.2 and proteomic assay chamber arrays 124.1 to 124.3 have heater elements 182 made from strips of titanium nitride. There are end-point liquid sensors 178 that detect when the flow has reached the end of the hybridization chamber array or proteomic assay chamber array and the heaters 182 are then activated after a time delay. The flow rate sensor 740 (see FIG. 277) is included in the pathogen incubation section 114.2 to determine the time delay.
• FIGS. 275 and 276 show the calibration chambers 382. They are used to calibrate the photodiodes 184 to adjust for system noise and background levels. The photodiode's response and electrical noise characteristics can vary with location and due to thermal variations. The output signal from calibration chambers 382, which do not contain any probes, closely approximates the noise and background in the output signal from all the chambers. Subtracting the calibration signal from the output signals generated by the other hybridization chambers substantially removes the noise and leaves the signal generated by the electrochemiluminescence (if any). Also, positive and negative control ECL probes 786 and 787 can be placed in some of the hybridization chambers 180 for assay quality control.
• Referring to FIG. 268, a humidifier 196, composed of the water reservoir 188 and evaporators 190, is located in the top left of the device. The position of the humidity sensor 232 is adjacent to the hybridization chamber array 110 where humidity measurement is most important to slow evaporation from the solution containing the exposed probes.
• By combining the leukocyte and pathogen output dialysis sections, three output streams are produced (leukocytes, erythrocytes, and pathogens and other biomolecules) which are processed separately to enable higher sensitivity and parallel analysis. The output from each stream is lysed and separately directed to the proteomic assay chamber arrays for protein detection. The lysed leukocytes and pathogens are also separately directed to the incubation 114 and amplification 112 sections for amplification, followed by hybridization for nucleic acid detection.
• LOC Device with Thermal Insulation Trench
• As best depicted in FIG. 280, a trench 896 is etched into the back of the silicon substrate 84. The purpose of the trench is to thermally insulate the amplification section 112 from the hybridization chamber array 110. The hybridization array contains detection probes that can degrade at high temperatures. The trench, when filled with air, has a thermal conductivity of the order of 6000 times less than that of the silicon substrate, thereby significantly reducing the heat flux into adjacent parts of the LOC device.
• This provides two main advantages: an increase in the heating efficiency in the amplification section 112; and a reduction in the undesirable temperature rise of the adjacent hybridization section 110. Improved heating efficiency means less power is required to heat the amplification section 112 and the temperature reaches its desired end-point temperature faster and with better spatial uniformity within the amplification section. A reduction in the temperature rise in the hybridization section 110 allows for a wider range of probe chemistries and superior signal quality.
• The trench can be placed around any region on the LOC device to thermally insulate the components in that region. The width and depth of the trench 896 are variable to suit the specific application.
• Microfluidic Device with Dialysis Device, LOC Device and Interconnecting Cap
• A microfluidic device 783 with dialysis device 784, LOC device 785 and interconnecting cap 51 provides for greater modularity and improved sensitivity. In contrast to LOC designs which combine all the functions (see, for example, LOC variant 729 in FIG. 268), separating the dialysis function from the LOC device allows different specialized dialysis devices 784 to be developed that select for different targets. These specialized dialysis devices 784 can be combined with a LOC device 785 and interconnecting cap 51 to form a complete assay system. Furthermore, different LOC devices 785 optimized for different assay approaches can also be developed and coordinated with different dialysis devices 784, thus providing an extremely powerful and flexible approach to system development. It is also possible to deploy the LOC device without a dialysis device for certain applications, or to combine multiple LOC devices 785.
• Each surface-micromachined chip constituent of the microfluidic device 783 can be fabricated with the optimal and most cost effective fabrication process. For example, the dialysis device 784 requires no CMOS circuitry and so can be manufactured utilizing less expensive materials and fewer process steps. Furthermore, a larger and optimized dialysis device 784 provides increased sensitivity, signal-to-noise ratio, and dynamic range for the assay system.
• As diagrammatically shown in FIG. 290, the microfluidic device 783 prepares the sample 288, and then extracts 290, incubates 291, amplifies 292, and detects 294 pathogenic DNA using twelve separate amplification chambers (112.1 to 112.12). The assembly employs multiple amplification chambers to increase assay sensitivity and improve the signal-to-noise ratio. This LOC device utilizes ECL in the hybridization chamber arrays 110.1 to 110.12 to detect probe-target hybrids. The system modularity provides for different dialysis devices 784 to be employed to detect other targets, for example, leukocytes, erythrocytes, pathogens, or molecules like free proteins or DNA within a liquid sample, and although the current description describes pathogenic DNA detection, the skilled worker will appreciate that the microfluidic device 783 is not limited to detecting this one target only.
• FIG. 291 shows the microfluidic device 783 with dialysis device 784, LOC device 785, and interconnecting cap 51. The interconnecting cap 51 consists of the reservoir layer 78, the cap channel layer 80, and the interface layer 594. The interface layer 594 is positioned between the cap channel layer 80 and the MST channel layer 100 of the CMOS+MST device 48. The interface layer 594 allows a more complex fluidic interconnection between the reagent reservoirs and the MST layer 87 without increasing the size of the silicon substrate 84. FIG. 292 superimposes the reservoirs, the top channels, and the interface channels to illustrate the more sophisticated plumbing achieved with the interface layer 594.
• Referring to FIGS. 292 and 293, the sample (for example, blood) enters the sample inlet 68 and capillary action draws it along the cap channel 94 to the anticoagulant surface tension valve 118. As best shown in FIG. 293, the anticoagulant surface tension valve 118 has two interface channels 596 and 598 in the interface layer 594. A reservoir-side interface channel 596 connects the reservoir outlet with the downtakes 92 and a sample-side interface channel 598 connects the uptakes 96 with the cap channel 94. Anticoagulant from the reservoir 54 flows through the MST channels 90 via the reservoir-side interface channel 596 until the meniscus is pinned at the uptakes 96. The sample flow along the cap channel 94 dips into the sample-side interface channel 598 to remove the meniscus so that the anticoagulant combines with the blood sample as it continues on to the pathogen dialysis section 70.
• Referring to FIGS. 292 and 293, the pathogen dialysis section 70 in this embodiment comprises an interface target channel 602 and an interface waste channel 604 fluidically coupled by a plurality of apertures of a predetermined threshold size. The aperture at the very upstream of the dialysis section 70 is different from the apertures downstream of it; the downstream apertures are holes with diameters of less than 8.0 microns selected to allow targets to pass through to the interface target channel 602. In the present embodiment the apertures are 3.0 micron diameter holes 164 selected to allow pathogens to pass through to the interface target channel 602. The pathogen dialysis section 70 is configured such that the sample flows through the channels and apertures under capillary action.
• Referring to FIGS. 292 and 293, the blood sample flows through the cap channel 94 to the upstream end of the interface waste cell channel 604. The interface waste cell channel 604 is open to the 3.0 micron diameter apertures 164 leading to the dialysis MST channels 204. Each of the dialysis MST channels 204 lead from the 3.0 micron diameter apertures 164 to respective dialysis uptake holes 168. The dialysis uptake holes 168 are open to the interface target channel 602. However the uptakes are configured to pin a meniscus rather than allow capillary driven flow to continue.
• The pathogen dialysis section 70 incorporates a bypass channel 600 for filling the flow channel structures without trapped air bubbles. The bypass channel 600 at the very upstream end of the pathogen dialysis section 70, has a CIF (capillary initiation feature) 202 to promote capillary driven flow from the bypass channel 600 into the interface target channel 602 (see FIGS. 292 and 293). The bypass channel also has a wide meander to lengthen the flow-path from the interface waste cell channel 604 to the interface target channel 602. The longer flow-path delays the sample flow such that it fills the interface target channel 602 after the meniscus forms at the most upstream dialysis MST channel 204. The sample flow starts at the upstream end and unpins the meniscus at each of the dialysis uptake holes 168 as the flow moves downstream along the interface waste channel 602. This ensures all the dialysis bottom channels fill with sample flow as the dialysis section fills. Without the bypass channel 600, or dialysis uptakes 168 configured to pin a meniscus, some dialysis MST channels 204 may not fill. Similarly, an air bubble may form in the interface target channel 602. In either case, flow through the dialysis section can be substantially throttled.
• The interface waste channel 604 feeds into the waste channel 72 and the interface target channel 602 feeds into the target channel 74 (see FIGS. 292 and 293). Five dialysis sections are joined in series by cap channels 72 and 74 to increase the efficiency of the dialysis process. At the exit of the fifth dialysis section, the sample flow with the target is drawn along the target channel 74 in the cap channel layer away from the dialysis device 784 and into the LOC device 785 by capillary driven flow. The waste channel 72 flows to the waste reservoir 76 (see FIG. 291).
• The LOC device 785 can also be employed as a standalone microfluidic device with an alternate cap suitable for a single device, and in this configuration optional anticoagulant reservoir 55 can be used to supply anticoagulant. One example of this standalone use of the LOC device 785 is its use for whole blood analysis. Referring to FIGS. 294 and 295, the target flows into LOC device 785 along cap channel 74. It passes via optional anticoagulant surface tension valve 117, which is used to add the contents of optional anticoagulant reservoir 55, and continues on until it reaches the lysis surface tension valve 128. In the configuration being described here, this optional reservoir and surface tension valves are unused and do not affect the sample flow or operation of the LOC device.
