US20110312076A1 - Microfluidic test module with flexible membrane for internal microenvironment pressure-relief - Google Patents
Microfluidic test module with flexible membrane for internal microenvironment pressure-relief Download PDFInfo
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- US20110312076A1 US20110312076A1 US13/149,943 US201113149943A US2011312076A1 US 20110312076 A1 US20110312076 A1 US 20110312076A1 US 201113149943 A US201113149943 A US 201113149943A US 2011312076 A1 US2011312076 A1 US 2011312076A1
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Definitions
- the present invention relates to diagnostic devices that use microsystems technologies (MST).
- MST microsystems technologies
- the invention relates to microfluidic and biochemical processing and analysis for molecular diagnostics.
- 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).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- 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.
- a nucleic acid based test has four distinct steps:
- 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.
- the specimen is mixed with an agent such as ethylenediaminetetraacetic acid (EDTA) prior to purification and concentration.
- EDTA ethylenediaminetetraacetic acid
- 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.
- 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.
- 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.
- 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.
- PCR polymerase chain reaction
- 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:
- pair of primers short single strands of DNA with around 10-30 nucleotides complementary to the regions flanking the target sequence
- DNA polymerase a thermostable enzyme that synthesizes DNA
- deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotides that are incorporated into the newly synthesized DNA strand
- PCR typically involves placing these reactants in a small tube ( ⁇ 10-50 microliters) 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.
- 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.
- 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.
- 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
- 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.
- Real-time PCR or quantitative PCR, is used to measure the quantity of a PCR product in real time.
- 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 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.
- cDNA complementary DNA
- 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 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.
- nickase enzymes which do not cut DNA in the traditional manner but produce a nick on one of the DNA strands, such as N. A1w1, N. BstNB1 and M1y1, are useful in this reaction.
- SDA has been improved by the use of a combination of a heat-stable restriction enzyme (Ava1) and heat-stable Exo-polymerase (Bst polymerase). This combination has been shown to increase amplification efficiency of the reaction from 10 8 fold amplification to 10 10 fold amplification so that it is possible using this technique to amplify unique single copy molecules.
- Ava1 heat-stable restriction enzyme
- Bst polymerase heat-stable Exo-polymerase
- TMA Transcription Mediated Amplification
- NASBA Nucleic Acid Sequence Based Amplification
- RNA polymerase 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.
- this primer hybridizes to the target ribosomal RNA (rRNA) at a defined site.
- rRNA target ribosomal RNA
- Reverse transcriptase creates a DNA copy of the target rRNA by extension from the 3′ end of the promoter primer.
- RNA in the resulting RNA:DNA duplex is degraded by the RNase activity of the reverse transcriptase if present or the additional RNase H.
- 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.
- RPA Recombinase Polymerase Amplification
- 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.
- HSA Helicase-dependent amplification
- a DNA helicase enzyme to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase.
- 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.
- dNTPs free deoxyribonucleoside triphosphates
- RCA Rolling Circle Amplification
- 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.
- the polymerase generates many thousands of copies of the circular template, with the chain of copies tethered to the original target DNA.
- 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 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.
- 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 10 9 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.
- the amplified product 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.
- a DNA size marker a DNA ladder
- 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.
- electrochemiluminescence ECL
- light is produced by luminophore molecules or complexes upon stimulation with an electric current.
- 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.
- a photosensor which is sensitive to the emission wavelength of the ECL species being employed.
- 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.
- 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.
- 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.
- fluorescent probes 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
- a point-of-care technology serving the physician's office, the hospital bedside or even consumer-based, at home, would offer many advantages including:
- LOC Lab-on-a-chip
- GBS001.1 This aspect of the invention provides a microfluidic device comprising:
- MST microsystems technologies
- the 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.
- 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.
- 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.
- the reservoir is in fluid communication with the channel via the MST layer and at least two of the fluidic connections.
- the channel and the reservoir are formed in a unitary layer of material.
- the channel is formed in one surface of the layer such that an outer surface of the MST layer encloses the channel.
- 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.
- 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.
- 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.
- the fluid is blood and the cap has a waste reservoir for collecting erythrocytes removed from the blood by the dialysis section.
- 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.
- the microfluidic device also has a nucleic acid amplification section for amplifying nucleic acid sequences in the fluid.
- the cap has a lysis reagent reservoir for containing a lysis reagent to lyse cells in the blood and release nucleic acid sequences within.
- 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.
- PCR polymerase chain reaction
- the cap has a polymerase reservoir containing a polymerase for mixing with the fluid prior to amplifying the nucleic acid sequences.
- 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.
- the probe nucleic acid sequences are contained in fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- the microfluidic device also has a plurality of heaters for controlling the temperature of the fluid.
- 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.
- test module comprising:
- a receptacle for receiving an unprocessed biological sample through an opening
- microfluidic device for processing the biological sample, the microfluidic device having a sample inlet;
- the receptacle is configured for fluid communication with the sample inlet such that the biological sample flows to the sample inlet by capillary action.
- the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
- the test module also has an outer casing for hand held portability.
- 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.
- LOC lab-on-a-chip
- MST microsystems technologies
- 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.
- the outer casing has a lancet for pricking a patient to obtain a blood sample for insertion in the sample inlet.
- the lancet is movable between a retracted and extended position
- 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.
- the cap is configured to draw fluid flow through the cap channels by capillary action.
- 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.
- 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.
- the constituents within the sample include cells and one of the two fluid flows has only cells smaller than a predetermined threshold size.
- 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.
- the cap has a laminar structure with the cap channels formed in an outer layer and the reservoir formed in the opposing outer layer.
- 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.
- the cap has a waste reservoir for collecting one of the two fluid flows.
- 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.
- the LOC device has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample fluid.
- PCR polymerase chain reaction
- 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.
- the cap has a PCR reagent reservoir containing dNTPs, buffer and primers for mixing with the fluid prior to amplifying the nucleic acid sequences.
- 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.
- test module comprising:
- 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.
- LOC lab-on-a-chip
- MST microsystems technologies
- 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.
- the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
- the MST layer incorporates heaters for heating fluid in the MST channels.
- 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.
- the outer casing has a lancet for pricking a patient to obtain a blood sample to insertion in the sample inlet.
- the lancet is movable between a retracted and extended position
- 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.
- the cap is configured to draw fluid flow through the cap channels by capillary action.
- 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.
- 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.
- the constituents within the sample include cells and one of the two fluid flows has only cells smaller than a predetermined threshold size.
- 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 cap has a laminar structure with the cap channels formed in an outer layer and the reservoir formed in the opposing outer layer.
- 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.
- the cap has a waste reservoir for collecting one of the two fluid flows.
- 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.
- the LOC device has a polymerase chain reaction (PCR) section for amplifying nucleic acid sequences in the sample fluid.
- PCR polymerase chain reaction
- 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.
- the cap has a PCR reagent reservoir containing dNTPs and primers for mixing with the fluid prior to amplifying the nucleic acid sequences.
- 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:
- MST microsystems technologies
- a photosensor adjacent the MST layer for detecting photons emitted from the MST layer.
- 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.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- the photodiodes in the array of photodiodes are positioned in registration with each of the hybridization chambers respectively.
- 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.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from: a ruthenium chelate;
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
- 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.
- the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action
- 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.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- the photodiodes are less than 249 microns from the FRET probes.
- 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.
- 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
- the flow-path is configured to draw the fluid sample from the sample inlet to the probes by capillary action.
- the microfluidic device also has:
- MST microsystems technologies
- the probes are configured to hybridize the target nucleic acid sequences to form probe-target hybrids that emit photons.
- 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.
- PCR polymerase chain reaction
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- 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.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
- 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.
- the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action
- 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.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- the photodiodes are less than 249 microns from the FRET probes.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- MST microsystems technologies
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- the microfluidic device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
- 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.
- the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action
- 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.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- the photodiodes are less than 249 microns from the FRET probes.
- 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:
- MST microsystems technology
- a temperature sensor for sensing the temperature at the array of hybridization chambers
- a hybridization heater for heating the array of hybridization chambers
- the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating a photosensor.
- the photosensor is an array of photodiodes positioned in registration with each of the hybridization chambers respectively.
- 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.
- the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
- 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.
- the photodiodes are less than 249 microns from the hybridization chambers.
- the hybridization chamber has a volume less than 900,000 cubic microns.
- the hybridization chamber has a volume less than 200,000 cubic microns.
- the hybridization chamber has a volume less than 40,000 cubic microns.
- the hybridization chamber has a volume less than 9000 cubic microns.
- the CMOS derives a single result from the photodiodes corresponding to the hybridization chambers that contain identical probes.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
- 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.
- the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
- PCR polymerase chain reaction
- 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.
- 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 LOC device has a cap in which the reagent reservoirs are defined.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- MST microsystems technology
- the hybridization chamber has a wall section that is optically transparent to the fluorescence signal.
- the microfluidic device also has a photosensor wherein the wall section is between the probes and the photosensor.
- the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating the photosensor.
- 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.
- 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.
- 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.
- 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.
- the photosensor positioned less than 249 microns from the hybridization chambers.
- the wall section is opaque to the excitation light.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
- 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.
- the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
- PCR polymerase chain reaction
- 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.
- the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
- the reagent reservoirs each have a volume less than 20,000,000 cubic microns.
- the microfluidic device also has a cap in which the reagent reservoirs are defined.
- 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.
- 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:
- MST microsystems technology
- the mass of the probes in each of the probe spots is less than 270 picograms.
- the mass of the probes in each of the probe spots is less than 60 picograms.
- the mass of the probes in each of the probe spots is less than 12 picograms.
- the mass of the probes in each of the probe spots is less than 2.7 picograms.
- 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.
- the microfluidic device also has CMOS circuitry positioned between the supporting substrate and the MST layer, the CMOS circuitry incorporating the array of photodiodes.
- each of the hybridization chambers has a volume less than 200,000 cubic microns.
- each of the hybridization chambers has a volume less than 40,000 cubic microns.
- each of the hybridization chambers has a volume less than 9000 cubic microns.
- 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.
- 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.
- 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.
- the photosensor is positioned less than 249 microns from the hybridization chambers.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers have an optical window positioned to expose the FRET probes to an excitation light.
- 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.
- the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences in the fluid.
- PCR polymerase chain reaction
- 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.
- the microfluidic device also has reagent reservoirs collectively containing all reagents for processing the fluid.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- MST microsystems technologies
- each of the hybridization chambers has a volume less than 900,000 cubic microns.
- each of the hybridization chambers has a volume less than 200,000 cubic microns.
- each of the hybridization chambers has a volume less than 40,000 cubic microns.
- each of the hybridization chambers has a volume less than 9000 cubic microns.
- the microfluidic device also has CMOS circuitry between the MST layer and the supporting substrate; wherein,
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
- 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.
- PCR polymerase chain reaction
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the sensors after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- 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.
- 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.
- the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action
- 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.
- 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
- MST microsystems technologies
- PCR polymerase chain reaction
- 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.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- 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
- the elongate heater element extends parallel with the longitudinal extent of the PCR chamber.
- 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.
- the microchannel is configured to draw liquid containing the nucleic acid sequences from the PCR inlet to the PCR outlet by capillary action.
- 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.
- each of the channel section along each of the wide meanders has a plurality of the elongate heaters.
- the plurality of elongate heaters are positioned end to end along the channel section.
- each of the plurality of elongate heaters is independently operable.
- the microfluidic device also has at least one temperature sensor for feedback control of the elongate heaters.
- 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.
- 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 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:
- each of the hybridization chambers having a chamber inlet for fluid communication between the sample flow-path and the probes;
- 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.
- the chamber inlet defines a tortuous flow-path.
- the tortuous flow-path has a serpentine configuration.
- the CMOS circuitry has an array of photodiodes for detecting hybridization of the probes.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the hybridization chambers each have an optical window to expose the FRET probes to an excitation light.
- 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.
- PCR polymerase chain reaction
- the sample flow-path is configured to draw the sample fluid through the PCR section and into the hybridization chambers by capillary action.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- 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 chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
- 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.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- 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;
- CMOS circuitry on the substrate, the CMOS circuitry having a photosensor for generating an output signal in response to the fluorescence signal;
- the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photosensor.
- the photosensor is a photodiode and the probe is spaced less than 249 microns from the photodiode.
- the microfluidic device also has an array of the probes and a corresponding array of the photodiodes.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
- PCR polymerase chain reaction
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the probe array has more than 1000 probes.
- the time delay is between 100 picoseconds and 10 milliseconds.
- 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.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- 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.
- 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.
- 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.
- 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.
- 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 having a reporting fluorophore for emitting a fluorescence signal in response to an excitation light
- 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.
- 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.
- 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.
- 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.
- the detection photodiodes are larger than the trigger photodiodes.
- the microfluidic device also has:
- MST microsystems technologies
- CMOS circuitry between the MST layer and the supporting substrate, the CMOS circuitry incorporating the array of detection photodiodes and the trigger photodiodes;
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the microfluidic device also has a polymerase chain reaction (PCR) section for amplifying the target nucleic acid sequences.
- PCR polymerase chain reaction
- 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.
- 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.
- 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.
- the CMOS circuitry has memory storing identity data for the different FRET probe types.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- 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 chambers each have a hybridization heater for providing thermal energy for hybridization, the hybridization heaters each being operatively controlled by the CMOS circuitry.
- 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.
- the fluid flow-path is configured to draw fluid from the PCR section to the liquid end point sensor by capillary action
- 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 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 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;
- the 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;
- the CMOS circuitry is configured to activate the photodiode when the trigger photodiode indicates the excitation light has deactivated.
- the CMOS circuitry provides a time delay between deactivation of the excitation light and activation of the photodiode.
- the microfluidic device also has an array of the hybridization chambers, each containing probes and a corresponding array of the photodiodes.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
- PCR polymerase chain reaction
- the hybridization chambers each have an optical window to expose the FRET probes to the excitation light.
- the CMOS circuitry has memory for identity data for the FRET probes.
- the probe array has more than 1000 probes.
- the time delay is between 100 picoseconds and 10 milliseconds.
- the microfluidic device also has:
- At least one calibration source configured to generate a calibration emission
- the CMOS circuitry has a differential circuit for subtracting the calibration photodiode output from the detection photodiode output.
- the microfluidic device also has:
- the calibration sources are calibration probes without a fluorophore.