• As with the anticoagulant surface tension valve 118 described above, the lysis surface tension valve 128 has a lysis reservoir-side interface channel 606 and a lysis sample-side interface channel 608 (see FIG. 295). Lysis reagent flows from the reservoir 56 to the lysis reservoir-side interface channel 606 via a cap channel 94. The reagent flows into the downtakes 92, through the MST channels 90 to the uptakes 96 where the reagents pin a meniscus (see FIG. 295). Sample flow from the target channel 74 fills the lysis sample-side interface channel 608. The sample flow removes the menisci at the uptakes 96 and the lysis reagent combines with the sample as it flows into the chemical lysis section 130.
• In the chemical lysis chamber section 130, the lysis reagent diffusively mixes through the flow to lyse the target cells and release the genetic material therein. The sample flow stops at the mixing section exit valve 206. The mixing section exit valve is a boiling-initiated valve 206. The liquid sensor 174 upstream of the valve provides feedback that the sample flow is about to reach the valve 206. If the CMOS circuitry 86 is programmed with a delay to ensure the target cells are completely lysed, the liquid sensor feedback initiates the delay period. After any delay period, the boiling-initiated valve 206 is activated and the downstream liquid sensor 174 registers that the flow has resumed along the MST channel 90.
• The lysed sample flow continues to the restriction enzyme, ligase and linker surface tension valve 132. The operation of the surface tension valve 132 is the same as described earlier for the anticoagulant surface tension valve 118. Referring to FIG. 295, when the lysed sample flow reaches the pinned-menisci at the surface tension valve 132, restriction enzymes, ligase and linker primers are released from the reservoir 58 and combine with the sample flow. The sample then flows through MST channel 90 to the heated microchannels of the incubation section 114. Referring to FIGS. 295 and 296, the incubation section 114 is composed of a serpentine microchannel 210 heated by heaters 154.
• Referring to FIG. 296, the sample flow is stopped at the incubator outlet valve 207 until sufficient time has passed. The incubator outlet valve 207 is a boiling-initiated valve similar to the mixing section outlet valve 206. The liquid sensor 174 at the beginning of the incubation section, in conjunction with the flow rate sensor 740 (see FIG. 300) and CMOS circuitry 86, initiates an incubation time delay. After sufficient incubation, the incubator outlet valve 207 activates and flow resumes flowing along the MST incubation exit channel 630 to the polymerase surface tension valve 140 (see FIG. 297). Polymerase from the reservoir 62 combines with the sample flow as it travels the serpentine path of the amplification input channel 632.
• Referring to FIGS. 296 and 297, the amplification input channel 632 directs the sample flow past the twelve amplification mix surface tension valves 138. Amplification mix in each of the amplification mix reservoirs 60.1-60.12 (see FIG. 297) flows through respective cap channels 94 to pin menisci at the amplification mix surface tension valves 138. The sample flow opens each of the surface tension valves in turn, and the amplification mix from the respective amplification mix reservoirs 60.1-60.12 entrains with the sample flow into each of the twelve amplification chambers 112.1-112.12. The LOC device has CMOS circuitry that allows operative control of amplification section via temperature sensors and heaters.
• Referring to FIG. 298, each of the twelve amplification chambers 112.1-112.12 has one of the amplification outlet valves 207, respectively. The amplification outlet valves 207 are boiling-initiated valves like the incubator outlet valve 207. The sample flow stops at each amplification outlet valve 207. After amplification, the amplification outlet valves 207 open for the amplicon to flow into the hybridization chamber arrays 110.1-110.12 containing probes configured to form probe-target hybrids with the target nucleic acid sequences, in this case pathogenic DNA. The sample is drawn along the flow-path 176 through each of the separate arrays 110.1-110.12 and into the individual hybridization chambers 180 via respective diffusion barrier inlets 175 (see FIG. 272 and FIG. 299).
• Referring to FIGS. 275 and 299, when the sample flow reaches the end-point liquid sensor 178, the hybridization heaters 182 are activated after a time delay to enhance the generation of probe-target hybrids. The flow rate sensor 740 (see FIG. 300) is included in the pathogen incubation section 114 to determine the time delay. After a suitable delay for hybridization, an excitation current applied to the ECL electrodes 860 and 870 (see FIGS. 299 and 302) causes the probe-target hybrids to emit photons of light that are detected with the photosensor 44 in the underlying CMOS circuitry 86. The photosensor is comprised of an array of photodiodes 184 positioned adjacent each of the hybridization chambers.
• FIGS. 301 and 302 show the calibration chambers 382. They are used to calibrate the photodiodes 184 to adjust for system noise and background levels as described elsewhere in this specification. Also, positive ECL control probes 787 and negative ECL control probes 786 are placed in some of the hybridization chambers 180 for assay quality control. A variant of the calibration chambers 382 with interdigitated ECL electrodes is shown in FIG. 276.
• A humidifier 196 and humidity sensor 232 are used to control evaporation and condensation in the LOC device 785, particularly the hybridization chamber array 110. FIG. 298 shows the major components of the humidifier 196, the water reservoir 188 and evaporators 190.
• Referring to FIG. 297, the evaporation-based telltale 189 indicates whether the packaging of the device has been damaged during storage and thus whether the integrity and reliability of the microfluidic device has been compromised. During manufacture, a small drop of liquid is applied to the liquid sensor 174 at the centre of the evaporation-based telltale 189. If the package seal is breached during storage then the drop of liquid will evaporate. The presence or absence of the liquid can be detected with the liquid sensor 174 and thus indicate the integrity of the seals on the microfluidic device.
• Test Module with Microfluidic Device Having Dialysis Device, LOC and Interconnecting Cap
• A test module 11 for analysing a sample fluid containing target molecules is shown in FIG. 260. The test module 11 comprises an outer casing 13 with a receptacle 24 for receiving the sample fluid, a removable sterile sealing tape 22 to cover the receptacle 24 prior to use, a membrane seal 408 with a membrane guard 410 forming part of the outer casing 13 to reduce dehumidification within the test module while providing pressure relief from small air pressure fluctuations with the membrane guard 410 protecting the membrane seal 408 from damage, a printed circuit board (PCB) 57, a microfluidic device 783, a porous element 49, a standard Micro-USB plug 14 for power, data and control, external power supply capacitors 32, and inductor 15.
• The microfluidic device 783 has a dialysis device 784 in fluid communication with the receptacle 24 and configured to separate the target molecules from other constituents of the sample, a LOC device 785 for analysing the target molecules and a cap 51 overlaying the LOC device 785 and the dialysis device 784 for establishing fluid communication between the LOC device 785 and the dialysis device 784.
Reagent Loading and Probe Spotting System
• Reagent reservoirs 54, 56, 58, 60 and 62 (see FIG. 6) are filled with reagents and water from a robotic, droplet ejection system shown in FIGS. 63 to 66. The robotic system also spots the oligonucleotide FRET probes 186 or ECL probes 237 into the hybridization chambers 180. Droplet dispensing technology is an inexpensive spotting technique, delivers small droplets with reproducible volumes and many droplets of different solutions can be dispensed simultaneously. This allows the LOC devices to be mass produced at extremely high throughput and low cost.
• The reagent and probe spotting system includes three robotic subsystems:
• 1. Reagent dispensing robot 256 (see FIG. 63)—microvials 258 (see FIG. 64), each with a droplet dispenser 262, dispense reagents into the reservoirs 54, 56, 58, 60 and 62 and water into the water reservoir 188 (see FIG. 6). It then applies the patterned upper seal 82 (if necessary) to the cap 46.
• 2. ONEC refill robot 274 (see FIG. 65)—microvials 258 with a droplet dispenser 262 dispense probes into the reservoirs 278 of an oligonucleotide ejector chip (ONEC) 272 (see FIGS. 85 and 86). The ONEC reservoirs 278 feed an array of thermal droplet generators 271. The ONEC is then used in the third robotic subsystem, the LOC spotting robot.
• 3. LOC spotting robot 289 (schematically shown in FIG. 66)—ONEC 272 spots each hybridization chamber 180 of the LOC device 30 with probes using a thermal droplet generator 271 (see FIG. 86).
Microvials
• The reagent dispensing robot 256 and the ONEC refill robot 274 both use microvials 258 as shown schematically in FIG. 64. Probes and reagents are ordered directly from the suppliers in macrovials (not shown). Liquids are micropipetted from the macrovials into a container 259 on each of the microvials 258 to form small aliquots (typically between 282 microliters and 400 microliters) that can be refrigerated along with the macrovials until required. Each microvial 258 has a piezoelectric droplet dispenser 262 and an enclosed quality assurance chip (i.e. integrated circuit) 266 with flash memory and electrical contacts 264 for power and data transmission. The droplet dispenser 262 has a piezo-electric actuator 261 configured to eject drops with a volume between 50 picoliters and 150 picoliters for reasonably quick reagent loading while maintaining accurate drop placement.
Probe and Reagent Identification Scheme
• The quality assurance chip 266 (see FIG. 64) has digital memory used to store, identify and track the specification data characterizing the reagent or oligonucleotide probe solution within the microvial 258. At the end of the spot and load process, the data from each microvial 258, along with other loading and spotting data, is downloaded and stored in the program and data flash memory 40 of the LOC device 30 via the control microprocessor 263 controlling the reagent dispensing robot or probe dispensing robot. This data is used for diagnostic information and processing tasks, quality control and auditing.
• Referring to FIG. 87, ONEC 272 also has digital memory such as flash memory 281 in the ONEC CMOS structure 285 to store oligonucleotide specification data such as probe identities, batch numbers and so on. As with the LOC device, the ONEC refill robot 274 downloads the specification data to the ONEC flash memory 281 from the quality assurance chips 266 on the microvials 258.
• Automated information transfer minimizes the possibility of errors occurring and in the event an incorrect microvial is used, the test module reader 12 or other system component identifies this error when processing the diagnostic information.
Reagent Dispensing Robot
• A simplified top and side view of the reagent dispensing robot 256 are shown in FIGS. 63 and 259. It includes:
• • microvials 258 containing reagents and molecular biology grade water (only some of the microvials are shown)
  • mechanical/electrical rack 286 (shown only in outline) which holds and provides electrical connectivity to microvials 258
  • XY stage 268 providing a surface for detachably mounting a partial-depth sawn silicon wafer 260 or other fixed array such as separable PCB wafer 720
  • Registration camera 270 providing feedback to the control microprocessor 263 for mapping the exact location of the piezoelectric droplet dispensers 262
• The piezoelectric droplet dispensers 262 on the microvials 258 are used to dispense the reagents and water directly into the LOC device reservoirs 54, 56, 58, 60 and 62 and the humidifier water reservoir 188 respectively.
ONEC Refill Robot
• The ONEC refill robot 274 is shown in FIG. 65. It is similar to the reagent dispensing robot 256 and includes:
• • 1080 microvials 258 containing solutions of oligonucleotide probes (for the purposes of illustration, not all microvials are shown)
  • mechanical/electrical rack 286 (shown only in outline)—holds and provides electrical connectivity to microvials 258
  • oligonucleotide ejector chip (ONEC) 272—with 1080 ONEC reservoirs 278 supplying respective ejectors 287 with four ONEC thermal droplet generators 271 each (see FIGS. 85 and 86)
  • XY stage 268: holds the oligonucleotide ejector chip/s (ONEC/s) 272
  • Registration camera 270 providing feedback to the control microprocessor 263 for mapping the exact location of the thermal droplet generators 271
• The ONEC 272 is moved under the mechanical/electrical rack 286. A unique probe solution is dispensed from each microvial 258 into each ONEC reservoir 278. The ONEC 272 is then used in the probe spotting robot 273 to spot the LOC device hybridization chambers 180 with a single droplet of probe solution.
ONEC
• FIGS. 85, 86 and 87 show the ONEC 272 in detail. The ONEC 272 is an oligonucleotide spotting device for contactless spotting of probes onto a surface such as the hybridization chamber array in any of the LOC devices. It has overall dimensions of 23,296 μm×1,760 μm and is fabricated using well-established high volume photolithography fabrication techniques. Each ONEC has 1080 reservoirs 278 etched into the reservoir side 277 of a monolithic silicon substrate 275 (see FIG. 87). With more than 1000 reservoirs 278, each ONEC has the complete assay of probes needed to spot the LOC devices described herein. This allows the spotting process of each LOC to be one-step in the sense that there is no need to use more than one ONEC to spot LOCs configured for each particular analysis. The ONEC reservoirs 278 have a rectangular base (96 μm×208 μm) with a depth of 200 μm. Each ONEC reservoir 278 feeds a probe suspension to a respective ejector 287. The liquid suspension of probes fill a common chamber 282 via a pair of chamber inlets 284 (see FIG. 86). The chamber inlets 284 are two 21 μm diameter holes from the reservoir 278 to the common chamber 282. One of four thermal droplet generators 271 ejects probe droplets through nozzles 283 in the ejector side 279 into the hybridization chambers 180 by heating the actuator 280 to generate a vapor bubble. Having four thermal droplet generators 271 allows for redundancy if there is a droplet generator failure.
LOC Probe Spotting Robot
• The LOC probe spotting robot 289 is shown in FIGS. 66 and 185. For clarity, components other than the LOC device 30 on the PCB wafer 720 are not shown. It includes the following:
• • ONEC 272—oligonucleotide ejector chip with 1080 reservoirs 278, each filled with a probe solution (see FIGS. 85 and 86)
  • XY stage 268: holds the partial-depth sawn silicon LOC wafer 260 (see FIG. 66) or alternatively the separable PCB wafer 720 (see FIG. 185)
  • Registration camera 270 providing feedback to the control processor 263 for mapping the exact location of the ONEC thermal droplet generators 271
• The LOC silicon wafer 260 or the separable PCB wafer 720 is detachably mounted to a stage that can translate along two orthogonal axes. The ONEC 272 is detachably held in a chuck 265 that is closely adjacent the stage with the ejectors 287 facing the stage (see FIG. 66). The LOC silicon wafer 260 or the separable PCB wafer 720 is moved relative to the ONEC 272 by the control processor 263. Each LOC device hybridization chamber 180 is spotted by the ejectors under the operative control of the control processor 263. Using volumes less than 100 picoliters reduces the reaction times and allows the density of the hybridization chamber array to increase. Spotting low-volume probe droplets has not been previously adopted because of the difficulty associated with ejecting very small droplets precisely and reliably. Misdirected drops can fail to spot the correct chamber and may contaminate an adjacent chamber.
• The ONEC 272 can be driven to generate a range of droplet volumes. For accurate dispensing, the droplets generated by the ONEC 272 would be less than 100 picoliters. To improve the accuracy of the probes and reagents dispensed (in terms of volume and position on the LOC device), the droplets generated by the ONEC can be reduced to less than 25 picoliters, and preferably less than 6 picoliters. The ONEC 272 dispenses probe solution into the 1080 hybridization chambers 180 in droplets with volumes between 0.1 picoliters and 1.6 picoliters and a high degree of positional accuracy.
• The hybridization chamber array 110 is configured as 24 rows with 45 adjacent chambers in each row (see FIG. 52). The sample flow-path 176 extends between every second row such that the overall array has a substantially square shape for approximately uniform illumination by the LED 26. As the hybridization chamber array 110 is confined to an area less than 1500 microns by 1500 microns, the spotting accuracy of the ONEC 272 is necessarily high. A registration camera 270 is used by the control processor 263 to determine the exact position of the ONEC thermal droplet generators 271 and the droplet generator drive pulses are synchronized with the XY stage 268 via the ONEC bond-pads 276.
• The LOC probe spotting robot 273 using the ONEC 272 and camera 270 can easily spot probes onto a surface (such as the hybridization chamber array 110) at a rate greater than 100 probes per second; in the vast majority of cases at a rate greater than 1,400 probes per second. Typically, the array of droplet generators spot the probes onto the surface at a rate greater than 20,000 probes per second and in many cases, the array of droplet generators spot the probes onto the surface at a rate between 300,000 probes per second and 1,000,000 probes per second.
• The array of droplet generators lithographically fabricated on a silicon substrate allows the ONEC 272 to spot oligonucleotides onto a surface at a density far greater than existing probe spotters. ONEC 272 easily spots at a density of more than 1 probe per square millimetre. In the vast majority of cases, the spotting density is greater than 8 probes per square millimetre. In most cases, the spotting density is more than 60 probes per square millimetre, and typically the density is between 500 probes per square millimetre and 1,500 probes per square millimetre.
• The LOC probe spotting robot 273, using the ONEC 272 as a biochemical deposition device, can easily deposit biochemicals onto a surface at a rate greater than 100 droplets per second, in the vast majority of cases at a rate greater than 1,400 droplets per second. Typically, the array of droplet generators spot the droplets onto the surface at a rate greater than 20,000 droplets per second, and in many cases, the array of droplet generators spot the droplets onto the surface at a rate between 300,000 droplets per second and 1,000,000 droplets per second.
• The LOC probe spotting robot 273, using the ONEC 272 as a biochemical deposition device, can easily deposit biochemicals onto a surface at a density of more than 1 droplet per square millimetre. In the vast majority of cases, the spotting density is greater than 8 droplets per square millimetre. In most cases, the spotting density is more than 60 droplets per square millimetre, and typically the density is between 500 droplets per square millimetre and 1,500 droplets per square millimetre.
Other Embodiments and Forms
• The following sections and associated figures highlight some examples of other embodiments and forms of the invention described herein.
Detection of Analytical Targets
• DNA—The user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts and amplifies the DNA, and via hybridization to oligonucleotide probes, analyzes the DNA (see FIG. 305).
• RNA—The user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts the RNA, generates cDNA via reverse transcription, amplifies the cDNA, and via hybridization of the cDNA to oligonucleotide probes, analyzes the RNA (see FIG. 306).
• Micro RNA (miRNA)—The user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts the total RNA in the sample, ligates a 3′ adaptor to the total RNA, generates cDNA via reverse transcription of the miRNA, amplifies the cDNA using suitable 5′ and 3′ adaptors, and, via hybridization of the cDNA to oligonucleotide probes, analyzes the miRNA (see FIG. 307).
• Small Interfering RNA (siRNA)—The user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts the total RNA in the sample, ligates a 3′ adaptor to the total RNA, generates cDNA from the siRNA, amplifies the cDNA using suitable 5′ and 3′ adaptors, and, via hybridization of the cDNA to oligonucleotide probes, analyzes the siRNA (see FIG. 308).
• Small Activating RNA (saRNA)—The user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts the total RNA in the sample, ligates a 3′ adaptor to the total RNA, generates cDNA from the saRNA, amplifies the cDNA using suitable 5′ and 3′ adaptors, and, via hybridization of the cDNA to oligonucleotide probes, analyzes the saRNA (see FIG. 309).
• Mitochondrial DNA—Most of the genetic information in eukaryotic cells is found in the genomic DNA of the nucleus (nuclear DNA). However, mitochondria carry their own small genomes. This mitochondrial DNA (mtDNA) can be detected and amplified using the same techniques as those used for nuclear DNA. Mitochondria are almost always passed through the maternal line, as they are inherited from the mitochondria of the ovum (sperm mitochondria are generally destroyed during fertilization). In mammals, mtDNA contains 37 genes, but mutations and repeat sequences in non-coding regions can also be detected. mtDNA is the basis for some heredity or evolutionary studies. As mtDNA molecules are small, and because frequently large numbers of copies are present in cells, mtDNA can be detectable in degraded samples where genomic DNA has deteriorated beyond usefulness. This has seen mtDNA being used in forensics, and studies of ancient or preserved tissues. For analysis of mtDNA, the user prepares the sample and deposits it in the LOC device sample receptacle. The LOC device extracts and amplifies the mitochondrial DNA, and, via hybridization to oligonucleotide probes, analyzes the mitochondrial DNA. Probes complementary to the mtDNA locations of interest are pre-designed and loaded to enable detection of variations and new mutations (see FIG. 310).
• Proteins (by DNA tag amplification)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target proteins in the sample conjugate, as substrates, with target-specific antibody proteins linked to unique ssDNA tags. Prior to the conjugation between the target proteins and their target-specific antibodies, the antibodies interfere with any PCR amplification of their linked ssDNA tags. After conjugation between the target proteins and their target-specific antibodies, the antibodies dissociate from their respective ssDNA tags, permitting PCR amplification of the ssDNA tags. Following PCR amplification, the tags are hybridized with complementary oligonucleotide probes, and the hybridization events provide the analytical results about the target proteins (see FIG. 311).