- 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.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- each of the calibration chambers are surrounded by a three-by-three square of the hybridization chambers.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- 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;
- the 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.
- the microfluidic device also has an array of hybridization chambers, each of the photodiodes corresponding to one of the hybridization chambers respectively.
- the hybridization chambers each have a volume less than 900,000 cubic microns.
- the CMOS circuitry provides a time delay between sensing deactivation of the excitation light and sensing the fluorescence signal.
- the photodiodes are spaced less than 249 microns from the probes in the corresponding hybridization chamber.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
- PCR polymerase chain reaction
- the hybridization chambers each have an optical window to expose the FRET probes to the excitation light.
- the CMOS circuitry has memory for identity data for the FRET probe.
- the probe array has more than 1000 probes.
- the time delay is between 100 picoseconds and 10 milliseconds.
- the microfluidic device also has:
- CMOS complementary metal-oxide-semiconductor
- the calibration sources are calibration probes without a fluorophore.
- 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.
- 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.
- 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;
- CMOS circuitry on the substrate, the CMOS circuitry having a photosensor for generating an output signal in response to the fluorescence signal;
- the CMOS circuitry is configured to initiate a time delay upon deactivation of the excitation light before activating the photosensor.
- 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.
- 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.
- the photosensor is a photodiode and the probe is spaced less than 249 microns from the photodiode.
- the microfluidic device also has an array of the probes and a corresponding array of the photodiodes.
- the microfluidic device also has an excitation sensor photodiode configured to indicate when the excitation light deactivates.
- 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.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the microfluidic device also has a polymerase chain reaction (PCR) section amplifying the target nucleic acid sequences from a biological sample.
- PCR polymerase chain reaction
- the CMOS circuitry has memory for identity data for the FRET probes.
- the probe array has more than 1000 probes.
- the time delay is between 100 picoseconds and 10 milliseconds.
- 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.
- the reagent in the reagent reservoirs are selected from:
- 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.
- 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 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
- CMOS circuitry for controlling the shunt transistor to remove carriers generated by absorption of photons of the excitation light in the photodiode.
- the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
- 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.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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 CMOS circuitry has memory for identity data for the FRET probes.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- 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.
- 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 having a fluorophore for generating fluorescence emissions in response to an excitation light
- the fluorophore has a fluorescence lifetime longer than 100 nanoseconds.
- 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.
- 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.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the FRET probes.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a quencher for quenching fluorescence emissions from the fluorophore.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the mass of the probes in each of the hybridization chambers is less than 60 picograms.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- a photodiode for detecting fluorescence emissions generated by hybridization of the probe in response to an excitation light
- 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;
- the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
- 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.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the FRET probes.
- the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the mass of the probes in each of the hybridization chambers is less than 60 picograms.
- 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 photodiode having an active area for detecting fluorescence emissions generated by the probe-target hybrid in response to an excitation light
- 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;
- the shunt transistor is encompassed by the active area.
- the shunt transistor is configured for turning on when the excitation light activates and turning off when the excitation light deactivates.
- 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.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- the mass of the probes in each of the hybridization chambers is less than 60 picograms.
- 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 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;
- the active area is less than 249 microns from the probe-target hybrids.
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the mass of the probes in each of the hybridization chambers is less than 60 picograms.
- the mass of the probes in each of the hybridization chambers is less than 12 picograms.
- 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.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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 CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photosensors after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- 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;
- 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°.
- the active area is less than 249 microns from the floor surface.
- the photodiode has a field of view such that the fluorescence signal from the probe-target hybrids is incident on the active area.
- 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.
- 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.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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 CMOS circuitry has memory for identity data for the FRET probes.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the quencher has no native emission in response to the excitation light.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- addition of the biological sample to the inlet prevents fluid communication between a fluid subsequently added to the inlet and the probe.
- 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.
- the CMOS circuitry is configured to deactivate the excitation light and subsequently activate the photodiode after a time delay.
- the probe is less than 249 microns from the photodiode.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- the microfluidic device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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 CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the quencher has no native emission in response to the excitation light.
- 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:
- the photosensor is configured for sensing which probes within the array of probes generated the fluorescence signal.
- the photosensor is an array of photodiodes positioned between the array of probes and the supporting substrate.
- the photosensor has a field of view such that the fluorescence signal from the probe-target hybrids is incident on the photosensor.
- 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.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- the probes are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from: a ruthenium chelate;
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the quencher has no native emission in response to the excitation light.
- 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:
- CMOS circuitry on the supporting substrate for operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- 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.
- the probe is less than 249 microns from the photodiode.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- the microfluidic device also has an array of the probes, the probes being fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the mass of the probes in each of the hybridization chambers is less than 270 picograms.
- the quencher has no native emission in response to the excitation light.
- 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.
- This aspect of the invention provides a lab-on-a-chip (LOC) device comprising:
- MST microsystems technologies
- FRET fluorescence resonance energy transfer
- a photosensor for detecting hybridization of probes within the array of probes.
- the photosensor is an array of photodiodes incorporated in CMOS circuitry between the MST layer and the supporting substrate.
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a predetermined delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- the LOC device also has a hybridization heater controlled by the CMOS circuitry for providing thermal energy for hybridization.
- 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.
- the fluid flow-path is configured to draw the fluid from the PCR section to the liquid end point sensor by capillary action
- 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.
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- the photodiodes are less than 249 microns from the FRET probes.
- 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.
- FRET fluorescence resonance energy transfer
- GAS027.1 This aspect of the invention provides a LOC device comprising:
- 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;
- CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- 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.
- the probe is less than 249 microns from the photodiode.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- 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 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;
- CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- 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.
- the probe is less than 249 microns from the photodiode.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- 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:
- 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;
- PCR polymerase chain reaction
- CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- 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.
- the probe is less than 249 microns from the photosensor.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- each of the hybridization chambers has a volume less than 9,000 cubic microns.
- 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.
- MST microsystems technology
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- 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 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;
- CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- 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.
- the probe is less than 249 microns from the photodiode.
- 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.
- the shunt transistor is configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization chambers during hybridization of the probes and the target nucleic acid sequences.
- 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.
- This aspect of the invention provides a LOC device for amplifying nucleic acid sequences and detecting target nucleic acid sequences, the LOC device comprising:
- 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
- PCR polymerase chain reaction
- dNTPs deoxyribonucleoside triphosphates
- the probes are FRET (fluorescence resonance energy transfer) probes which change their fluorescent response to an excitation light when hybridized into probe-target hybrids.
- FRET fluorescence resonance energy transfer
- the LOC device also has photodiodes for each of the FRET probes respectively.
- the LOC device also has CMOS circuitry on the supporting substrate, the CMOS circuitry having operative control of the excitation light.
- the operative control includes activation and deactivation of the excitation light.
- the CMOS circuitry is configured to deactivate the excitation light and subsequently activate the photodiodes after a time delay.
- the FRET probes are less than 249 microns from the photodiodes.
- 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.
- the shunt transistors are configured to turn on when the excitation light activates and turn off when the excitation light deactivates.
- 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.
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- a reagent reservoir containing a reagent for addition to the biological sample
- a hybridization chamber containing probes having a nucleic acid sequence for hybridization with the target nucleic acid sequence to form probe-target hybrids
- 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.
- 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.
- 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.
- 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.
- the probe-target hybrids are configured to emit a fluorescence signal in response to exposure to an excitation light.
- the microfluidic device also has a photodiode for sensing the fluorescent response.
- 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.
- the operative control includes activation and deactivation of the excitation light, and conditioning of power supplied to the excitation light.
- the probes are less than 249 microns from the photodiode.
- 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.
- 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.
- FRET fluorescence resonance energy transfer
- 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.
- PCR polymerase chain reaction
- 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.
- MST microsystems technology
- 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.
- the CMOS circuitry is configured to enable the photodiodes after a delay following the excitation light being extinguished.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the quencher has no native emission in response to the excitation light.
- 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 provide for the low probe and reagent volumes, which in turn provide for low probe and reagent costs and the inexpensive assay system.
- 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:
- probe-target hybrids 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
- a negative control probe configured to never emit a fluorescence signal
- 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
- a negative control photodiode for exposure to the negative control probes.
- the negative control probe lacks a reporting fluorophore.
- 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.
- the LOC device also has:
- MST microsystems technologies
- 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;
- the probes within the probe array are fluorescence resonance energy transfer (FRET) probes.
- FRET fluorescence resonance energy transfer
- the LOC device also has a nucleic acid amplification section for amplifying the nucleic acid sequences.
- 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.
- 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.
- 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.
- the CMOS circuitry has memory for identity data for the different FRET probe types.
- the FRET probes each have a fluorophore and quencher, the fluorophore having a fluorescence lifetime greater than 100 nanoseconds.
- the fluorophore is a transition metal-ligand complex.
- the fluorophore is a lanthanide metal-ligand complex.
- the fluorophore is selected from:
- the quencher has no native emission in response to the excitation light.
- the CMOS circuitry is configured for temperature control of the hybridization section during hybridization of the probes and the target nucleic acid sequences.
- 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.
- 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.
- the fluid flow-path is configured to draw the fluid from the nucleic acid amplification section to the liquid sensor by capillary action
- 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.
- 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.
- 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.
- This aspect of the invention provides a microfluidic device comprising:
- the array of probes includes a control probe for hybridization with a target sequence known to be always present in the biological sample.
- 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.
- the photosensor is a charge coupled device (CCD) array positioned between the array of probes and the supporting substrate.
- CCD charge coupled device
- the photosensor is an array of photodiodes positioned on the supporting substrate in registration with the array of probes.
- the array of photodiodes is less than 249 microns from the array of probes.
- 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.
- the CMOS circuitry is configured to trigger a time delay when the excitation light is deactivated before activating the photodiode.
- 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.
Abstract
A microfluidic test module having 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.
Description
- 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.
- 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 GAS104US 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 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.
- 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.
- 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 microliters) 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. A1w1, N. BstNB1 and M1y1, are useful in this reaction. SDA has been improved by the use of a combination of a heat-stable restriction enzyme (Ava1) 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
- 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:
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- 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.
- 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.
- 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.
- 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.
- GAS002.5 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.
- 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,
- minors 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 80K 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 inFIG. 6 ; -
FIG. 14 is an enlarged view of Inset AB shown inFIG. 6 ; -
FIG. 15 is an enlarged view of Inset AE shown inFIG. 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 inFIG. 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 inFIG. 31 ; -
FIG. 33 is an enlarged view of Inset AF shown inFIG. 15 ; -
FIG. 34 is a schematic section view of the upstream end of the dialysis section taken through line 35-35 shown inFIG. 33 ; -
FIG. 35 is an enlarged view of Inset AC shown inFIG. 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 inFIG. 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 inFIG. 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 inFIG. 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 inFIG. 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 inFIG. 6 ; -
FIG. 56 is an enlarged view of Inset AD shown inFIG. 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 an enlarged plan view of the humidity sensor shown in Inset AH ofFIG. 6 ; -
FIG. 64 is a schematic showing part of the photodiode array of the photo sensor; -
FIG. 65 is a circuit diagram for a single photodiode; -
FIG. 66 is a timing diagram for the photodiode control signals; -
FIG. 67 is an enlarged view of the evaporator shown in Inset AP ofFIG. 55 ; -
FIG. 68 is a schematic section view through a hybridization chamber with a detection photodiode and trigger photodiode; -
FIG. 69 is a diagram of linker-primed PCR; -
FIG. 70 is a schematic representation of a test module with a lancet; -
FIG. 71 is a diagrammatic representation of the architecture of LOC variant VII; -
FIG. 72 is a diagrammatic representation of the architecture of LOC variant VIII; -
FIG. 73 is a schematic illustration of the architecture of LOC variant XIV; -
FIG. 74 is a schematic illustration of the architecture of LOC variant XLI; -
FIG. 75 is a schematic illustration of the architecture of LOC variant XLIII; -
FIG. 76 is a schematic illustration of the architecture of LOC variant XLIV; -
FIG. 77 is a schematic illustration of the architecture of LOC variant XLVII; -
FIG. 78 is a diagram of a primer-linked, linear fluorescent probe during the initial round of amplification; -
FIG. 79 is a diagram of a primer-linked, linear fluorescent probe during a subsequent amplification cycle; -
FIGS. 80A to 80F diagrammatically illustrate thermal cycling of a primer-linked fluorescent stem-and-loop probe; -
FIG. 81 is a schematic illustration of the excitation LED relative to the hybridization chamber array and the photodiodes; -
FIG. 82 is a schematic illustration of the excitation LED and optical lens for directing light onto the hybridization chamber array of the LOC device; -
FIG. 83 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. 84 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. 85 is a plan view showing all the features superimposed on each other, and showing the location of Insets DA to DK; -
FIG. 86 is an enlarged view of Inset DG shown inFIG. 85 ; -
FIG. 87 is an enlarged view of Inset DH shown inFIG. 85 ; -
FIG. 88 shows one embodiment of the shunt transistor for the photodiodes; -
FIG. 89 shows one embodiment of the shunt transistor for the photodiodes; -
FIG. 90 shows one embodiment of the shunt transistor for the photodiodes; -
FIG. 91 is a circuit diagram of the differential imager; -
FIG. 92 schematically illustrates a negative control fluorescent probe in its stem-and-loop configuration; -
FIG. 93 schematically illustrates the negative control fluorescent probe ofFIG. 92 in its open configuration; -
FIG. 94 schematically illustrates a positive control fluorescent probe in its stem-and-loop configuration; -
FIG. 95 schematically illustrates the positive control fluorescent probe ofFIG. 94 in its open configuration; -
FIG. 96 shows a test module and test module reader configured for use with ECL detection; -
FIG. 97 is a schematic overview of the electronic components in the test module configured for use with ECL detection; -
FIG. 98 shows a test module and alternative test module readers; -
FIG. 99 shows a test module and test module reader along with the hosting system housing various databases; - 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, 96 and 97, the system has the following top level components: -
Test modules Test modules device 30 preloaded with reagents and typically more than 1000 probes for the molecular diagnostic assay (seeFIGS. 1 and 96 ).Test module 10 schematically shown inFIG. 1 uses a fluorescence-based detection technique to identify target molecules, whiletest module 11 inFIG. 96 uses an electrochemiluminescence-based detection technique. TheLOC device 30 has an integratedphotosensor 44 for fluorescence or electrochemiluminescence detection (described in detail below). Bothtest modules Micro-USB plug 14 for power, data and control, both have a printed circuit board (PCB) 57, and both have externalpower supply capacitors 32 and aninductor 15. Thetest modules - The
outer casing 13 has amacroreceptacle 24 for receiving the biological sample and a removablesterile sealing tape 22, preferably with a low tack adhesive, to cover the macroreceptacle prior to use. Amembrane seal 408 with amembrane guard 410 forms part of theouter casing 13 to reduce dehumidification within the test module while providing pressure relief from small air pressure fluctuations. Themembrane guard 410 protects themembrane seal 408 from damage. -
Test module reader 12 powers thetest module Micro-USB port 16. Thetest module reader 12 can adopt many different forms and a selection of these are described later. The version of thereader 12 shown inFIGS. 1 , 3 and 96 is a smart phone embodiment. A block diagram of thisreader 12 is shown inFIG. 3 .Processor 42 runs application software fromprogram storage 43. Theprocessor 42 also interfaces with thedisplay screen 18 and user interface (UI)touch screen 17 andbuttons 19, acellular radio 21,wireless network connection 23, and asatellite navigation system 25. Thecellular radio 21 andwireless 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 thetouch screen 17 orbuttons 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 andprogram storage 43 may be shared in a common memory facility. Application software installed on thetest 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 thetest module reader 12. Thesterile sealing tape 22 is peeled back and the biological sample (in a liquid form) is loaded into thesample macroreceptacle 24. Pressingstart button 20 initiates testing via the application software. The sample flows into theLOC 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 theLED 26 housed in thecasing 13 provides the necessary excitation light to induce fluorescence emission from the hybridized probes (seeFIGS. 1 and 2 ). In test module 11 (which uses electrochemiluminescence (ECL) detection), theLOC device 30 is loaded with ECL probes (discussed above) and theLED 26 is not necessary for generating the luminescent emission. Instead, electrodes 860 and 870 provide the excitation electrical current (seeFIG. 97 ). The emission (fluorescent or luminescent) is detected using aphotosensor 44 integrated into CMOS circuitry of each LOC device. The detected signal is amplified and converted to a digital output which is analyzed by thetest 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 test module reader 12 and disposed of appropriately. -
FIGS. 1 , 3 and 96 show thetest module reader 12 configured as a mobile phone/smart phone 28. In other forms, the test module reader is a laptop/notebook 101, adedicated reader 103, anebook reader 107, atablet computer 109 ordesktop computer 105 for use in hospitals, private practices or laboratories (seeFIG. 98 ). 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. 99 , the data generated by thetest module 10 can be used to update, via thereader 12 andnetwork 125, the epidemiological databases hosted on the hosting system forepidemiological data 111, the genetic databases hosted on the hosting system forgenetic 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 forepidemiological data 111, the genetic data hosted on the hosting system forgenetic 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, vianetwork 125 and thereader 12, the digital memory in theLOC 30 of thetest module 10. - Referring back to
FIGS. 1 , 2, 96 and 97 thereader 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. AMicro-USB port 16 is provided for connection to a computer or mains power supply for battery recharge. -
FIG. 70 shows an embodiment of thetest 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-onlymodule 47 is adequate. No other reader or application software is necessary. Anindicator 45 on the USB power/indicator-onlymodule 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. 70 shows an embodiment of the test module incorporating a spring-loaded,retractable lancet 390 andlancet release button 392 for convenience. A satellite phone can be used in remote areas. -
FIGS. 2 and 97 are block diagrams of the electronic components in thetest modules LOC device 30 has aUSB device driver 36, acontroller 34, a USB-compatible LED driver 29,clock 33,power conditioner 31,RAM 38 and program anddata flash memory 40. These provide the control and memory for theentire test module photosensor 44, thetemperature sensors 170, theliquid sensors 174, and thevarious heaters drivers power supply capacitors 32 and theMicro-USB plug 14 are external to theLOC device 30. TheLOC devices 30 include bond-pads for making connections to these external components. TheRAM 38 and the program anddata 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 oftest module 11 configured for ECL detection, there is no LED 26 (seeFIGS. 96 and 97 ). Data is encrypted by theLOC device 30 for secure storage and secure communication with an external device. TheLOC 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.