• Proteins (direct detection)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target proteins in the sample conjugate with protein (antibody or immunoglobulin) probes spotted at specific locations in an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target proteins (see FIG. 312).
• Antibodies (immunoglobulins)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target antibodies (immunoglobulins) in the sample conjugate with probes (antigens) spotted at specific locations in an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target antibodies (immunoglobulins) (see FIG. 313).
• Antigens—The user prepares the sample and deposits it in the LOC device sample receptacle. The target antigens in the sample conjugate with antibody (immunoglobulin) probes spotted at specific locations in the form of an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target antigens (see FIG. 314).
• Antigens—The user prepares the sample and deposits it in the LOC device sample receptacle. The target antigens (haptens) in the sample conjugate with antibody probes linked to conjugation-sensitive enzymes that are spotted at specific locations in the form of an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes), as determined via the interaction of conjugation-sensitive enzymes with a suitable substrate, provide the analytical results about the target antigens (see FIG. 315).
• Sugars—The user prepares the sample and deposits it in the LOC device sample receptacle. The target sugars in the sample undergo chemical or enzymatic reactions (e.g., with glucose oxidase, glucose dehydrogenase, hexokinase, or others), with the reaction outcomes providing the analytical results about the target sugars (see FIG. 316).
• Salts—The user prepares the sample and deposits it in the LOC device sample receptacle. The target salts in the sample are analyzed via discrete LED spectroscopy, selective precipitation, and/or conductivity measurement (see FIG. 317).
• Alcohols—The user prepares the sample and deposits it in the LOC device sample receptacle. The target alcohols in the sample undergo chemical or enzymatic reactions (e.g., with alcohol dehydrogenase), with the reaction outcomes providing the analytical results about the target alcohols (see FIG. 318).
• Drugs (illicit)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target drugs in the sample conjugate with antibody (immunoglobulin) probes spotted at specific locations in an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target drugs (see FIG. 319).
• Pharmaceuticals (overdose, interactions)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target pharmaceuticals in the sample conjugate with antibody (immunoglobulin) probes spotted at specific locations in an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target pharmaceuticals (see FIG. 320).
• Toxicants (e.g., snake venoms, chemicals)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target toxicants in the sample conjugate with antibody (immunoglobulin) probes spotted at specific locations in an array on the LOC substrate. The locations of target-probe conjugation events (immunocomplexes) provide the analytical results about the target toxicants (see FIG. 321).
• Metals (e.g., arsenic, mercury, lead)—The user prepares the sample and deposits it in the LOC device sample receptacle. The target metals in the sample are chelated by chelating agents spotted at specific locations in an array on the LOC substrate or bind to proteins also spotted on the array, in either case forming antigens detectable by target-specific antibodies. The locations of antigen-antibody conjugation events (immunocomplexes) provide the analytical results about the target metals (see FIG. 322).
Sample Variants
• Blood, Blood Products and Blood Cultures—The subject's finger/heel is sterilized using an alcohol wipe and allowed to dry. A standard fingerstick/heelstick lancet device and procedure is used to obtain a whole blood sample. A single drop of blood is allowed to drip directly from the lanced finger/heel into the LOC device sample receptacle. Blood products and cultures must be obtained by conventional methods and sampled using a micropipette to collect a small volume from the storage vial and deposit it into the sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 323).
• Saliva—The subject is instructed not to eat, brush or floss at least one hour before the test. Saliva is collected directly from the subject's mouth using a micropipette and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 324).
• Cerebrospinal Fluid—The subject prepares for lumbar puncture and cerebrospinal fluid (CSF) sample is collected in a vial via a standard lumbar puncture procedure. A micropipette is used to collect the CSF from the vial and a single droplet is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 325).
• Urine—The subject is instructed to collect a midstream urine sample into a sterile container. A micropipette is used to draw a small volume of urine from the container and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 326).
• Feces—The subject is instructed to obtain a stool sample in a sterile container. A small amount of sample is removed and suspended in PCR grade water in a centrifuge tube. The fecal suspension is processed using standard protocol to produce a clarified supernatant. A micropipette is used to draw a small volume of the fecal supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 327).
• Buccal Cells—The subject is instructed not to eat, drink, or smoke for 45 minutes before the test. A cytology brush is used to obtain a buccal cell sample. The brush is placed in a tube filled with PCR grade water and agitated to displace the buccal cells from the brush. A micropipette is used to draw a small volume of the buccal cell suspension from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 328).
• Skin—The site on the subject's skin to be sampled is sterilized with an alcohol or saline wipe and allowed to dry. A skin scraping is collected from the site using a sterilized scalpel blade. The scalpel blade is then placed in placed in a Petri dish with PCR-grade water and agitated to dislodge the skin cells from the scalpel. A micropipette is used to deposit a droplet of the skin cell suspension into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 329).
• Semen—The subject is instructed to collect a semen sample in a sterile container. Following liquefaction of the semen, a micropipette is used to deposit a single drop of the semen into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 330).
• Biopsies (solid tissue)—Collect solid tissue biopsy as per a standard protocol. Solid tissue biopsies must be processed to produce a liquidized form. Processing procedure depends on the sample type and standard protocols can be followed. A micropipette is used to deposit a single drop of the liquidized biopsy sample into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 331).
• Fingerprints—A sterile scalpel blade is used to collect the cells left behind from a fingerprint. The scalpel blade is then placed in placed in a Petri dish with PCR grade water and agitated to dislodge the skin cells from the scalpel. A micropipette is used to deposit a droplet of the skin cell suspension into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 332).
• Hair—A single hair is collected from the subject or forensic site. Where possible, hair with the root bulb attached is preferred. The hair is placed in a centrifuge tube with a suitable enzyme, and processed to produce a supernatant using a standard protocol. A micropipette is used to deposit a droplet of the digested hair into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 333).
• Nails—A finger/toenail clipping is collected from the subject or from the forensic site. The nail is placed in a centrifuge tube with a suitable enzyme, and processed to produce a supernatant using standard protocol. A micropipette is used to deposit a droplet of the digested nail into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 334).
• Synovial Fluid—The subject prepares for standard arthrocentesis procedure as per a standard protocol. Synovial fluid sample is collected in a vial. A micropipette is used to collect the synovial fluid from the vial and a single droplet is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 335).
• Vaginal Swab—The subject prepares for vaginal swab as per a standard protocol. A vaginal swab is obtained using a sterile cotton swab. The swab is placed in a centrifuge tube with phosphate-buffered saline (PBS) and is processed using a standard protocol to produce a supernatant. A micropipette is used to draw a small volume of the supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 336).
• Cervical Swab—The subject prepares for cervical swab as per a standard protocol. A cervical swab is obtained using a cytology brush. The brush is placed in a centrifuge tube with phosphate-buffered saline (PBS) and is processed using standard protocol to produce a supernatant. A micropipette is used to draw a small volume of the supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 337).
• Vesicle Aspirate—The vesicle site is sterilized with saline and allowed to dry. Fluid from a vesicle is aspirated using a needle and syringe as per a standard protocols. A single drop of the vesicle aspirate is deposited from the syringe into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 338).
• Bone Marrow—The subject is prepared for bone marrow aspiration or trephine biopsy and bone marrow is aspirated using a needle and syringe as per a standard protocol. A single droplet of the bone marrow aspirate is deposited from the syringe into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 339).
• Vomitus—The vomitus sample is collected in a vial. A small volume of the sample is transferred to a centrifuge tube with phosphate-buffered saline (PBS) and is processed using standard protocol to produce a supernatant. A micropipette is used to draw a small volume of the supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 340).
• Amniotic Fluid—The subject is prepared for amniocentesis and amniotic fluid is aspirated using a needle and syringe as per a standard protocol. A single droplet of the amniotic fluid is deposited from the syringe into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 341).
• Umbilical Cord Blood—The umbilical cord is prepared and a small volume of blood is drawn into a collection vial as per a standard protocol. A micropipette is used to deposit a single drop of the cord blood into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 342).
• Breast Milk—The subject is instructed to collect a breast milk sample in a sterile container. A micropipette is used to draw a small volume of breast milk from the container and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 343).
• Sweat—The subject is instructed to wear a sweat patch for up to 14 days. Following removal of the sweat patch, the gauze pad is removed and processed using a standard protocol to produce a supernatant. A micropipette is used to draw a small volume of the supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 344).
• Fetal/Embryonic Tissue—Fetal/embryonic tissue is collected and the sample is processed in a centrifuge tube with suitable enzymes to produce a supernatant using a standard protocol. A micropipette is used to deposit a single drop of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 345).
• Placental Tissue (chorionic villus sampling)—The subject is prepared for chorionic villus sampling and chorionic villi are sampled as per a standard protocol. The sample is processed in a centrifuge tube with a suitable enzyme to produce a supernatant. A single droplet of the supernatant is deposited from the syringe into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 346).
• Vitreous Humor—The subject is prepared and undergoes a vitrectomy as per a standard protocol. The sample is placed in a centrifuge tube with a suitable enzyme and processed to produce a supernatant. A micropipette is used to deposit a single drop of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 347).
• Pleural Effusion—The subject is prepared and undergoes a thoracentesis as per a standard protocol. The sample is placed in a centrifuge tube with a suitable enzyme and processed to produce a supernatant. A micropipette is used to deposit a single drop of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 348).
• Tears—Prepare the subject for tear collection as per a standard protocol. The tear wash collection technique is used to obtain a tear sample in a micropipette. A single drop of the sample is placed into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 349).
• Wound and Ulcer Drainage—The site of the wound or ulcer is sterilized with saline and allowed to dry. Drainage is aspirated using a needle and syringe as per a standard protocol. The sample is placed in a centrifuge tube with a suitable enzyme and processed to produce a supernatant. A micropipette is used to deposit a single drop of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 350).
• Gastric Fluid—The subject is prepared and undergoes a gastric fluid aspiration as per a standard protocol. A micropipette is used to deposit a single drop of the gastric fluid into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 351).
• Pericardial Fluid—The subject is prepared and undergoes a pericardiocentesis as per a standard protocol. A micropipette is used to collect the pericardial fluid from the vial and a single droplet is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 352).
• Peritoneal Fluid—The subject is prepared and undergoes a peritoneocentesis as per a standard protocol. A micropipette is used to collect the peritoneal fluid from the vial and a single droplet is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 353).
• Sputum—A sputum sample is collected into a sterilized container. A small volume of the sample is transferred to a centrifuge tube with mucolytic agent and is processed using a standard protocol to produce a supernatant. A micropipette is used to draw a small volume of the supernatant from the tube and a single drop is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 354).
• Exhaled Breath Condensate—The subject is instructed to breathe normally into a standard condensation chamber for 5 minutes to collect exhaled breath condensate (EBC). The EBC is transferred from the chamber into a tube according to the user instructions. A micropipette is used to deposit a single drop of EBC into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 355).
• Teeth—A tooth is collected from the subject or from the forensic site. The tooth is placed in a centrifuge tube with a suitable enzyme, and processed to produce a supernatant using a standard protocol. A micropipette is used to deposit a droplet of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 356).
• Water—A water sample is collected and a micropipette is used to deposit a single drop into the LOC device sample receptacle to test for contaminants, e.g. cholera. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 357).
• Food—A food sample is collected and processed to produce a liquidized form. Processing procedure depends on the sample type and standard protocols can be followed. A micropipette is used to deposit a single drop of the liquidized sample into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 358).
• Drinks—Drink to be tested is collected in a container. A micropipette is used to deposit a single droplet into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 359).
• Plant Sap—Plant sap collected in a vial. A sample is placed in a centrifuge tube with a suitable enzyme, and processed to produce a supernatant using a standard protocol. A micropipette is used to deposit a droplet of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 360).
• Plant Matter—Plant matter collected in a bag/container. A sample is placed in a centrifuge tube with a suitable enzyme, and processed to produce a supernatant using a standard protocol. A micropipette is used to deposit a droplet of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 361).
• Soil—The soil sample is collected and crushed to a fine powder with an agate mortar and pestle. Distilled water is added and the suspension centrifuged to produce a supernatant using a standard protocol. A micropipette is used to deposit a droplet of the supernatant into the LOC device sample receptacle. The LOC device performs an analysis of the sample for the analytical targets of interest (see FIG. 362).
• Amplicon—The amplicon sample is collected in a micropipette and a droplet is deposited into the LOC device sample receptacle. The LOC device performs an analysis of the amplicon for the analytical targets of interest (see FIG. 363).
MST and Electronics Integration Level
• Monolithic Chip with MST and Electronics
• Referring to FIG. 364, the MST structures 4010 and the electronic circuitry 4011 are fabricated as an integrated unit on the same silicon substrate 4012.
Bonded Separately-Fabricated MST and Electronic Chips
• Referring to FIG. 365, the MST structures 4020 are fabricated on substrate 4021 and the electronic circuitry 4022 fabricated on substrate 4023 and the two units then bonded together.
Image Sensor Chip Coupled to MST Chip
• Referring to FIG. 366, the MST structures 4030 are fabricated on substrate 4031 and the image sensor 4032 fabricated on substrate 4033 and the two units then bonded together.
Fabrication Processes Etched Chamber
• Referring to FIG. 367, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS circuitry on the silicon wafer 5010 up to and including the deposition of passivation layer 5011.
  2. Deposit wall material 5012 and pattern it to form chamber 5013. The wall material can be made of silicon dioxide, silicon nitride, silicon oxynitride or any other suitable material known in the art.
  3. Deposit sacrificial layer 5014 and CMP it.
  4. Deposit the roof layer 5015. The wall material can be made of silicon dioxide, silicon nitride, silicon oxynitride or any other suitable material known in the art.
  5. Etch the MEMS contacts 5016 down to the top of metal layer 5017.
  6. Use electroless plating to plate the contact material into the contact openings, and CMP it to form contacts 5016.
  7. Deposit the heater material, and pattern it to form heaters 5018.
  8. Etch the roof layer 5015 to open the roof openings 5019.
  9. Ash the sacrificial layer 5014.
Suspended Heater
• Referring to FIG. 368, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS on silicon wafer 5020 to completion with the deposition of passivation layer 5021 and etching the passivation layer 5021 to open the passivation windows 5022 on the top of the CMOS top metal 5023.
  2. Deposit resist 5024 and pattern it.
  3. Deposit heater material 5025 and pattern it.
  4. Deposit resist 5026 and pattern it.
  5. Deposit wall material 5027 and pattern it to form openings 5028.
  6. Ash resists 5024 and 5026.
Bonded Heater
• Referring to FIG. 369, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS on silicon wafer 5030 to completion with the deposition of passivation layer 5031 and etching the passivation layer 5031 to open the passivation windows 5032 on the top of the CMOS top metal 5033.
• 2. Deposit heater material 5034 and pattern it.
  3. Deposit resist 5035 and pattern it.
  4. Deposit wall material 5036 and pattern it to form openings 5037.
  5. Ash resist 5035.
Resist Chamber
• Referring to FIG. 370, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS on silicon wafer 5040 to completion with the deposition of passivation layer 5041 and etching the passivation layer 5041 to open the passivation windows 5042 on the top of the CMOS top metal 5043.
  2. Deposit the heater material and pattern it to form heaters 5044.
  3. Deposit the wall resist 5045 and expose to pattern it to for chambers 5046.
  4. Deposit roof 5047 resist and expose to pattern it for openings 5048.
  5. Develop both resist layers.
Sacrificial Material
• Referring to FIG. 371, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS on silicon wafer 5050 to completion with the deposition of passivation layer 5051 and etching the passivation layer 5051 to open the passivation windows 5052 on the top of the CMOS top metal 5053.
  2. Deposit heater material 5054 and pattern it.
  3. Deposit sacrificial layer 5055 and pattern it. The sacrificial layer may be made of silicon dioxide, aluminium, polymer or any other suitable material known in the art.
  4. Deposit roof material 5056 and pattern it to form openings 5057.
  5. Etch the sacrificial layer 5055.
Fluorescence Detection Subsystem
• External Detector with Optics
• Referring to FIG. 372, the excitation light outputted by an external light source 6021 is focused by optical train consisting of lenses 60221, 60222, and 60223 onto each of the hybridization chambers 6023, and then the light emitted by the fluorophores is imaged by optical train consisting of lenses 60223, 60224, and 60225 onto the photosensor 6024. Wavelength dependent filtering is provided by the dichroic mirror 6025 and the emission filter 6026. Pinhole 6027 minimize the excitation light reaching any region outside of the hybridization chamber 6023, and pinhole 6028 limits fluorescence originating outside of the hybridization chamber 6023 impinging on the photosensor 6024.
Valve Mechanisms
• Thermal Valve with Droplet Ejection Initiation
• Referring to FIG. 373, prior to actuation, the liquid enters through the valve inlet 7041 and gets pinned at lip 7042 of opening 7047. Upon actuation, the heater 7043 is pulsed on, heating the liquid in the proximity of the heater 7043 into the liquid's film-boiling region, generating an expanding bubble that ejects the liquid out of the opening 7047. After a sufficient number of ejections which results in the liquid bridging between openings 7047 and 7045, the liquid moves via capillary action through cavity 7044 and opening 7045 to valve outlet 7046.
• Thermal Valve with Combined Surface Tension Control and Droplet Ejection Initiation
• Referring to FIG. 374, prior to actuation, the liquid enters through the valve inlet 7051 and gets pinned at lip 7052 of opening 7057. Upon actuation, the heater 7053 is initially turned on, heating the liquid in the proximity of the lip 7052. The heating lowers the surface tension and viscosity of the liquid. The lowered surface tension results in the liquid gaining a tendency to break away from the lip 7052. The heater 7053 is then pulsed on, heating the liquid in the proximity of the heater 7053 into the liquid's film-boiling region, generating an expanding bubble that ejects the liquid out of the opening 7057, with the lowered liquid viscosity facilitating the ejection process. After a sufficient number of ejections which results in the liquid bridging between openings 7057 and 7055, the liquid moves via capillary action through cavity 7054 and opening 7055 to valve outlet 7056.