- 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 LOC device 30 has numerous different embodiments tailored to the target(s) of interest. One form of theLOC device 30 isLOC 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 ofLOC device 301 is now described in detail with reference toFIGS. 4 to 26 and 27 to 57. -
FIG. 4 is a schematic representation of the architecture of theLOC device 301. For convenience, process stages shown inFIG. 4 are indicated with the reference numeral corresponding to the functional sections of theLOC 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 andpreparation 288,extraction 290,incubation 291,amplification 292 anddetection 294. The various reservoirs, chambers, valves and other components of theLOC device 301 will be described in more detail later. -
FIG. 5 is a perspective view of theLOC device 301. It is fabricated using high volume CMOS and MST (microsystems technology) manufacturing techniques. The laminar structure of theLOC device 301 is illustrated in the schematic (not to scale) partial section view ofFIG. 12 . TheLOC device 301 has asilicon substrate 84 which supports the CMOS+MST chip 48, comprisingCMOS circuitry 86 and anMST layer 87, with acap 46 overlaying theMST 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 theCMOS circuitry 86, but excluding the features of thecap 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 theMST layer 87 superimposed with the features of the cap. Insets AA to AD, AG and AH shown inFIG. 6 are enlarged inFIGS. 13 , 14, 35, 56, 55 and 63, respectively, and described in detail below for a comprehensive understanding of each structure within theLOC device 301.FIGS. 7 to 10 show the features of thecap 46 in isolation whileFIG. 11 shows the CMOS+MST device 48 structures in isolation. -
FIGS. 12 and 22 are sketches that diagrammatically show the laminar structure of the CMOS+MST device 48, thecap 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 thesample inlet 68 andFIG. 22 is a schematic section through thereservoir 54. As best shown inFIG. 12 , the CMOS+MST device 48 has asilicon substrate 84 which supports theCMOS circuitry 86 that operates the active elements within theMST layer 87 above. Apassivation layer 88 seals and protects theCMOS layer 86 from the fluid flows through theMST layer 87. - Fluid flows through both the
cap channels 94 and the MST channels 90 (see for exampleFIGS. 7 and 16 ) in thecap layer 46 andMST channel layer 100, respectively. Cell transport occurs in thelarger channels 94 fabricated in thecap 46, while biochemical processes are carried out in thesmaller MST channels 90. Cell transport channels are sized so as to be able to transport cells in the sample to predetermined sites in theMST 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 andMST channel 90 are generic references andparticular 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 aMST channel layer 100 deposited on thepassivation layer 88 and patterned with photoresist. TheMST channels 90 are enclosed by aroof 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 thereservoir 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 acap channel layer 80 in which thecap channels 94 are etched and thereservoir layer 78 in which thereservoirs - 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 thecap channels 94 and theupper seal layer 82 encloses thereservoirs - The five
reservoirs -
- 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. 69, 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 thelower seal 64 and theroof layer 66. These openings are referred to asuptakes 96 anddowntakes 92 depending on whether fluid is flowing from theMST channels 90 to thecap channels 94 or vice versa. - 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 toFIG. 4 , there are five major steps involved in analysing a biological sample, comprising sample input andpreparation 288,nucleic acid extraction 290,nucleic acid incubation 291,nucleic acid amplification 292 and detection andanalysis 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 thepathogen dialysis section 70. As best shown inFIGS. 7 and 12 , the blood sample enters the device via thesample inlet 68. Capillary action draws the blood sample along thecap channel 94 to thereservoir 54. Anticoagulant is released from thereservoir 54 as the sample blood flow opens its surface tension valve 118 (seeFIGS. 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 thereservoir 54 by capillary action and into theMST channel 90 via thedowntake 92. Thedowntake 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 thedowntake 92. Vent holes 122 in theupper seal 82 allows air to replace the anticoagulant 116 as it is drawn out of thereservoir 54. - The
MST channel 90 shown inFIG. 22 is part of asurface tension valve 118. The anticoagulant 116 fills thesurface tension valve 118 and pins ameniscus 120 to theuptake 96 to ameniscus anchor 98. Prior to use, themeniscus 120 remains pinned at theuptake 96 so the anticoagulant does not flow into thecap channel 94. When the blood flows through thecap channel 94 to theuptake 96, themeniscus 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 inFIG. 13 . As shown inFIGS. 15 , 16 and 17, thesurface tension valve 118 has threeseparate MST channels 90 extending between respective downtakes 92 anduptakes 96. The number ofMST 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 ofapertures 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 ofapertures 164 or the smaller cells that pass through the apertures, are redirected to awaste 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 3micron diameter holes 164 fluidically connecting the input flow in thecap channel 94 to atarget channel 74. The 3micron diameter apertures 164 and the dialysis uptake holes 168 for thetarget channel 74 are connected by a series of dialysis MST channels 204 (best shown inFIGS. 15 and 21 ). Pathogens are small enough to pass through the 3micron diameter apertures 164 and fill thetarget channel 74 via thedialysis MST channels 204. Cells larger than 3 microns, such as erythrocytes and leukocytes, stay in thewaste channel 72 in thecap 46 which leads to a waste reservoir 76 (seeFIG. 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 thetarget channel 74 to thesurface tension valve 128 of thelysis reagent reservoir 56. Thesurface tension valve 128 has sevenMST channels 90 extending between thelysis reagent reservoir 56 and thetarget channel 74. When the menisci are unpinned by the sample flow, the flow rate from all seven of theMST channels 90 will be greater than the flow rate from theanticoagulant reservoir 54 where thesurface 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 thechemical lysis section 130. A boiling-initiatedvalve 126 stops the flow until sufficient time has passed for diffusion and lysis to take place, releasing the genetic material from the target cells (seeFIGS. 6 and 7 ). The structure and operation of the boiling-initiated valves are described in greater detail below with reference toFIGS. 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 amixing section 131 for pre-amplification restriction digestion and linker ligation. - Referring to
FIG. 13 , restriction enzymes, linkers and ligase are released from thereservoir 58 when the flow unpins the menisci at thesurface tension valve 132 at the start of themixing section 131. The mixture flows the length of themixing section 131 for diffusion mixing. At the end of themixing section 131 is downtake 134 leading into theincubator inlet channel 133 of the incubation section 114 (seeFIG. 13 ). Theincubator inlet channel 133 feeds the mixture into a serpentine configuration ofheated microchannels 210 which provides an incubation chamber for holding the sample during restriction digestion and ligation of the linkers (seeFIGS. 13 and 14 ). -
FIGS. 23 , 24, 25, 26, 27, 28 and 29 show the layers of theLOC device 301 within Inset AB ofFIG. 6 . Each figure shows the sequential addition of layers forming the structures of the CMOS+MST layer 48 and thecap 46. Inset AB shows the end of theincubation section 114 and the start of theamplification section 112. As best shown inFIGS. 14 and 23 , the flow fills themicrochannels 210 of theincubation section 114 until reaching the boiling-initiatedvalve 106 where the flow stops while diffusion takes place. As discussed above, themicrochannel 210 upstream of the boiling-initiatedvalve 106 becomes an incubation chamber containing the sample, restriction enzymes, ligase and linkers. Theheaters 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 theamplification 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 theamplification section 112. Referring toFIGS. 31 and 32 , the mixture fills the serpentine configuration ofheated microchannels 158, which form one or more amplification chambers, until it reaches the boiling-initiatedvalve 108. As best shown in the schematic section view ofFIG. 30 , amplification mix (dNTPs, primers, buffer) is released fromreservoir 60 and polymerase is subsequently released fromreservoir 62 into theintermediate MST channel 212 connecting the incubation and amplification sections (114 and 112 respectively). -
FIGS. 35 to 51 show the layers of theLOC device 301 within Inset AC ofFIG. 6 . Each figure shows the sequential addition of layers forming the structures of the CMOS+MST device 48 and thecap 46. Inset AC is at the end of theamplification section 112 and the start of the hybridization anddetection section 52. The incubated sample, amplification mix and polymerase flow through themicrochannels 158 to the boiling-initiatedvalve 108. After sufficient time for diffusion mixing, theheaters 154 in themicrochannels 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-initiatedvalve 108 opens and flow resumes into the hybridization anddetection section 52. The operation of boiling-initiated valves is described in more detail later. - As shown in
FIG. 52 , the hybridization anddetection section 52 has an array ofhybridization chambers 110.FIGS. 52 , 53, 54 and 56 show thehybridization chamber array 110 andindividual hybridization chambers 180 in detail. At the entrance to thehybridization chamber 180 is adiffusion barrier 175 which prevents diffusion of the target nucleic acid, probe strands and hybridized probes between thehybridization chambers 180 during hybridization so as to prevent erroneous hybridization detection results. Thediffusion 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 thephotodiodes 184 corresponding to thehybridization 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 theCMOS circuitry 86 signals theLED 26 located in thetest 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 aphotodiode 184 in theCMOS circuitry 86 underlying each hybridization chamber 180 (see hybridization chamber description below). Thephotodiodes 184 for all hybridization chambers and associated electronics collectively form the photosensor 44 (seeFIG. 64 ). In other embodiments, the photosensor may be an array of charge coupled devices (CCD array). The detected signal from thephotodiodes 184 is amplified and converted to a digital output which is analyzed by thetest module reader 12. Further details of the detection method are described later. - The
LOC device 301 has many functional sections, including thereagent reservoirs dialysis section 70,lysis section 130,incubation section 114, andamplification 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 thefirst amplification section 112 of a tandem amplification assay system, with the chemicallysis reagent reservoir 56 being used to add the first amplification mix of primers, dNTPs and buffer andreagent reservoir 58 being used for adding the reverse transcriptase and/or polymerase. A chemical lysis reagent can also be added to thereservoir 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 ofreservoir 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 thereagent reservoir 58 andincubation 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 thelysis section 130 to theamplification 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. Thedialysis section 70 can be located at the beginning of the fluidic system within the microfluidic device as shown inFIG. 4 or can be located anywhere else within the microfluidic device. For example, dialysis after theamplification phase 292 to remove cellular debris prior to the hybridization anddetection 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 incorporateadditional amplification sections 112 to enable multiple targets to be amplified in parallel or in series prior to being detected in thehybridization chamber arrays 110 with specific nucleic acid probes. For analysis of samples like whole blood, in which dialysis is not required, thedialysis section 70 is simply omitted from the sample input andpreparation section 288 of the LOC design. In some cases, it is not necessary to omit thedialysis 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 thedialysis 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.
- 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 thehybridization 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.
- Referring to
FIGS. 1 and 12 , the sample is added to themacroreceptacle 24 of thetest module 10. Themacroreceptacle 24 is a truncated cone which feeds into theinlet 68 of theLOC device 301 by capillary action. Here it flows into the 64 μm wide×60 μmdeep cap channel 94 where it is drawn towards theanticoagulant reservoir 54, also by capillary action. - 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 theLOC device 301 shown in the drawings, less than 20,000,000 cubic microns. - Referring to
FIGS. 15 to 21 , 33 and 34, thepathogen 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 theroof layer 66 filter the target cells from the bulk of the sample. As the sample flows past the 3micron diameter apertures 164, microbial pathogens pass through the holes into a series of dialysis MSTchannels 204 and flow back up into thetarget channel 74 via 16 μm dialysis uptake holes 168 (seeFIGS. 33 and 34 ). The remainder of the sample (erythrocytes and so on) stay in thecap channel 94. Downstream of thepathogen dialysis section 70, thecap channel 94 becomes thewaste channel 72 leading to thewaste reservoir 76. For biological samples of the type that generate a substantial amount of waste, a foam insert or otherporous element 49 within theouter casing 13 of thetest module 10 is configured to be in fluid communication with the waste reservoir 76 (seeFIG. 1 ). - The
pathogen dialysis section 70 functions entirely on capillary action of the fluid sample. The 3micron diameter apertures 164 at the upstream end of thepathogen dialysis section 70 have capillary initiation features (CIFs) 166 (seeFIG. 33 ) so that the fluid is drawn down into thedialysis MST channel 204 beneath. Thefirst uptake hole 198 for thetarget channel 74 also has a CIF 202 (seeFIG. 15 ) to avoid the flow simply pinning a meniscus across the dialysis uptake holes 168. - The small
constituents dialysis section 682 schematically shown inFIG. 74 can have a similar structure to thepathogen 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 awaste reservoir 766. Thus, the LOC device 30 (seeFIGS. 1 and 96 ) 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. - 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 thelysis reservoir 56 mixes with the sample flow in thetarget channel 74 downstream of thesurface tension valve 128 for thelysis 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. TheLOC device 301 accommodates this with theheated microchannels 210 of theincubation section 114. The sample flow fills theincubation section 114 and stops at the boiling-initiatedvalve 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 thechemical lysis section 130. - As discussed above, the
LOC device 301 has three boiling-initiatedvalves FIG. 6 .FIG. 31 is an enlarged plan view of the boiling-initiatedvalve 108 in isolation at the end of theheated microchannels 158 of theamplification section 112. - The
sample flow 119 is drawn along theheated microchannels 158 by capillary action until it reaches the boiling-initiatedvalve 108. The leadingmeniscus 120 of the sample flow pins at ameniscus anchor 98 at thevalve inlet 146. The geometry of themeniscus anchor 98 stops the advancing meniscus to arrest the capillary flow. As shown inFIGS. 31 and 32 , themeniscus anchor 98 is an aperture provided by an uptake opening from theMST channel 90 to thecap channel 94. Surface tension in themeniscus 120 keeps the valve closed. Anannular heater 152 is at the periphery of thevalve inlet 146. Theannular heater 152 is CMOS-controlled via the boiling-initiatedvalve heater contacts 153. - To open the valve, the
CMOS circuitry 86 sends an electrical pulse to thevalve heater contacts 153. Theannular heater 152 resistively heats until theliquid sample 119 boils. The boiling unpins themeniscus 120 from thevalve inlet 146 and initiates wetting of thecap channel 94. Once wetting thecap channel 94 begins, capillary flow resumes. Thefluid sample 119 fills thecap channel 94 and flows through the valve downtake 150 to thevalve outlet 148 where capillary driven flow continues along the amplificationsection exit channel 160 into the hybridization anddetection 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 thetest module 10 are single-use devices, re-closing the valves is unnecessary. -
FIGS. 6 , 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show theincubation section 114 and theamplification section 112. Theincubation section 114 has a single,heated incubation microchannel 210 etched in a serpentine pattern in theMST channel layer 100 from the downtake opening 134 to the boiling-initiated valve 106 (seeFIGS. 13 and 14 ). Control over the temperature of theincubation section 114 enables enzymatic reactions to take place with greater efficiency. Similarly, theamplification section 112 has aheated amplification microchannel 158 in a serpentine configuration leading from the boiling-initiatedvalve 106 to the boiling-initiated valve 108 (seeFIGS. 6 and 14 ). These valves arrest the flow to retain the target cells in the heated incubation oramplification microchannels - In the
incubation section 114 and theamplification section 112, the sample cells and the reagents are heated by theheaters 154 controlled by theCMOS circuitry 86 using pulse width modulation (PWM). Each meander of the serpentine configuration of theheated incubation microchannel 210 andamplification microchannel 158 has three separatelyoperable heaters 154 extending between their respective heater contacts 156 (seeFIG. 14 ) which provides for the two-dimensional control of input heat flux density. As best shown inFIG. 51 , theheaters 154 are supported on theroof layer 66 and embedded in thelower seal 64. The heater material is TiAl but many other conductive metals would be suitable. Theelongate heaters 154 are parallel with the longitudinal extent of each channel section that forms the wide meanders of the serpentine shape. In theamplification 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 inamplification 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 theLOC device 301, between 2 nanoliters and 30 nanoliters. - 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 theamplification 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 theamplification 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.
- 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 anddetection section 52 via the boiling-initiatedvalve 108. -
FIGS. 52 , 53, 54, 56 and 57 show thehybridization chambers 180 in thehybridization chamber array 110. The hybridization anddetection section 52 has a 24×45array 110 ofhybridization chambers 180, each with hybridization-responsive FRET probes 186,heater element 182 and anintegrated photodiode 184. Thephotodiode 184 is incorporated for detection of fluorescence resulting from the hybridization of a target nucleic acid sequence or protein with the FRET probes 186. Eachphotodiode 184 is independently controlled by theCMOS circuitry 86. Any material between the FRET probes 186 and thephotodiode 184 must be transparent to the emitted light. Accordingly, thewall section 97 between theprobes 186 and thephotodiode 184 is also optically transparent to the emitted light. In theLOC device 301, thewall section 97 is a thin (approximately 0.5 micron) layer of silicon dioxide. - Incorporation of a
photodiode 184 directly beneath eachhybridization chamber 180 allows the volume of probe-target hybrids to be very small while still generating a detectable fluorescence signal (seeFIG. 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 theLOC 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. InLOC 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 (seeFIGS. 52 and 56 ). An end-point liquid sensor 178 indicates when thehybridization chambers 180 are filled with amplicon and theheaters 182 can be activated. - After sufficient hybridization time, the LED 26 (see
FIG. 2 ) is activated. The opening in each of thehybridization chambers 180 provides anoptical window 136 for exposing the FRET probes 186 to the excitation radiation (seeFIGS. 52 , 54 and 56). TheLED 26 is illuminated for a sufficiently long time in order to induce a fluorescence signal from the probes with high intensity. During excitation, thephotodiode 184 is shorted. After a pre-programmed delay 300 (seeFIG. 2 ), thephotodiode 184 is enabled and fluorescence emission is detected in the absence of the excitation light. The incident light on theactive area 185 of the photodiode 184 (seeFIG. 54 ) is converted into a photocurrent which can then be measured usingCMOS circuitry 86. - The
hybridization chambers 180 are each loaded with probes for detecting a single target nucleic acid sequence. Eachhybridization 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 thehybridization 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 thehybridization chamber array 110, depending on the assay specification. - Inset AG of
FIG. 6 indicates the position of thehumidifier 196. The humidifier prevents evaporation of the reagents and probes during operation of theLOC device 301. As best shown in the enlarged view ofFIG. 55 , awater reservoir 188 is fluidically connected to threeevaporators 190. Thewater reservoir 188 is filled with molecular biology-grade water and sealed during manufacturing. As best shown inFIGS. 55 and 67 , water is drawn into threedowntakes 194 and along respectivewater supply channels 192 by capillary action to a set of threeuptakes 193 at theevaporators 190. A meniscus pins at eachuptake 193 to retain the water. The evaporators have annular shaped heaters 191 which encircle theuptakes 193. The annular heaters 191 are connected to theCMOS circuitry 86 by theconductive columns 376 to the top metal layer 195 (seeFIG. 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 inFIG. 6 . However, as best shown in the enlarged view of Inset AH inFIG. 63 , the humidity sensor has a capacitive comb structure. A lithographically etchedfirst electrode 296 and a lithographically etchedsecond 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 theCMOS circuitry 86. As the humidity increases, the permittivity of the air gap between the electrodes increases, so that the capacitance also increases. Thehumidity sensor 232 is adjacent thehybridization chamber array 110 where humidity measurement is most important to slow evaporation from the solution containing the exposed probes. - Temperature and liquid sensors are incorporated throughout the
LOC device 301 to provide feedback and diagnostics during device operation. Referring toFIG. 35 , ninetemperature sensors 170 are distributed throughout theamplification section 112. Likewise, theincubation section 114 also has ninetemperature sensors 170. These sensors each use a 2×2 array of bipolar junction transistors (BJTs) to monitor the fluid temperature and provide feedback to theCMOS circuitry 86. TheCMOS 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, theCMOS circuitry 86 uses thehybridization heaters 182 as temperature sensors (seeFIG. 56 ). The electrical resistance of thehybridization heaters 182 is temperature dependent and theCMOS circuitry 86 uses this to derive a temperature reading for each of thehybridization chambers 180. - The
LOC device 301 also has a number of MSTchannel liquid sensors 174 and capchannel liquid sensors 208.FIG. 35 shows a line of MSTchannel liquid sensors 174 at one end of every other meander in theheated microchannel 158. As best shown inFIG. 37 , the MSTchannel liquid sensors 174 are a pair of electrodes formed by exposed areas of thetop metal layer 195 in theCMOS 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 capchannel liquid sensors 208. Opposing pairs ofTiAl electrodes roof layer 66. Between theelectrodes gap 222 to hold the circuit open in the absence of liquid. The presence of liquid closes the circuit and theCMOS circuitry 86 uses this feedback to monitor the flow. - 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. - 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 inFIG. 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 thepathogen dialysis section 70 inFIG. 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 inFIG. 6 , a dialysis section which effectively concentrates pathogens small enough to be passed into theamplification section 292 is employed upstream of thesample extraction section 290, and rejects larger cells to awaste 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. - 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 theheaters 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 thereagent reservoir 58 to store and dispense the buffers, primers, dNTPs and reverse transcriptase and theincubation section 114 for the reverse transcription step followed by amplification in the normal way in theamplification section 112. - 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 reagent reservoir 60 contains amplification buffer, primers and dNTPs andreagent 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, withreagent reservoir 62 containing a strand displacing DNA polymerase such as Bsu. Similarly, for HDA,reagent reservoir 60 contains amplification buffer, primers and dNTPs andreagent 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 andreagent 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 theheaters 154 in the amplification microchannels 158 (seeFIG. 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 inFIGS. 75 , 76 and 77, respectively. Direct isothermal amplification may also be combined with one or more pre-amplification dialysis steps 70, 686 or 682 as shown inFIGS. 75 and 77 and/or apre-hybridization dialysis step 682 as indicated inFIG. 76 to help partially concentrate the target cells in the sample before nucleic acid amplification or remove unwanted cellular debris prior to the sample entering thehybridization 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. 71 , 72 and 73, multiplexed and some methods of isothermal nucleic acid amplification, such as LAMP, are compatible with an initial reverse transcription step to amplify RNA. -
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 asingle FRET probe 236 prior to hybridization with a targetnucleic acid sequence 238. The probe has aloop 240,stem 242, afluorophore 246 at the 5′ end, and aquencher 248 at the 3′ end. Theloop 240 consists of a sequence complementary to the targetnucleic acid sequence 238. Complementary sequences on either side of the probe sequence anneal together to form thestem 242. - In the absence of a complementary target sequence, the probe remains closed as shown in
FIG. 58 . Thestem 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 theexcitation light 244. -
FIG. 59 shows theFRET probe 236 in an open or hybridized configuration. Upon hybridization to a complementary targetnucleic acid sequence 238, the stem-and-loop structure is disrupted, the fluorophore and quencher are spatially separated, thus restoring the ability of thefluorophore 246 to fluoresce. Thefluorescence 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, 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. -
FIGS. 78 and 79 show a primer-linkedlinear probe 692 during the initial round of nucleic acid amplification and in its hybridized configuration during subsequent rounds of nucleic acid amplification, respectively. Referring toFIG. 78 , the primer-linkedlinear probe 692 has a double-strandedstem segment 242. One of the strands incorporates the primer linkedprobe sequence 696 which is homologous to a region on the targetnucleic acid 696 and is labelled on its 5′ end withfluorophore 246, and linked on its 3′ end to anoligonucleotide primer 700 via anamplification blocker 694. The other strand of thestem 242 is labelled at its 3 end with aquencher 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, theoligonucleotide primer 700 anneals to the target DNA 238 (FIG. 78 ) and is then extended, forming a DNA strand containing both the probe sequence and the amplification product. Theamplification blocker 694 prevents the polymerase from reading through and copying theprobe region 696. Upon subsequent denaturation, theextended oligonucleotide primer 700/template hybrid is dissociated and so is the double strandedstem 242 of the primer-linked linear probe, thus releasing thequencher 248. Once the temperature decreases for the annealing and extension steps, the primer linkedprobe sequence 696 of the primer-linked linear probe curls around and hybridizes to the amplifiedcomplementary 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. -