Magnetic Valve
• Referring to FIG. 375, channel 7060 is normally closed off by membrane 7061, being forced by block 7062, which in turn is forced by spring 7063 with support 7064. Upon actuation, the solenoid 7065 is energized pulling the block 7062 against the springs force, opening the valve.
Externally Actuated Membrane Deformation Valve
• Referring to FIG. 376, a channel 7070 is blocked as necessary by deformation of membrane 7071 via the force applied externally on it by, for example, an actuated block 7072.
Electrowetting Valve
• Referring to FIG. 377, the liquid enters through the valve inlet 7080 gets pinned at lip 7081 prior to actuation. For actuation, a suitable voltage is applied between electrodes 7083 and 7084, changing the contact angle of the liquid meniscus 7082 around the lip 7081 forcing the liquid to break away from the lip 7081 and to move via capillary action through opening 7085 to valve outlet 7086.
Excitation Methods
• Xenon, or other noble gas, flash tube with filter
• Referring to FIG. 378, the light generated by xenon, or other noble gas, flash tube 10060 is filtered for the excitation wavelength by filter 10065 and is then directed via the optical train 10061 onto the hybridization array 10062 of the lab-on-a-chip 10064. Gathering the hybridization data is effected by pulsing the flash tube 10060 on and then acquiring the fluorescence emissions via the photodiode array 10063.
Nucleic Acid Amplification Temperature Control Techniques Flow-Through PCR
• The PCR mix is transported cyclically through the various zones of a PCR chamber with a number of different temperature zones, each corresponding to either the dsDNA denaturing, the primer annealing, or primer extension temperatures. This cycling is repeated the required number of times until the desired level of PCR amplification is obtained (see FIG. 379).
Mixer Alternatives
• AC electrophoresis
• Referring to FIGS. 380, two liquid streams enter the mixer through inlets 13020 and 13021, and mixing occurs via the interaction of charged specimens in the flow with an AC electric field, as the flow moves through the region between electrodes 13022 and 13023. The mix leaves the mixer via outlet 13024. The AC electric field between the electrodes 13022 and 13023 can be created by applying an alternating voltage to the electrodes.
Rotating Mixer
• Referring to FIG. 381, two liquid streams to be mixed enter the mixer through inlets 13050 and 13051, and out of outlet 13053. Mixing occurs when the flow interacts with the rotor 13052.
DNA Polymerases
• Any DNA polymerase capable of amplifying DNA sequences can be used in the LOC device. A few examples of these include, but are not limited to: Tag DNA polymerase isolated from Thermus aquaticus, Tfi DNA polymerase isolated from Thermus filiformis, Tfl DNA polymerase isolated from Thermus flavus, KOD NA polymerase isolated from Thermococcus kodakaraensis, Tth DNA polymerase isolated from Therms thermophilus, Pfu DNA polymerase isolated from Pyrococcus furiosus, DyNAzyme DNA polymerase isolated from Therms brockianus, Phire DNA polymerase, a modified form of Taq polymerase, Primestar DNA polymerase, a modified form of Taq polymerase, Vent DNA polymerase isolated from Thermococcus litoralis, DNA polymerase isolated from Pyrococcus abyssi, Chy DNA polymerase isolated from Carboxydothermus hydrogenoformans, Phusion DNA Polymerase which is a fusion of Pfu DNa polymerase and the small DNA-binding protein Sso7d isolated from Sulfolobus solfataricus, Phage DNA polymerase isolated from bacteriophages such as bacteriophage lambda, bacteriophage T7, bacteriophage M13, or bacteriophage RB69, Herculase DNA Polymerase which is a fusion of Pfu DNA polymerase and a high affinity double-stranded DNA binding domain, including an element which converts dUTP to dUMP.
• The type of DNA polymerase selected depends on the PCR application. For example, the polymerase isolated from Pyrococcus abyssi, is suitable for high fidelity applications since the polymerase has proofreading ability, whereas Chy is suitable for reverse transcriptase PCR.
• The procedure for using these example polymerases and others in the test module is as follows. The DNA template is prepared, the PCR buffer, dNTPs, primers, and the polymerase suited to the PCR application are added, and PCR temperature cycling is initiated (as described in detail earlier).
Integration Level of the Subsystem for Reagent Storage and Large-Scale Fluid Transport In Reservoirs Etched in Substrate
• Referring to FIG. 382, the reagents are stored in reservoirs such as 16020 etched in substrate 16022 containing other functional lab-on-a-chip subunits, and the reagents utilize channels such as 16021 also etched in the substrate 16022 for transport. MST is shown as 16023.
• In Reservoirs in a Base which Connects to Channels Etched in Substrate
• Referring to FIG. 383, the reagents are stored in reservoirs such as 16030 fabricated in a base 16032 bonded to the substrate 16033 containing other functional lab-on-a-chip subunits, and the reagents utilize channels such as 16031 etched in the substrate 16033 for transport. MST is shown as 16034.
In-Situ Probe Synthesis
• The oligonucleotide probes can be synthesized directly in the LOC device hybridization chambers using, for example, a solid-phase synthesis process based on phosphoramidite chemistry, with the reagents and precursors spotted into the hybridization chambers. This process begins by spotting a starter compound to bind the oligonucleotide strand to the chamber base. The nucleotides are then printed one-by-one in the 3′ to 5′ direction according to the pre-programmed sequence in a cycle of four chemical reactions per base as follows:
• 1. Deprotection—The first base is at first inactive because the active 5′ hydroxyl site is protected with a trityl group. To add the next base, a deprotecting agent (e.g. dichloroacetic acid) is spotted into the chamber which activates the 5′-hydroxyl group. The chambers are then washed to remove excess acid and by-products.
  2. Coupling—The next base is spotted and activated by tetrazole which removes the protective (iPr)2N group on the phosphate group. The chambers are then washed to remove unbound base and by-products.
  3. Capping—About 1% of the 5′-hydroxyl group groups do not react with the new base and need to be blocked from further reaction. This is done by spotting a protective group in the form of acetic anhydride and 1-methylimidazole which react with the free 5′-hydroxyl group groups via acetylation. The chambers are then washed to remove excess reagents.
  4. Stabilization—The phosphite linkage between the first and second base is stabilised by making the phosphate group pentavalent. This is achieved by spotting iodine and water which leads to the oxidation of the phosphite into phosphate. The chambers are then washed to remove excess reagents. This cycle of four steps is repeated for each base until all bases have been added. The process finishes with a final deprotection step, wash and vacuum dry (see FIG. 384).
Diagnostic and Analytical Objectives
• A general flow-chart for the diagnostic and analytical objectives for the LOC device is shown in FIG. 385, but a few examples of the individual applications are provided below.
Infectious Disease Detection
• The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample for infectious disease pathogens. The suspected infectious disease agent may be a bacteria, virus, fungus, protozoan, endoparasite or ectoparasite. If the result is positive the analysis is complete, but if the result is negative and still other suitable sample types can be obtained, then an analysis on the new sample types can be performed. If the result for every possible sample type is negative then it can be concluded that the LOC analysis has failed to return a positive result.
Identification of Hereditary Disorders
• The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample for hereditary disorders DNA or RNA signatures. The nucleic acid signatures for such disorders can be found within nuclear or mitochondrial nucleic acids.
• Disorders associated with mitochondrial DNA include: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS, associated with genes MT-ND1, MT-NDS, MT-TH, MT-TL1, and MT-TV); Leber hereditary optic neuropathy (associated with genes MT-ND1, MT-ND4, MT-ND4L, and MT-ND6); nonsyndromic hearing loss (associated with a large number of genes, ACTG1, ATP2B2, CDH23, CLDN14, COCH, COL11A2, DFNA5, DFNB31, DFNB59, ESPN, EYA4, GJB2, GJB3, GJB6, KCNQ4, LHFPL5, MT-RNR1, MT-TS1, MYO15A, MYO1A, MYO6, MYO7A, OTOF, PCDH15, POU3F4, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP, USH1C, and WFS1); myoclonic epilepsy with ragged-red fibers (associated with genes MT-TK, MT-TL1, MT-TH, and MT-TS1); and neuropathy, ataxia, and retinitis pigmentosa (NARP, associated with gene MT-ATP6).
Genetic Traits
• The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample's DNA, or RNA, with the objective of the identification of organism's genetic traits. The DNA analyzed can be either nuclear or mitochondrial DNA.
• In the case of mitochondrial DNA, the traits which can be detected include mutations arising in the organism being tested and inherited mitochondrial characteristics. Mitochondrial DNA traits can be of clinical or diagnostic interest, such as those responsible for mitochondrial disorders; they may also be used in forensic or paleontological applications, since mitochondrial DNA is often able to be amplified successfully from degraded sample materials. Mutations, whether they are new or inherited, can also be used for evolutionary (inheritance tree) studies.
Metabolic Disorders
• Metabolic disorders may be detected via changes in RNA expression, protein levels or salt imbalances in the body. The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an RNA expression analysis, proteomic analysis and/or an analysis of the salt content of the sample for diagnosis of metabolic disorders.
Forensics
• The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample's DNA, alcohol, drug, pharmaceutical, toxicant, and metal contents with respect to forensic objectives. DNA used for identification can be nuclear or mitochondrial. Mitochondrial DNA is relevant to forensics because it can often be successfully amplified and detected in degraded sample material.
Biological Warfare
• The user obtains a suitable sample type and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample's genomic DNA, genomic RNA, protein, and toxin contents with the objective of the identification of possible biological warfare agents.
Other Applications
• A few examples of potential other applications of the LOC device include, but are not limited to: toxicant detection and identification, drug screening, species identification, IVF screening (blastomere screening), autoimmune disease detection (via detection of autoantibodies), food/water contamination screening, environmental testing, animal husbandry, domestic animal breeding, drug testing, paternity screening, and oncology screening (via screening miRNA expression and other relevant markers).
• For any of the applications, the user obtains a suitable sample type or sample types and deposits it in the LOC device sample receptacle. The LOC device performs an analysis of the sample\s for the target\s of interest (see FIG. 385).
Heater Configuration Under Chamber