FIGS. 80A to 80F show the operation of a primer-linked stem-and-loop probe 704. Referring toFIG. 80A , the primer-linked stem-and-loop probe 704 has astem 242 of complementary double-stranded DNA and aloop 240 which incorporates the probe sequence. One of thestem strands 708 is labelled at its 5′ end withfluorophore 246. Theother strand 710 is labelled with a 3′-end quencher 248 and carries both theamplification blocker 694 andoligonucleotide primer 700. During the initial denaturation phase (seeFIG. 80B ), the strands of the targetnucleic 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 (seeFIG. 80C ), theoligonucleotide primer 700 on the primer-linked stem-and-loop probe 704 hybridizes to the targetnucleic acid sequence 238. During extension (seeFIG. 80D ) thecomplement 706 to the targetnucleic acid sequence 238 is synthesized forming a DNA strand containing both theprobe sequence 704 and the amplified product. Theamplification blocker 694 prevents the polymerase from reading through and copying theprobe region 704. When the probe next anneals, following denaturation, the probe sequence of theloop segment 240 of the primer-linked stem-and-loop probe (seeFIG. 80F ) anneals to thecomplementary sequence 706 on the extended strand. This configuration leaves thefluorophore 246 relatively remote from thequencher 248, resulting in a significant increase in fluorescence emission. - The
hybridization chamber array 110 includes somehybridization chambers 180 with positive and negative control probes used for assay quality control.FIGS. 92 and 93 schematically illustrate negative control probes without afluorophore 796, andFIGS. 94 and 95 are sketches of positive control probes without aquencher 798. The positive and negative control probes have a stem-and-loop structure like the FRET probes described above. However, afluorescence signal 250 will always be emitted frompositive control probes 798 and nofluorescence signal 250 is ever emitted fromnegative control probes 796, regardless of whether the probes hybridize into an open configuration or remain closed. - Referring to
FIGS. 92 and 93 , thenegative control probe 796 has no fluorophore (and may or may not have a quencher 248). Hence, whether the targetnucleic acid sequence 238 hybridizes with the probe (seeFIG. 93 ), or the probe remains in its stem-and-loop configuration (seeFIG. 92 ), the response to theexcitation light 244 is negligible. Alternatively, thenegative control probe 796 could be designed so that it always remains quenched. For example, by synthesizing theloop 240 to have a probe sequence that will not hybridize to any nucleic acid sequence within the sample under investigation, thestem 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 fromhybridization 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 inFIGS. 94 and 95 . Nothing quenches thefluorescence emission 250 from thefluorophore 246 in response to theexcitation light 244 regardless of whether thepositive control probe 798 hybridizes with the targetnucleic acid sequence 238. -
FIG. 52 shows a possible distribution of the positive and negative control probes (378 and 380 respectively) throughout thehybridization chamber array 110. The control probes 378 and 380 are placed inhybridization chambers 180 positioned in a line across thehybridization 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). - 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 λem 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 thequencher 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 thequencher 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 Calif., 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. - 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. 81 , theLED 26 is positioned directly above thehybridization chamber array 110 on an external surface of theLOC device 301. On the opposing side of thehybridization chamber array 110, is the photosensor 44, made up of an array of photodiodes 184 (seeFIGS. 53 , 54 and 64) for detection of fluorescence signals from each of the chambers. -
FIGS. 82 , 83 and 84 schematically illustrate other embodiments for exposing the probes to excitation light. In theLOC device 30 shown inFIG. 82 , theexcitation light 244 generated by theexcitation LED 26 is directed onto thehybridization chamber array 110 by thelens 254. Theexcitation LED 26 is pulsed and the fluorescence emissions are detected by thephotosensor 44. - In the
LOC device 30 shown inFIG. 83 , theexcitation light 244 generated by theexcitation LED 26 is directed onto thehybridization chamber array 110 by thelens 254, a firstoptical prism 712 and secondoptical prism 714. Theexcitation LED 26 is pulsed and the fluorescence emissions are detected by thephotosensor 44. - Similarly, the
LOC device 30 shown inFIG. 84 , theexcitation light 244 generated by theexcitation LED 26 is directed onto thehybridization chamber array 110 by thelens 254, afirst minor 716 andsecond minor 718. Again, theexcitation LED 26 is pulsed and the fluorescence emissions are detected by thephotosensor 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) @ 1 A 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 - 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. - The
LED driver 29 drives theLED 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. -
FIG. 54 shows thephotodiode 184 integrated into theCMOS circuitry 86 of theLOC device 301. Thephotodiode 184 is fabricated as part of theCMOS 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 itsactive 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 thephotodiode 184 and hence the number ofhybridization chambers 180 in the hybridization and detection section 52 (seeFIG. 52 ). The size and number of chambers are technical parameters that are limited by the dimensions of the LOC device (in the case of theLOC device 301, the dimensions are 1760 μm×5824 μm) and the real estate available after other functional modules such as thepathogen 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. - 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 ormore 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 bycalibration chambers 382 in thehybridization 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. 92 to 95 can be used in calibration chambers. However, as shown inFIGS. 86 and 87 , which are enlarged views of insets DG and DH of LOC variant X 728 shown inFIG. 85 , another option is to fluidically isolate thecalibration 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 thatcalibration 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 ifphotodiodes 184 are calibrated by acalibration chamber 382 that is relatively proximate. Referring toFIG. 56 , thehybridization chamber array 110 has onecalibration chamber 382 for every eighthybridization chambers 180. That is, acalibration chamber 382 is positioned in the middle of every three by three square ofhybridization chambers 180. In this configuration, thehybridization chambers 180 are calibrated by acalibration chamber 382 that is immediately adjacent. -
FIG. 91 shows adifferential imager circuit 788 used to subtract the signal from thephotodiode 184 corresponding to thecalibration chamber 382 as a result of excitation light, from the fluorescence signal from the surroundinghybridization chambers 180. Thedifferential imager circuit 788 samples the signal from thepixel 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 thepixel 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 andMD4 801 transistors are turned on.Switches pixel 790 and “dummy”pixel 792 are stored onpixel capacitor 803 anddummy 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 switchedcapacitor amplifier 815 at the output amplifies thedifferential signal 817. - The
photodiode 184 needs to be suppressed during excitation by theLED 26 and enabled during fluorescence.FIG. 65 is a circuit diagram for asingle photodiode 184 andFIG. 66 is a timing diagram for the photodiode control signals. The circuit hasphotodiode 184 and six MOS transistors,M shunt 394,M tx 396,M reset 398,M sf 400,M read 402 andM bias 404. At the beginning of the excitation cycle, t1, thetransistors M shunt 394, andM reset 398 are turned on by pulling the Mshunt gate 384 and thereset gate 388 high. During this period, the excitation photons generate carriers in thephotodiode 184. These carriers have to be removed, as the amount of generated carriers can be sufficient to saturate thephotodiode 184. During this cycle,M shunt 394 directly removes the carriers generated inphotodiode 184, whileM 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 pullingtx gate 386 high, which turns on thetransistor M tx 396 and transfers any accumulated carriers on thephotodiode 184 to node ‘NS’ 406. The duration of the capture cycle can be as long as the fluorophore emits. The outputs from allphotodiodes 184 in thehybridization 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 (seeFIG. 52 ) separately following the capture cycle. Thefirst photodiode 184 to be read will have the shortest delay before the read cycle, while thelast photodiode 184 will have the longest delay before the read cycle. During the read cycle,transistor M read 402 is turned on by pulling theread 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:
-
FIGS. 88 , 89 and 90 show threepossible configurations FIG. 88 , the Mshunt gate 384 is configured to be on the edge of thephotodiode 184. Optionally, as shown inFIG. 89 , the Mshunt gate 384 may be configured to surround thephotodiode 184. A third option is to configure the Mshunt gate 384 inside thephotodiode 184, as shown inFIG. 90 . Under this third option there would be less photodiodeactive area 185. - These three
configurations photodiode 184 to the Mshunt gate 384. InFIG. 88 , the Mshunt gate 384 is on one side of thephotodiode 184. This configuration is simplest to fabricate and impinges the least on the photodiodeactive area 185. However, any carriers lingering on the remote side of thephotodiode 184 would take longer to propagate through to the Mshunt gate 384. - In
FIG. 89 , the Mshunt gate 384 surrounds thephotodiode 184. This further reduces the average path length for carriers in thephotodiode 184 to the Mshunt gate 384. However, extending the Mshunt gate 384 about the periphery of thephotodiode 184 imposes a greater reduction of the photodiodeactive area 185. Theconfiguration 782 inFIG. 90 positions the Mshunt gate 384 within theactive area 185. This provides the shortest average path length to the Mshunt gate 384 and hence the shortest transition time. However, the impingement on theactive area 185 is greatest. It also poses a wider leakage path. - 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. 68 is a schematic section view through ahybridization chamber 180 showing aphotodiode 184 andtrigger photodiode 187 embedded in theCMOS circuitry 86. A small area in the corner of thephotodiode 184 is replaced with thetrigger photodiode 187. Atrigger photodiode 187 with a small area is sufficient as the intensity of the excitation light will be high in comparison with the fluorescence emission. Thetrigger photodiode 187 is sensitive to theexcitation light 244. Thetrigger photodiode 187 registers that theexcitation light 244 has extinguished and activates thephotodiode 184 after a short time delay Δt 300 (seeFIG. 2 ). This delay allows thefluorescence photodiode 184 to detect the fluorescence emission from the FRET probes 186 in the absence of theexcitation light 244. This enables detection and improves the signal to noise ratio. - Both
photodiodes 184 and triggerphotodiodes 187 are located in theCMOS circuitry 86 under eachhybridization chamber 180. The array of photodiodes combines, along with appropriate electronics, to form the photosensor 44 (seeFIG. 64 ). Thephotodiodes 184 are pn-junction fabricated during CMOS structure manufacturing without additional masks or steps. During MST fabrication, the dielectric layer (not shown) above thephotodiodes 184 is optionally thinned using the standard MST photolithography techniques to allow more fluorescent light to illuminate theactive area 185 of thephotodiode 184. Thephotodiode 184 has a field of view such that the fluorescence signal from the probe-target hybrids within thehybridization chamber 180 is incident on the sensor face. The fluorescent light is converted into a photocurrent which can then be measured usingCMOS circuitry 86. - Alternatively, one or
more hybridization chambers 180 can be dedicated to atrigger photodiode 187 only. These options can be used in these in combination with 2a and 2b above. - 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 Ie 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:
-
- where c is the molar concentration of fluorophores, E is the molar extinction coefficient, νe is the excitation frequency, and h=6.62606896(10)−34 Js is the Planck constant.
This differential equation has the general form: -
- which has the solution:
-
- Using this now to solve equation (1),
-
- Now at time t1, [S1](t1)=0, and from (3):
-
- Substituting (4) into (3):
-
- At time t2:
-
- For t≧t2, the excited states decay exponentially and this is described by:
-
[S1](t)=[S1](t 2)e −(t-t2 )/τf (6) - Substituting (5) into (6):
-
- The fluorescence intensity is given by the following equation:
-
- where νf is the fluorescence frequency, η is the quantum yield and 1 is the optical path length.
- Now from (7):
-
- Substituting (9) into (8):
-
- Therefore, we can write the following approximate equation which describes the fluorescence intensity decay after a sufficiently long excitation pulse (t2−t1>>τf):
-
- In the previous section, we concluded that for t2−t1>>τf,
-
- From the above equation, we can derive the following:
-
{dot over ({dot over ({dot over (n)}f(t)={dot over ({dot over ({dot over (n)}e ∈clηe −(t-t2 )/τ f (12) - where
-
- is the number of fluorescent photons per unit time per unit area and
-
- is the number of excitation photons per unit time per unit area.
- Consequently,
-
- 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): -
- Now, the number of fluorescent photons that reach the photodiode per unit time per unit area, {dot over ({dot over ({dot over (n)}s(t), is given by the following:
-
{dot over ({dot over ({dot over (n)}s(t)={dot over ({dot over ({dot over (n)}f(t)φ0 (15) - where φ0 is the light gathering efficiency of the optical system.
- Substituting (12) into (15) we find
-
{dot over ({dot over ({dot over (n)}s(t)=φ0{dot over ({dot over ({dot over (n)}e ∈clηe −(t-t2 )/τf (16) - Similarly, the number of fluorescence photons that reach the photodiode per unit fluorescent area {umlaut over (n)}s, will be as follows:
-
- and substituting in (16) and integrating:
-
{umlaut over (n)} s=φ0{dot over ({dot over ({dot over (n)}e ∈clητ f e −(t3 -t2 )/τf -
Therefore, -
n 2=φ0 {dot over (n)} e ∈clητ f e −Δt/τf (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 thephotodiode 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:
-
{dot over ({dot over (ė)}f(t)=φf{dot over ({dot over ({dot over (n)}s(t) - where φf is the quantum efficiency of the sensor at the fluorescence wavelength.
- Substituting in (17) we have:
-
{dot over ({dot over (ė)}f(t)=φfφ0{dot over ({dot over ({dot over (n)}e τclηe −(t-t2 )/τf (18) - Similarly, the rate of sensor output electrons per unit fluorescent area due to the excitation photons is:
-
{dot over ({dot over (ė)}e(t)=φe{dot over ({dot over (ė)}e e −(t-t2 )/τe (19) - where φe is the quantum efficiency of the sensor at the excitation wavelength, and τe 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 If(t), thus we are taking a conservative approach.
- Now, as mentioned earlier, the optimal value of t3 is when:
-
{dot over ({dot over (ė)}f(t 3)={dot over ({dot over (ė)}e(t 3) - Therefore, from (18) and (19) we have:
-
φfφ0 {dot over ({dot over ({dot over (n)} e ∈clηe −(t3 -t2 )/τf =φe {dot over ({dot over ({dot over (n)} e e −(t3 -t2 )/τe - and rearranging we find:
-
- From the previous two sections, we have the following two working equations:
-
- where F=∈clη and Δt=t3−t2. We also know that, in practice, t2−t1>>τf.
- 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 1=8(10)−6 m. - 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,
-
- 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 τe=0.5 ns and using Pulsar 650 specifications, Δt can be determined:
-
- 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:
-
{dot over ({dot over ({dot over (n)}l ={dot over ({dot over ({dot over (n)} l0 cos(θ) - where {dot over ({dot over ({dot over (n)}l 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 {dot over ({dot over ({dot over (n)}l0 is the valve of {dot over ({dot over ({dot over (n)}l in the forward axial direction.
- The total number of photons emitted by the LED per unit time is:
-
- Now,
-
- Substituting this into (24):
-
- Rearranging, we have:
-
- The LED's output power is 0.515 W and νe=6.52(10)14 Hz, therefore:
-
- Substituting this value into (26) we have:
-
- Referring to
FIG. 61 , theoptical centre 252 and thelens 254 of theLED 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 theLED 26 to thephotodiode 184 is approximately: -
- It will be appreciated that the
central photodiode 184 of thephotodiode 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:
-
- Now referring to equation (29):
-
n s=φ0 {dot over (n)} e Fτ f e −Δt/τf -
n s=(0.5)[1.22(10)13][3.42(10)−5][1(10)−6 ]e −4.32(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 ID=20 mA, the LED has a minimum optical power output of p1=240 μW centred at λe=340 nm (the absorption wavelength of the terbium chelate). Driving the LED at ID=200 mA would increase the output power linearly to p1=2.4 mW. By placing the LED'soptical centre 252, 17.5 mm away from thehybridization 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. -
- Using
equation 28, we have: -
- Now, recalling
equation 22 and using the Tb chelate properties listed previously, -
- 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.
- 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 toFIG. 54 . - Utilizing a terbium chelate fluorophore and a SET UVTOP335TO39BL LED, we calculated 11600 photons reaching our 16 micron×16
micron photodiode 184 from therespective hybridization chamber 180. In performing this calculation we assumed that the light-collecting region of ourhybridization chamber 180 has a base area which is the same as our photodiodeactive area 185, and half of the total number of the hybridization photons reaches thephotodiode 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(a2/(4d0 2+a2)), 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. - 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 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.
2. The microfluidic test module according to claim 1 wherein the outer casing has a guard for protecting the membrane seal from damaging contact.
3. The microfluidic test module according to claim 2 further comprising a sensor and a functional unit for adjusting the microenvironment in response to feedback from the sensor.
4. The microfluidic test module according to claim 3 wherein the sensor and the functional unit are fabricated in the microfluidic device.
5. The microfluidic test module according to claim 4 wherein the sensor is a humidity sensor and the functional unit is a humidifier.
6. The microfluidic test module according to claim 5 wherein 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.
7. The microfluidic test module according to claim 6 wherein 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.
8. The microfluidic test module according to claim 6 wherein 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.
9. The microfluidic test module according to claim 8 wherein the heater is annular and positioned about the aperture.
10. The microfluidic test module according to claim 5 wherein 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.
11. The microfluidic test module according to claim 10 wherein the evaporator has a plurality of the supply channels, a corresponding plurality of the apertures and a corresponding plurality of the heaters.
12. The microfluidic test module according to claim 1 wherein 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.
13. The microfluidic test module according to claim 12 wherein the cap has a plurality of reagent reservoirs for different reagents.
14. The microfluidic test module according to claim 4 wherein 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.
15. The microfluidic test module according to claim 14 wherein the cover is a sealing tape with a low tack adhesive for sealing the opening when in the closed position.
16. The microfluidic test module according to claim 9 wherein the MST layer incorporates heaters for heating fluid in the MST channels.
17. The microfluidic test module according to claim 16 wherein 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.
18. The microfluidic test module according to claim 14 wherein the outer casing has a lancet for finger pricking a patient to obtain a blood sample for insertion into the receptacle.
19. The microfluidic test module according to claim 18 wherein 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.
20. The microfluidic test module according to claim 19 wherein 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.
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US13/150,119 Abandoned US20110312763A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with in-loc storage of all required reagents |
US13/150,113 Abandoned US20110312757A1 (en) | 2010-06-17 | 2011-06-01 | Reagent microvial with digital memory |
US13/149,936 Abandoned US20110312646A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for separating constituents of intermediate size from larger and smaller constituents in a biological sample |
US13/150,038 Abandoned US20110312540A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescent probes and calibration probes lacking a luminophore |
US13/149,927 Abandoned US20110312075A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel incubation and parallel dna and rna amplification functionality |
US13/149,969 Abandoned US20110312672A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic assembly with test module and detachable indicator module |
US13/150,175 Abandoned US20110312601A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with digital memory for secure storage of data |
US13/149,950 Abandoned US20110312655A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr, probe hybridization and electrochemiluminescent detection of probe-target hybrids |
US13/150,112 Abandoned US20110312756A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low reagent volumes |
US13/150,120 Abandoned US20110311418A1 (en) | 2010-06-17 | 2011-06-01 | Microvial with digital memory for storage of reagent specification data |
US13/149,924 Abandoned US20110312551A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis which performs nucleic acid amplification before removing non-nucleic acid constituents in a dialysis section |
US13/150,107 Abandoned US20110312586A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for chemically and thermally lysing cells |
US13/149,934 Abandoned US20110312644A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for simultaneous detection of multiple conditions in a patient |
US13/149,898 Expired - Fee Related US8349277B2 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having LOC and dialysis device for separating pathogens from other constituents in a biological sample |
US13/149,921 Abandoned US20110312550A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section |
US13/150,021 Abandoned US20110312565A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using hybridization chamber array and negative control chamber containing probes without electrochemiluminescent reporter |
US13/150,027 Abandoned US20110312568A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,037 Abandoned US20110312704A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr amplification using low pcr mixture volume |
US13/150,269 Abandoned US20110312838A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with electrochemiluminescent probes and integrated photosensor for detection of target molecules |
US13/150,233 Abandoned US20110312806A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with humidifier |
US13/150,265 Abandoned US20110312834A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection using a ruthenium organic complex |
US13/150,099 Abandoned US20110312746A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with chemical lysis section |
US13/150,082 Abandoned US20110312735A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with nucleic acid amplification section |
US13/149,979 Abandoned US20110312555A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting hybridization of target nucleic acid sequences with electrochemiluminescent resonant energy transfer, primer-linked, stem-and-loop probes |
US13/149,959 Abandoned US20110312664A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a dedicated reader |
US13/150,248 Abandoned US20110312819A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescence of a luminophore in the presence of an electrochemical coreactant |
US13/150,018 Abandoned US20110312697A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled pcr section |
US13/150,065 Abandoned US20110312726A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with controllable shunts inside integrated photodiodes |
US13/149,939 Abandoned US20110312648A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for genetic and mitochondrial analysis of a biological sample |
US13/150,159 Abandoned US20110312775A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with digital memory |
US13/150,242 Abandoned US20110308313A1 (en) | 2010-06-17 | 2011-06-01 | Humidity sensor |
US13/150,229 Abandoned US20110312803A1 (en) | 2010-06-17 | 2011-06-01 | System for variable loading of reagents into microfluidic device for genetic analysis |
US13/150,126 Abandoned US20110312541A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with primer-linked linear probes |
US13/149,970 Abandoned US20110312673A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device with multi-layer structure |
US13/149,956 Abandoned US20110312661A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with array of chambers and corresponding diffusion barriers for electrochemiluminescent detection of targets |
US13/149,929 Abandoned US20110311411A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic thermal bend actuated surface tension valve |
US13/150,261 Abandoned US20110312612A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences with probes between a working electrode and a photosensor |
US13/150,040 Abandoned US20110312705A1 (en) | 2010-06-17 | 2011-06-01 | Test module for pcr amplification using low pcr mixture volume |
US13/150,087 Abandoned US20110312615A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with parallel nucleic acid amplification section |
US13/149,953 Abandoned US20110312658A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for concentrating nucleated cells in a biological sample |
US13/150,061 Expired - Fee Related US8388910B2 (en) | 2010-06-17 | 2011-06-01 | Portable test module for excitation of electrochemiluminescent probes |
US13/150,266 Abandoned US20110312835A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with electrochemiluminescent probes and integrated photosensor for detection of target sequences |
US13/150,243 Abandoned US20110312814A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module with excitation source |
US13/149,991 Abandoned US20110312556A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with trigger photodiode in each hybridization chamber |
US13/150,163 Abandoned US20110312777A1 (en) | 2010-06-17 | 2011-06-01 | Test module with digital memory |
US13/150,202 Abandoned US20110312794A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates epidemiological databases with location data |
US13/150,093 Abandoned US20110312741A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for analysis of mitochondrial dna |
US13/150,055 Abandoned US20110312717A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, incubation and nucleic acid amplification |
US13/150,251 Abandoned US20110312822A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for electrochemiluminescent detection of target nucleic acid sequences |
US13/150,143 Abandoned US20110312842A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device |
US13/150,022 Abandoned US20110312699A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with on-chip semiconductor controlled pcr section |
US13/150,184 Expired - Fee Related US8425845B2 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis LOC with hybridization array with calibration chamber containing probe that lacks a reporter |
US13/150,020 Abandoned US20110312564A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and nucleic acid amplification |
US13/149,962 Abandoned US20110312666A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with triggered photodetection of fluorescing probe-target hybrid |
US13/150,218 Abandoned US20110312609A1 (en) | 2010-06-17 | 2011-06-01 | Test module for orientation-independent operation |
US13/150,044 Abandoned US20110312573A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and parallel nucleic acid amplification |
US13/149,952 Abandoned US20110312657A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a mobile telephone |
US13/150,271 Abandoned US20110312840A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sample inlet, electrochemiluminescent probes and integrated photosensor for detection of target sequences |
US13/149,966 Abandoned US20110312670A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with an ebook reader |
US13/150,270 Abandoned US20110312839A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for electrochemiluminescent detection of target sequences with working electrode in contact with photosensor |
US13/150,264 Abandoned US20110312833A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences using transparent electrodes |
US13/150,160 Abandoned US20110312845A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with laminar structure |
US13/150,075 Abandoned US20110312731A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with large angle of collection of emission light |
US13/150,253 Abandoned US20110312824A1 (en) | 2010-06-17 | 2011-06-01 | Test module with waste storage incorporating porous element |
US13/150,056 Abandoned US20110312718A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using recombinase polymerase amplification |
US13/150,272 Abandoned US20110308945A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with thick electrodes for electrochemiluminescent detection of target sequences |
US13/149,943 Abandoned US20110312076A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with flexible membrane for internal microenvironment pressure-relief |
US13/150,096 Abandoned US20110312616A1 (en) | 2010-06-17 | 2011-06-01 | Test module with parallel dna and rna amplification sections |
US13/150,255 Abandoned US20110312825A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection with electrode pairs having complementary and mutually interdigitated finger formations |
US13/150,000 Abandoned US20110312690A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section having two-dimensional control of input heat flux density |
US13/149,964 Abandoned US20110312668A1 (en) | 2010-06-17 | 2011-06-01 | Loc with dialysis section for retaining insoluble sample constituents after amplification and passing soluble constituents to a detection section |
US13/149,997 Abandoned US20110312558A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and tandem nucleic acid amplification |
US13/150,244 Abandoned US20110312815A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with humidity sensor |
US13/149,897 Abandoned US20110312626A1 (en) | 2010-06-17 | 2011-06-01 | Test module incorporating spectrometer |
US13/149,920 Abandoned US20110312639A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating leukocytes and pathogens from blood |
US13/150,221 Abandoned US20120004145A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for wafer-scale spotting of locs |
US13/150,258 Abandoned US20110312828A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection using electrode pairs optically coupled to photodiode |
US13/150,154 Abandoned US20110312596A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface tension valve at reagent reservoir outlet |
US13/150,006 Abandoned US20110312538A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes for detecting targets in a fluid and a positive control probe for detecting a nucleic acid sequence known to be present |
US13/150,236 Abandoned US20110312808A1 (en) | 2010-06-17 | 2011-06-01 | Test module with controlled exposure of fluorophores to excitation light source |
US13/150,134 Abandoned US20110312770A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with nucleic acid amplification using primers covalently attached to linear probes |
US13/149,907 Abandoned US20110312632A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section and diffusion mixer |
US13/150,059 Abandoned US20110312575A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using a nicking enzyme and a dna polymerase |
US13/150,165 Abandoned US20110312778A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control chambers incorporating probes designed to be noncomplementary to nucleic acid sequences in the amplicon |
US13/150,071 Abandoned US20110312728A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with non-imaging optics |
US13/150,081 Abandoned US20110312527A1 (en) | 2010-06-17 | 2011-06-01 | Method of analysing the nucleic acid content of biological fluid |
US13/150,048 Abandoned US20110312711A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with controllable shunts peripheral to integrated photodiodes |
US13/150,199 Abandoned US20110312851A1 (en) | 2010-06-17 | 2011-06-01 | Device for high density spotting of oligonucleotides |
US13/150,057 Expired - Fee Related US8383065B2 (en) | 2010-06-17 | 2011-06-01 | Test module with integral photosensor for electrochemiluminescent detection of hybridization |
US13/150,153 Abandoned US20120028842A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating probes that hybridize for any amplicon |
US13/150,257 Abandoned US20110312827A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for detection of target sequences with electrochemiluminescent luminophore and functional moiety for quenching photon emissions |
US13/150,127 Abandoned US20120053088A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for biochemical processing and analysis |
US13/150,053 Expired - Fee Related US8394340B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low mass electrochemiluminescent probe spots |
US13/150,122 Abandoned US20110312587A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with primer-linked stem-and-loop probes |
US13/150,110 Abandoned US20110312754A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of mitochondrial dna via electrochemiluminescence modulated hybridization |
US13/150,083 Abandoned US20110312736A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow rate sensor |
US13/149,906 Abandoned US20110312631A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with non-specific nucleic acid amplification section and subsequent specific amplification of particular sequences in a separate section |
US13/150,150 Abandoned US20110312773A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with fault-tolerant multiple valve assembly |
US13/150,051 Abandoned US20110312714A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for amplification of nucleic acids using dna polymerases of thermophiles |
US13/150,117 Abandoned US20110312761A1 (en) | 2010-06-17 | 2011-06-01 | Test module for chemically and thermally lysing cells |
US13/149,890 Abandoned US20110312622A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume hybridization chambers for electrochemiluminescent detection of target sequences |
US13/150,262 Abandoned US20110312831A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequence with electrochemiluminescent metalorganic complex |
US13/149,978 Abandoned US20110312678A1 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having dialysis device, loc and interconnecting cap |
US13/150,180 Abandoned US20110312785A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device for spotting fixed array of locs |
US13/150,168 Abandoned US20110312779A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for operation under external microprocessor control |
US13/149,910 Abandoned US20110312549A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with multi-layer dialysis section |