• Referring to FIG. 386, the heater 22012 can be located under the chamber 22011 where the mixture 22010 is heated to the required temperature by the heater 22012.
Inside Chamber
• Referring to FIG. 387, the heater 22022 may be located inside the chamber 22021 where the mixture 22020 is heated to the required temperature by the heater 22022.
Beside Chamber
• Referring to FIG. 388, the heater 22042 may be positioned beside the chamber 22041 where the mixture 22040 is heated to the required temperature by the heater 22042.
Methods of Spotting the LOCs
• Micropositioned Array of Microvials with Droplet Generators into PCB Array of LOC devices
• Referring to FIG. 389, an array 23030 of microvials 23031 eject picoliter drops of target material to specific hybridization chamber sites of an array of LOC devices 23032 mounted accurately on a separable PCB array 23033.
• Pipelined Array of Individual Microvials with Droplet Generators into LOC devices
• Referring to FIG. 390, a pipelined array 23040 of individual microvials 23041 with droplet generators eject picoliter drops of target material to specific hybridization chamber sites of LOC devices 23042 brought into position by, for example, a conveyer system 23043.
• Array of Microvials with Droplet Generators into Micro-positioned LOC devices
• Referring to FIG. 391, an array 23050 of individual microvials 23051 eject picoliter drops of target material to specific hybridization chamber sites of LOC devices 23052 micro-positioned by, for example, a micrometer stage 23053.
• Microprobe Array Spotting into LOC devices
• Referring to FIG. 392, a microprobe array 23060 has of a number of microprobes such as 23061 to spot picoliter quantities of target material to specific hybridization chamber sites of LOC devices.
Fluidic Propulsion External Pump
• Referring to FIG. 393, the liquid is propelled within the fluidic structures of the LOC device 24021 via an external pump 24020 connected to the LOC device's fluidic structures via connector 24022. The pump 24020 shown here, as an example, is a peristaltic pump.
Downstream Jetpump
• Referring to FIG. 394, the liquid is propelled within the fluidic structures of the LOC device from location 24030 to location 24031, for example, via an upstream jetpump 24032, where the jetpump is an upstream droplet generator ejecting upstream sacrificial portions of the liquid mix, with the droplets 24033 of this sacrificial mix being aimed at an absorbent waste pad 24034.
On-Chip Peristaltic Flexure Array
• Referring to FIG. 395, the liquid is propelled within the fluidic structures of the LOC device by an on-chip peristaltic flexure array consisting of a linear array of thermal bend actuated paddles 24040. The paddles get deflected downward, via the expansion of an electrothermally heated layer 24041, displacing the liquid. The cyclic actuation of the paddles provides the required propulsion of the liquid from array inlet 24042 to array outlet 24043.
On-Chip Rotary Pump
• Referring to FIG. 396, the liquid is propelled within the fluidic structures of the LOC device by an on-chip rotary pump which propels the liquid from pump inlet 24050 to pump outlet 24051 via an electromagnetically rotated impeller 24052.
External Flexure Peristaltic Pump
• Referring to FIG. 397, the liquid is propelled via peristalsis with external actuators, of, for example, the rotary type represented by 24062. The membrane 24063 is deformed in a cyclic manner to force the liquid from the pump inlet 24060 to its outlet 24061.
On-Chip Bubble Peristaltic Pump
• Referring to FIG. 398, the liquid is propelled via peristalsis with heater elements 24070 generating bubbles, represented as 24071, in a cyclic manner to force the liquid from the pump inlet 24072 to its outlet 24073.
Bubble Squeeze Pump
• Referring to FIG. 399, the liquid is propelled via the impulse generated via a heater element 24080 generating a sequence of bubbles to force the liquid from the pump inlet 24081 to its outlet 24082 with rectifying valves 24083 and 24084 assuring the correct net flow direction.
Electrophoresis
• Referring to FIG. 400, the charged specimens in the solution are propelled via electrophoresis due to their interaction with the electric field created by the application of a suitable voltage between the electrodes 24090 and 24091.
Dialysis Designs Pathogens Optimally Selected by Shape
• Referring to FIG. 401, the blood enters through inlet 25040 and moves through the channel 25041 towards the outlet 25044. The channel 25041 is separated from a second channel 25042 by a wall 25047 with apertures 25048. The apertures 25048 are of a specific geometric shape that facilitates the crossing of the desired pathogens of that geometry, and any cell or molecule smaller than the threshold size, into the second channel 25042. The second channel 25042 is separated from a third channel 25043 by a wall 25049 with apertures of a different size and geometry 250492. These apertures 250492 are of a predetermined size which prevent the desired pathogens being able to cross into the third channel 25043, but enable the smaller cells or molecules to pass through the apertures 250492 into the third channel where they travel along the channel and are collected at the outlet 25046 to be treated as waste. All the cells too large to cross through the first apertures 25048, stay in the first channel 25041 until they exit it via outlet 25044 and are also treated as waste. The desired pathogens move along the second channel 25042 and are collected at the outlet 25045 to be further processed and analyzed.
Nucleated Cells Selectively Retained in Sample
• Referring to FIG. 402, the sample enters through the inlet 25050 and moves through the first channel 25051 towards the outlet 25053. The first channel 25051 is separated from a second channel 25052 by a wall 25055 with apertures 25056. The apertures 25056 are of a predetermined size so that smaller constituents such as pathogens can cross into the second channel 25052. The smaller constituents then move along the second channel and are collected at the outlet 25054 to be treated as waste. The nucleated cells, too large to cross through the apertures 25056, stay in the first channel 25051 until they exit it via outlet 25053 to be further processed and analyzed.
• Sample types sorted by this type of dialysis section may include any of the sample types previously described in order to selectively retain leukocytes, stem cells, cervical cells, ova and spores.
Poly(Dimethylsiloxane) (PDMS) Utilized for Wall Material
• Referring to FIG. 403, the microfluidic device is fabricated according to the following process:
• 1. Process the CMOS on silicon wafer 27050 to completion with the deposition of passivation layer 27051 and etching the passivation layer 27051 to open the passivation windows 27052 on the top of the CMOS top metal 27053.
  2. Deposit the heater material and pattern it to form heaters 27054.
  3. Separately form the walls 27055 from poly(dimethylsiloxane) (PDMS) with voids for chambers 27056.
  4. Form the roof 27057 from PDMS with voids for roof openings 27058; plasma-activate the PDMS layer; and press-bond it to the top of the layer with walls 27055.
  5. Plasma-activate the stack with walls 27055 and roof 27057, and thermally bond it to the top of the wafer.
Photosensor Shunting Photosensor Reset by Charge Transfer
• Referring to FIG. 404, the charge signals in the photosensor wells are removed via a deliberate charge transfer process analogous to the steps performed during sensor readout. In this scheme, any charge signals which are not due to the desired emission from the sample are rejected from the data for analysis. This rejection of unwanted signals is achieved by running a first dummy exposure cycle prior to a 2^nd analytical exposure cycle which measures emission from the sample. The charge transfer steps at the end of the 1^st cycle remove the charge which may have been generated in the photosensor from sources such as dark current or the excitation light from a fluorescence measurement. This will result in higher signal quality for the analytical data.
• It will be obvious to one skilled in the art that the photosensor could be a charge coupled device (CCD) or a CMOS Image Sensor (CIS).
Photosensor Reset by Interline Charge Transfer
• Referring to FIG. 405, the charge signals in the photosensor wells are removed via an interline charge transfer process analogous to the steps performed during sensor readout. In this scheme, any charge signals which are not due to the desired emission from the sample are rejected from the data for analysis. This rejection of unwanted signals is achieved by running a first dummy exposure cycle prior to a 2^nd analytical exposure cycle which measures emission from the sample. The interline charge transfer steps which are the start of readout out the charge from the 1^st cycle remove the charge from the sensor wells which may have been generated in the photosensor from sources such as dark current or the excitation light from a fluorescence measurement. The interline charge transfer moves the charge from sensing wells to parallel rows of non-sensing pixels in a single operation lasting one or a few clock cycles. After the interline transfer steps, the photosensor is ready to begin the 2nd (analytical) exposure cycle. The remaining pixel-by-pixel of the 1^st exposure readout (or simply charge dump), which takes several clock steps per pixel along each row, then proceeds in parallel with the 2^nd exposure integration in the sensing pixels.
• The charge transfer scheme will result in higher signal quality for the analytical data. The interline transfer variant significantly decreases the time required for the charge transfer of the dummy exposure signal.
• It will be obvious to one skilled in the art that the photosensor could be a charge coupled device (CCD) or a CMOS Image Sensor (CIS).
• LOC without Controller
• The lab-on-a-chip (LOC) device 31020 may utilize an external controller 31022, powered by an external power supply 31021, for controlling the operation of the LOC device and analysing the output of the LOC, as shown in FIG. 406. In this configuration, the LOC CMOS contains only logic for operating sensors 31024 and activating drivers 31026. The CMOS logic 31025 is controlled by the external controller 31022. The CMOS logic 31025 performs some functions independent of the external controller 31022, such as temperature stabilization at a predetermined temperature set point, while for other functions information from on-chip sensors is fed back to the external controller 31022 which then activates on-chip drivers via the CMOS logic 31025. The drivers 31026 operate internal functional units 31027 integrated into the LOC device, such as heaters and ECL electrodes, and also functional units external to the LOC device 31028, such as an excitation LED and output devices. The output device, which may consist simply a series of LEDs or LCD display, may be required to indicate to the user the device status, for example, in progress or complete, and the outcome of the analysis. The controller 31022 is also connected to peripheral devices 31023, such as a display screen 18, which may be used for input and output of information.
CONCLUSION
• The devices, systems and methods described here facilitate molecular diagnostic tests at low cost with high speed and at the point-of-care.
• The system and its components described above are purely illustrative and the skilled worker in this field will readily recognize many variations and modifications which do not depart from the spirit and scope of the broad inventive concept.