US13/149,914 Abandoned US20110312636A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating leukocytes from blood |
US13/150,135 Expired - Fee Related US8383064B2 (en) | 2010-06-17 | 2011-06-01 | Genetic test module with low oligonucleotide probe mass and reagent volumes |
US13/150,109 Abandoned US20110312753A1 (en) | 2010-06-17 | 2011-06-01 | Loc with integral led driver for excitation led |
US13/150,152 Abandoned US20110312844A1 (en) | 2010-06-17 | 2011-06-01 | Biochemical deposition device |
US13/150,220 Abandoned US20110312610A1 (en) | 2010-06-17 | 2011-06-01 | Test module with long fluorescence lifetime probes |
US13/150,224 Abandoned US20110312855A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for high density spotting of oligonucleotides |
US13/150,193 Abandoned US20110312789A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with flash memory |
US13/150,124 Abandoned US20110312766A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with feedback controlled incubation section |
US13/150,128 Abandoned US20110312767A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubation section having temperature feedback |
US13/149,981 Abandoned US20110312680A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting hybridization of target nucleic acid sequences with electrochemiluminescent resonant energy transfer, primer-linked, linear probes |
US13/149,975 Abandoned US20110312677A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of targets with electrochemiluminescent resonant energy transfer probes |
US13/150,213 Abandoned US20110312800A1 (en) | 2010-06-17 | 2011-06-01 | Test module for gravity-independent operation |
US13/150,235 Abandoned US20110312807A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with a membrane seal to prevent dehumidification of the mixture |
US13/150,179 Abandoned US20110312603A1 (en) | 2010-06-17 | 2011-06-01 | Test module with loc having on-chip electronics for module control |
US13/150,079 Abandoned US20110312733A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with nucleic acid amplification section |
US13/150,036 Abandoned US20110312571A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and parallel nucleic acid amplification |
US13/150,240 Abandoned US20110312812A1 (en) | 2010-06-17 | 2011-06-01 | Genetic test module with feedback-controlled humidifier |
US13/149,908 Abandoned US20110312548A1 (en) | 2010-06-17 | 2011-06-01 | Test module with diffusive mixing in small cross sectional area microchannel |
US13/150,097 Abandoned US20110312744A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for amplifying mitochondrial dna in a biological sample |
US13/149,984 Abandoned US20110312682A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for amplifying and detecting target nucleic acid sequences using electrochemiluminescent resonant energy transfer, stem-and-loop probes with covalently attached primers |
US13/150,267 Abandoned US20110312836A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for electrochemiluminescent detection of target sequences |
US13/150,178 Abandoned US20110312784A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting targets with probes, detection photodiodes and a calibration photodiode |
US13/150,035 Abandoned US20110312703A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for rapid pcr amplification |
US13/149,992 Abandoned US20110312557A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and parallel nucleic acid amplification |
US13/150,003 Abandoned US20110312693A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with feedback controlled pcr section |
US13/149,965 Abandoned US20110312669A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with electrochemiluminescent probes and photosensor with large angle of collection for probe emittted light |
US13/150,200 Abandoned US20110312793A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low mass of probes |
US13/150,068 Abandoned US20110312577A1 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume hybridization chambers and reagent reservoir for electrochemiluminescent detection of target nucleic acid sequences |
US13/149,895 Abandoned US20110312625A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification |
US13/150,070 Abandoned US20110312579A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel incubation and parallel nucleic acid amplification functionality |
US13/150,130 Abandoned US20110312768A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with pcr amplification using primers covalently attached to stem-and-loop probes |
US13/149,925 Abandoned US20120052562A1 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having laminar structure and sample receptacle |
US13/150,046 Abandoned US20110312709A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescent probes and calibration probes with detection photosensors and calibration photosensors |
US13/150,167 Abandoned US20110312599A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with a pcr section with single activation, outlet valve |
US13/150,085 Abandoned US20110312582A1 (en) | 2010-06-17 | 2011-06-01 | Test module with nucleic acid amplification section |
US13/150,234 Abandoned US20110312856A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for dispensing reagents, loading oligonucleotide spotting devices and spotting oligonucleotide probes |
US13/150,042 Abandoned US20110312707A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for pcr amplification of nucleic acids |
US13/150,249 Abandoned US20110312820A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and prisms for simultaneous excitation of oligonucleoutide probes |
US13/150,101 Abandoned US20110312747A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for biochemical processing and analysis |
US13/150,033 Abandoned US20110312077A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences in a fluid with calibration chamber containing probes designed to be non-complementary with any nucleic acid sequences in the fluid |
US13/150,195 Abandoned US20110312790A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low-volume hybridization chamber |
US13/150,151 Abandoned US20110312595A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with mixing section |
US13/150,209 Abandoned US20110309275A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly |
US13/150,194 Abandoned US20110312850A1 (en) | 2010-06-17 | 2011-06-01 | Biochemical deposition device with high deposition rate |
US13/150,142 Abandoned US20110312591A1 (en) | 2010-06-17 | 2011-06-01 | Loc with low-volume hybridization chamber and reagent reservoir for genetic analysis |
US13/149,995 Abandoned US20110312687A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with low volume hybridization chambers and reagent reservoirs for genetic analysis using electrochemiluminescent target detection |
US13/150,156 Abandoned US20110312617A1 (en) | 2010-06-17 | 2011-06-01 | Monolithic microsystems technology device for oligonucleotide spotting |
US13/150,155 Abandoned US20110312774A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for diffusive mixing in small cross sectional area microchannel |
US13/150,245 Abandoned US20110312816A1 (en) | 2010-06-17 | 2011-06-01 | Test module with led for simultaneous excitation of oligonucleoutide probes |
US13/150,225 Abandoned US20110312801A1 (en) | 2010-06-17 | 2011-06-01 | Test module with lanthanide metal-ligand complex fluorophore |
US13/149,972 Abandoned US20110312675A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device with flow-channel structure for capillary-driven fluidic propulsion without trapped air bubbles |
US13/149,999 Abandoned US20110312689A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sensor-triggered photodetection of fluorescent probe-target hybrid |
US13/150,030 Abandoned US20110312701A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences with calibrated photodetection of probes in hybridization array |
US13/150,147 Abandoned US20110312593A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubator having two-dimensional control of input heat flux |
US13/150,211 Abandoned US20110312799A1 (en) | 2010-06-17 | 2011-06-01 | Usb-interfaceable portable test module for detection of hybridized probes |
US13/150,076 Abandoned US20110312581A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with nucleic acid amplification chamber heater bonded to chamber interior |
US13/150,131 Expired - Fee Related US8354074B2 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume reagent reservoir |
US13/150,161 Abandoned US20110312776A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating reporters |
US13/150,223 Abandoned US20110312611A1 (en) | 2010-06-17 | 2011-06-01 | Test module with transition metal-ligand complex fluorophore |
US13/149,894 Pending US20110312624A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure having active valve for capillary-driven fluidic propulsion without trapped air bubbles |
US13/150,141 Abandoned US20110312082A1 (en) | 2010-06-17 | 2011-06-01 | Dispensing apparatus for wafer-scale dispensing of reagents |
US13/149,947 Abandoned US20110312653A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume hybridization chambers |
US13/150,252 Abandoned US20110312823A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and mirrors for simultaneous excitation of oligonucleoutide probes |
US13/150,041 Abandoned US20110312706A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with hybridization chambers containing probes for electrochemiluminescent detection of target nucleic acid sequences in a fluid and calibration chamber containing probes sealed from the fluid |
US13/149,912 Abandoned US20110312635A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure for capillary-driven fluidic propulsion without trapped air bubbles |
US13/150,174 Abandoned US20110312783A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control comprising empty chambers |
US13/149,893 Abandoned US20110312545A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification |
US13/149,902 Abandoned US20110312629A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section having stomata tapering counter to flow direction |
US13/149,900 Abandoned US20110312628A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with mst layer and overlying cap |
US13/150,177 Abandoned US20110312602A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal bend actuated surface tension valve |
US13/150,263 Abandoned US20110312832A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of target sequences with electrochemiluminescent probes in hybridization chambers |
US13/150,019 Abandoned US20110312698A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section having short thermal cycle times |
US13/150,108 Abandoned US20110312752A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume reagent reservoir |
US13/150,091 Abandoned US20110312584A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module with driver for excitation of electrochemiluminescent luminophores |
US13/150,115 Abandoned US20110312759A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with reagent reservoir |
US13/150,139 Abandoned US20110312590A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with elongate incubation chamber |
US13/150,204 Abandoned US20110312795A1 (en) | 2010-06-17 | 2011-06-01 | Diagnostic test module with a loc with integral photosensor and excitation led for detection of hybridization assay results |
US13/149,935 Abandoned US20110312645A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled hybridization chambers for electrochemiluminescent detection of targets |
US13/149,903 Abandoned US20110312547A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by number of active outlet valves |
US13/150,121 Abandoned US20110312764A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubator |
US13/150,089 Abandoned US20110312740A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with capillary meniscus marching velocity sensor |
US13/150,146 Abandoned US20110311394A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal bend actuated surface tension valve |
US13/150,067 Abandoned US20110312727A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel nucleic acid amplification functionality |
US13/150,004 Abandoned US20110312694A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with delay-triggered photodetection of fluorescent probe-target hybrid |
US13/150,116 Abandoned US20110312760A1 (en) | 2010-06-17 | 2011-06-01 | Reagent microvial with authentication integrated circuit |
US13/150,182 Abandoned US20110312604A1 (en) | 2010-06-17 | 2011-06-01 | Loc having on-chip electronics for use in a test module to control module communications |
US13/150,259 Abandoned US20110312829A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis and electrochemiluminescent detection of target sequences |
US13/149,931 Abandoned US20110312641A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sample inlet and probe hybridization section |
US13/150,125 Abandoned US20110312069A1 (en) | 2010-06-17 | 2011-06-01 | Microvial with digital memory for storage of oligonucleotide specification data |
US13/149,892 Abandoned US20110312623A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis, nucleic acid amplification and prehybridization filtering |
US13/150,183 Abandoned US20110312848A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for wafer-scale spotting of locs |
US13/150,228 Abandoned US20110312802A1 (en) | 2010-06-17 | 2011-06-01 | Test module with probes suspended in fluid |
US13/150,054 Abandoned US20110312716A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using an isothermal reaction |
US13/149,958 Abandoned US20110312663A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with time delayed detection of fluorescence from hybridized probes |
US13/150,028 Abandoned US20110312569A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with small cross sectional area microchannel |
US13/150,077 Abandoned US20110312732A1 (en) | 2010-06-17 | 2011-06-01 | Test module using lanthanide metal-ligand complex, electrochemiluminescent luminophores |
US13/150,066 Abandoned US20110312576A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for multi-stage amplification of nucleic acid sequences |
US13/149,990 Abandoned US20110312685A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pcr using adaptor primers and target detection using electrochemiluminescent resonant energy transfer probes |
US13/149,891 Abandoned US20110312841A1 (en) | 2010-06-17 | 2011-06-01 | Fabrication system for lab-on-a-chip (loc) devices with differing application specific functionality |
US13/150,172 Abandoned US20110312782A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with digital memory |
US13/150,049 Abandoned US20110312712A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for pcr amplification of nucleic acids from whole blood |
US13/150,192 Abandoned US20110312607A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration photosensor output subtracted in a differential circuit from the output of hybridization photosensors |
US13/149,985 Abandoned US20110312683A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with tablet computer |
US13/150,074 Abandoned US20110312730A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel dna and rna amplification functionality |
US13/149,941 Abandoned US20110312649A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with optically transparent hybridization chambers |
US13/150,227 Abandoned US20110309276A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal boiling-initiated valve |
US13/150,164 Abandoned US20110312846A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with fluidics on both sides of supporting substrate |
US13/150,260 Abandoned US20110312830A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with electrochemiluminescent probes having a functional moiety for quenching photon emissions configured to change proximity to a luminophore upon forming a probe-target hybrid |
US13/150,208 Abandoned US20110312852A1 (en) | 2010-06-17 | 2011-06-01 | Robotic system for loading oligonucleotides into spotting devices |
US13/150,238 Abandoned US20110312810A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for detection of hybridization of targets with oligonucleotide probes |
US13/150,222 Abandoned US20110311415A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal bend-actuator surface tension valve |
US13/150,078 Abandoned US20110312526A1 (en) | 2010-06-17 | 2011-06-01 | Method of analysing the nucleic acid content of a blood sample |
US13/149,937 Abandoned US20110312647A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled hybridization chambers |
US13/150,189 Abandoned US20110312618A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with high spotting rate |
US13/149,917 Abandoned US20110312637A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating pathogens from a biological sample |
US13/149,942 Abandoned US20110312650A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with optically transparent hybridization chambers for electrochemiluminescent detection of targets |
US13/149,968 Abandoned US20110312671A1 (en) | 2010-06-17 | 2011-06-01 | Single use microfluidic device with photosensor for electrochemiluminescent detection of targets |
US13/149,944 Abandoned US20110312651A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low mass probe spots |
US13/150,032 Abandoned US20110312702A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and nucleic acid amplification |
US13/150,084 Abandoned US20110312737A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for electrochemiluminescent detection of targets |
US13/150,118 Abandoned US20110312762A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with fluorescence resonance energy transfer (fret) probes |
US13/149,955 Abandoned US20110312660A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a laptop computer |
US13/150,007 Abandoned US20110312559A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and parallel nucleic acid amplification |
US13/150,137 Abandoned US20110312081A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus for array of microfluidic devices |
US13/150,230 Abandoned US20110312804A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with aperture with geometry to promote unpinned flow-through of fluid |
US13/150,148 Abandoned US20110312843A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device for complete assay spotting of locs |
US13/149,960 Abandoned US20110312553A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with non-imaging optics for electrochemiluminescent detection of targets |
US13/150,268 Abandoned US20110312837A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis and electrochemiluminescent detection of target sequences |
US13/150,029 Abandoned US20110312570A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting target nucleic acid sequences with probes having long fluorescence lifetime fluorophores |
US13/150,105 Abandoned US20110312750A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with total reagent storage |
US13/150,196 Abandoned US20110312791A1 (en) | 2010-06-17 | 2011-06-01 | Test module with fault-tolerant multiple valve assembly |
US13/150,050 Expired - Fee Related US8398939B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low-volume hybridization chambers for electrochemiluminescent detection of target nucleic acid sequences in a fluid |
US13/149,913 Abandoned US20110312071A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with large channels for cell transport and small channels suitable for biochemical processes |
US13/150,073 Abandoned US20110312729A1 (en) | 2010-06-17 | 2011-06-01 | Test module using transition metal-ligand complex, electrochemiluminescent luminophores |
US13/149,986 Abandoned US20110312537A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for amplifying and detecting target nucleic acid sequences using electrochemiluminescent resonant energy transfer, linear probes with covalently attached primers |
US13/150,111 Abandoned US20110312755A1 (en) | 2010-06-17 | 2011-06-01 | Test module with chemical lysis section |