Claims (20)

1. A microfluidic device for detecting target nucleic acid sequences in a sample, the microfluidic device comprising:

an array of hybridization chambers, each containing electrochemiluminescent (ECL) probes for hybridization with the target nucleic acid sequences, and electrode pairs for receiving an electrical pulse, the ECL probes being configured to emit a photon of light when hybridized with one of the nucleic acid targets and activated by one of the electrodes; wherein,

the hybridization chambers each have a volume less than 900000 cubic microns.

2. A microfluidic device according to claim 1 wherein the hybridization chambers each have a volume less than 200000 cubic microns.

3. A microfluidic device according to claim 1 wherein the hybridization chambers each have a volume less than 40000 cubic microns.

4. A microfluidic device according to claim 1 wherein the hybridization chambers each have a volume less than 9000 cubic microns.

5. A microfluidic device according to claim 1 wherein the hybridization chambers each have a wall section that is optically transparent to the light emitted by the ECL probes.

6. A microfluidic device according to claim 5 further comprising a photosensor for detecting the light emitted by the ECL probes, wherein the wall section is positioned between the ECL probes and the photosensor.

7. A microfluidic device according to claim 6 further comprising a supporting substrate for the photosensor and the array of hybridization chambers wherein the photosensor is between the hybridization chambers and the supporting substrate, and the wall section is a layer incorporating silicon dioxide.

8. A microfluidic device according to claim 1 wherein the probes each have an ECL luminophore that emits a photon when in an excited state, and a functional moiety for quenching photon emission from the ECL luminophore by resonant energy transfer.

9. A microfluidic device according to claim 8 wherein the probes are configured such that the functional moiety for quenching photon emission from the ECL luminophore is further from the ECL luminophore when the probe forms a probe-target hybrid.

10. A microfluidic device according to claim 1 further comprising CMOS circuitry configured to provide an electrical pulse to the electrodes.

11. A microfluidic device according to claim 10 wherein the electrical pulse has a duration less than 0.69 seconds.

12. A microfluidic device according to claim 11 wherein the electrical pulse has a current of 0.1 nanoamperes to 69.0 nanoamperes.

13. A microfluidic device according to claim 12 wherein the electrodes have an anode and a cathode each having fingers configured such that the fingers of the anode are interdigitated with the fingers of the cathode.

14. A microfluidic device according to claim 13 wherein the anode and the cathode are separated by a dielectric gap between 0.4 microns and 2.0 microns wide.

15. A microfluidic device according to claim 10 further comprising a cap having reagent reservoirs for addition to the sample prior to detection of the target sequences, wherein the electrodes and the probes are between the cap and the CMOS circuitry.

16. A microfluidic device according to claim 15 wherein the reagent reservoirs each have an outlet valve for retaining liquid reagent in the reservoir until reagent addition to the sample is required.

17. A microfluidic device according to claim 7 wherein the layer is less than 1600 microns thick.

18. A microfluidic device according to claim 6 wherein the photosensor is an array of photodiodes positioned in registration with the hybridization chambers such that each of the hybridization chambers corresponds to one of the photodiodes respectively.

19. A microfluidic device according to claim 18 wherein the photodiodes have a planar active surface area for receiving the light from the luminophore, each of the active surface areas being coplanar, and the electrodes are a layer of conductive material patterned to form the separate anodes and cathodes, the layer extending in a plane parallel to that of the active surface areas of the photodiodes.

20. A microfluidic device according to claim 18 wherein one of the electrodes in each of the electrode pairs is a working electrode which causes oxidation or reduction of the luminophore to generate an excited species that emits a photon, the working electrode being positioned such that the probes are between the photodiode and the working electrode.

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