US13/149,930 Abandoned US20110312552A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with conductivity sensor |
US13/150,002 Abandoned US20110312692A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and nucleic acid amplification |
US13/150,205 Abandoned US20110311407A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic boiling-initiated valve |
US13/150,149 Abandoned US20110312594A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization probes including positive and negative control probes |
US13/150,024 Abandoned US20110312567A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences using hybridization chamber array and negative control chamber without probes |
US13/150,144 Abandoned US20110312592A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubation chamber between supporting substrate and heater |
US13/150,072 Abandoned US20110312580A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with nucleic acid amplification section and thermal insulation trench |
US13/150,207 Abandoned US20110312797A1 (en) | 2010-06-17 | 2011-06-01 | Portable test module for fluorescence excitation of probe nucleic acid sequences |
US13/149,922 Abandoned US20110312074A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with sample receptacle |
US13/149,971 Abandoned US20110312674A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral photosensor for electrochemiluminescence based detection of targets |
US13/149,946 Abandoned US20110312652A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume electrochemiluminescence-based probe spots |
US13/150,201 Expired - Fee Related US8398938B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic thermal bend actuated pressure pulse valve |
US13/149,957 Abandoned US20110312662A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for removing cell debris from a biological sample |
US13/150,047 Abandoned US20110312710A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and nucleic acid amplification |
US13/150,063 Abandoned US20110312724A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with incubation, nucleic acid amplification and prehybridization filtering |
US13/150,016 Abandoned US20110312562A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and parallel nucleic acid amplification |
US13/150,197 Abandoned US20110312792A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates epidemiological databases |
US13/149,976 Abandoned US20110312068A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device for separating nucleated cells in a biological sample from other constituents |
US13/150,176 Abandoned US20110312847A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device with stored oligonucleotide specification data |
US13/150,062 Abandoned US20110312723A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using nucleic acid sequence based amplification |
US13/150,045 Abandoned US20110312708A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for isothermal amplification of nucleic acids |
US13/150,206 Abandoned US20110312796A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates medical databases |
US13/150,188 Abandoned US20110312542A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration chamber containing chamber with a blocked inlet spotted with reporter |
US13/150,060 Abandoned US20110312721A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, incubation, and nucleic acid amplification |
US13/150,031 Abandoned US20110312070A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr chamber for high rate of temperature change |
US13/149,967 Abandoned US20110312554A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis device, loc and interconnecting cap |
US13/150,095 Abandoned US20110312743A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of target nucleic acid sequences using electrodes configured for electrochemiluminescence of luminophores without a coreactant |
US13/150,246 Abandoned US20110312817A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with humidity sensor |
US13/150,191 Abandoned US20110312606A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with digital memory |
US13/149,954 Abandoned US20110312659A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with hybridization chambers and corresponding diffusion barriers |
US13/150,129 Abandoned US20110311408A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus |
US13/150,166 Abandoned US20110312079A1 (en) | 2010-06-17 | 2011-06-01 | Loc with digital memory to store epidemiological updates |
US13/150,138 Abandoned US20110312772A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with pcr amplification using linker primers |
US13/149,993 Abandoned US20110312686A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with elongate pcr chambers |
US13/150,170 Abandoned US20110312781A1 (en) | 2010-06-17 | 2011-06-01 | Loc with digital memory to store genetic data updates |
US13/150,247 Abandoned US20110312818A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and lens for simultaneous excitation of oligonucleoutide probes |
US13/150,094 Abandoned US20110312742A1 (en) | 2010-06-17 | 2011-06-01 | Single-use microfluidic device |
US13/150,086 Abandoned US20110312738A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with liquid sensor |
US13/150,136 Abandoned US20110312771A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pwm controlled incubation section |
US13/150,133 Abandoned US20110311409A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus with automatic collection and storage of reagent data |
US13/149,983 Abandoned US20110312681A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for removing erythrocytes from blood |
US13/149,904 Abandoned US20110312630A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,226 Abandoned US20110312620A1 (en) | 2010-06-17 | 2011-06-01 | System for variable microarray spotting and genetic analysis |
US13/149,980 Abandoned US20110312679A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface-micromachined dialysis section |
US13/150,080 Abandoned US20110312734A1 (en) | 2010-06-17 | 2011-06-01 | Test module with suspended electrochemiluminescent probes |
US13/150,025 Abandoned US20110312700A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pwm controlled pcr heater |
US13/150,181 Abandoned US20110312786A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration chamber containing probe designed to be noncomplementary to nucleic acid sequences in the amplicon |
US13/150,090 Abandoned US20110312583A1 (en) | 2010-06-17 | 2011-06-01 | Test module with parallel nucleic acid amplification sections |
US13/150,241 Abandoned US20110312813A1 (en) | 2010-06-17 | 2011-06-01 | Single-use genetic test module |
US13/150,098 Abandoned US20110312745A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with photosensor |
US13/150,102 Abandoned US20110312748A1 (en) | 2010-06-17 | 2011-06-01 | Loc with integral photosensor for detection of hybridization assay results |
US13/149,899 Abandoned US20110312546A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,232 Abandoned US20110312621A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for dispensing reagents and loading oligonucleotide spotting devices |
US13/149,996 Abandoned US20110312688A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr chamber between supporting substrate and heater |
US13/150,214 Abandoned US20110311413A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with liquid detector sensor feedback |
US13/150,219 Abandoned US20110311414A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal bend-actuator pressure pulse valve |
US13/150,009 Abandoned US20110312560A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and tandem nucleic acid amplification |
US13/150,173 Abandoned US20110312600A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal bend actuated pressure pulse valve |
US13/149,961 Abandoned US20110312665A1 (en) | 2010-06-17 | 2011-06-01 | Loc with dialysis section for removing insoluble sample constituents from a nucleic acid mixture |
US13/150,239 Abandoned US20110312811A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences with electrodes profiled for greater peripheral edge length |
US13/150,023 Abandoned US20110312566A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and parallel nucleic acid amplification |
US13/150,123 Abandoned US20110312765A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with low oligonucleotide probe mass and low reagent volume |
US13/150,186 Abandoned US20110312849A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for ejecting low volume droplets |
US13/150,250 Abandoned US20110312821A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with waste storage |
US13/149,951 Abandoned US20110312656A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr and probe hybridization |
US13/150,058 Abandoned US20110312720A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis and nucleic acid amplification |
US13/149,918 Abandoned US20110312638A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification |
US13/150,169 Abandoned US20110312780A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control chambers incorporating probes with no reporters |
US13/150,217 Abandoned US20110312854A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for spotting arrays of locs |
US13/150,162 Abandoned US20110312598A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by outlet valve numbers |
US13/150,092 Abandoned US20110312585A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with parallel dna and rna amplification section |
US13/149,909 Abandoned US20110312633A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section |
US13/150,158 Abandoned US20110311395A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with active valve at reagent reservoir outlet |
US13/150,008 Abandoned US20110312539A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes for detecting targets in a fluid and a positive control probe without a quencher for luminophore emissions |
US13/149,974 Abandoned US20110312067A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device for separating pathogens from a biological sample |
US13/150,140 Abandoned US20110311393A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal bend actuated pressure pulse valve |
US13/150,114 Abandoned US20110312758A1 (en) | 2010-06-17 | 2011-06-01 | Test module with thermal lysis section |
US13/149,973 Abandoned US20110312676A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral driver for excitation of electrochemiluminescent luminophores |
US13/150,157 Abandoned US20110312597A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating probes with no quenchers |
US13/150,216 Abandoned US20110312608A1 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume hybridization chamber and low-volume reagent reservoir |
US13/150,011 Abandoned US20110312695A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with hybridization chamber array with positive control chamber containing electrochemiluminescent reporter |
US13/150,132 Expired - Fee Related US8394339B2 (en) | 2010-06-17 | 2011-06-01 | LOC device with on-chip semiconductor controlled incubation section |
US13/149,916 Abandoned US20110312072A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface micro-machined chips and interconnecting cap |
US13/150,256 Abandoned US20110312826A1 (en) | 2010-06-17 | 2011-06-01 | Test module with laser for excitation of oligonucleoutide probes |
US13/150,052 Abandoned US20110312574A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and parallel nucleic acid amplification |
US13/150,187 Abandoned US20110312605A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral controller |
US13/150,104 Abandoned US20110312749A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal lysis section |
US13/150,190 Abandoned US20110312788A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal boiling-initiated valve |
US13/150,237 Abandoned US20110312809A1 (en) | 2010-06-17 | 2011-06-01 | Test module with humidifier |
US13/150,203 Abandoned US20110312619A1 (en) | 2010-06-17 | 2011-06-01 | Device for high-density deposition of biochemicals |
US13/149,963 Abandoned US20110312667A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a desktop computer |
US13/150,069 Abandoned US20110312578A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for non-specific nucleic acid amplification prior to specific amplification of particular sequences |
US13/149,933 Abandoned US20110312643A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of hybridization of nucleic acid targets |
US13/150,088 Abandoned US20110312739A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for pcr amplification of targets and electrochemiluminescent detection of targets |
US13/150,001 Abandoned US20110312691A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes including positive and negative control probes |
US13/150,212 Abandoned US20110312853A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot |
US13/150,039 Abandoned US20110312572A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and nucleic acid amplification |
US13/150,106 Abandoned US20110312751A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of mitochondrial dna via fluorescence modulated by hybridization |
US13/150,014 Abandoned US20110312561A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with photodiodes with controllable shunts to detect fluorescing hybridized probes |
US13/150,210 Abandoned US20110312798A1 (en) | 2010-06-17 | 2011-06-01 | Test module with inbuilt lancet |
US13/149,928 Abandoned US20110312640A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with photosensor |
US13/150,231 Abandoned US20110312805A1 (en) | 2010-06-17 | 2011-06-01 | Test module with time delayed detection of fluorescence from hybridized probe |
US13/149,911 Abandoned US20110312634A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with laminar structure |
US13/150,012 Abandoned US20110312696A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and nucleic acid amplification |
US13/149,989 Abandoned US20110312684A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and nucleic acid amplification |
US13/150,185 Abandoned US20110312787A1 (en) | 2010-06-17 | 2011-06-01 | Loc having usb device driver for use in a test module to control usb connection |
US13/150,017 Abandoned US20110312563A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences in a fluid using hybridization chamber array and negative control chamber containing electrochemiluminescent probe designed to be non-complementary to any sequence in the fluid |
US13/150,064 Expired - Fee Related US8398940B2 (en) | 2010-06-17 | 2011-06-01 | USB-interfaceable portable test module for electrochemiluminescent detection of targets |
US13/149,932 Abandoned US20110312642A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of nucleic acid targets with electrochemiluminescent probes |
US13/150,100 Abandoned US20110312078A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting target nucleic acid sequences in mitochondrial dna |
US13/149,948 Abandoned US20110312654A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for loading oligonucleotide spotting devices and spotting oligonucleotide probes |
US13/149,919 Abandoned US20110312073A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module incorporating surface micro-machined chips and interconnecting cap |
US13/685,105 Abandoned US20130079254A1 (en) | 2010-06-17 | 2012-11-26 | Microfluidic dialysis device |
Family Applications Before (66)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/150,119 Abandoned US20110312763A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with in-loc storage of all required reagents |
US13/150,113 Abandoned US20110312757A1 (en) | 2010-06-17 | 2011-06-01 | Reagent microvial with digital memory |
US13/149,936 Abandoned US20110312646A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for separating constituents of intermediate size from larger and smaller constituents in a biological sample |
US13/150,038 Abandoned US20110312540A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescent probes and calibration probes lacking a luminophore |
US13/149,927 Abandoned US20110312075A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel incubation and parallel dna and rna amplification functionality |
US13/149,969 Abandoned US20110312672A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic assembly with test module and detachable indicator module |
US13/150,175 Abandoned US20110312601A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with digital memory for secure storage of data |
US13/149,950 Abandoned US20110312655A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr, probe hybridization and electrochemiluminescent detection of probe-target hybrids |
US13/150,112 Abandoned US20110312756A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low reagent volumes |
US13/150,120 Abandoned US20110311418A1 (en) | 2010-06-17 | 2011-06-01 | Microvial with digital memory for storage of reagent specification data |
US13/149,924 Abandoned US20110312551A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis which performs nucleic acid amplification before removing non-nucleic acid constituents in a dialysis section |
US13/150,107 Abandoned US20110312586A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for chemically and thermally lysing cells |
US13/149,934 Abandoned US20110312644A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for simultaneous detection of multiple conditions in a patient |
US13/149,898 Expired - Fee Related US8349277B2 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having LOC and dialysis device for separating pathogens from other constituents in a biological sample |
US13/149,921 Abandoned US20110312550A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis which performs nucleic acid amplification after sample preparation in a dialysis section |
US13/150,021 Abandoned US20110312565A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using hybridization chamber array and negative control chamber containing probes without electrochemiluminescent reporter |
US13/150,027 Abandoned US20110312568A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,037 Abandoned US20110312704A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr amplification using low pcr mixture volume |
US13/150,269 Abandoned US20110312838A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with electrochemiluminescent probes and integrated photosensor for detection of target molecules |
US13/150,233 Abandoned US20110312806A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with humidifier |
US13/150,265 Abandoned US20110312834A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection using a ruthenium organic complex |
US13/150,099 Abandoned US20110312746A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with chemical lysis section |
US13/150,082 Abandoned US20110312735A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with nucleic acid amplification section |
US13/149,979 Abandoned US20110312555A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting hybridization of target nucleic acid sequences with electrochemiluminescent resonant energy transfer, primer-linked, stem-and-loop probes |
US13/149,959 Abandoned US20110312664A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a dedicated reader |
US13/150,248 Abandoned US20110312819A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescence of a luminophore in the presence of an electrochemical coreactant |
US13/150,018 Abandoned US20110312697A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled pcr section |
US13/150,065 Abandoned US20110312726A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with controllable shunts inside integrated photodiodes |
US13/149,939 Abandoned US20110312648A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for genetic and mitochondrial analysis of a biological sample |
US13/150,159 Abandoned US20110312775A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with digital memory |
US13/150,242 Abandoned US20110308313A1 (en) | 2010-06-17 | 2011-06-01 | Humidity sensor |
US13/150,229 Abandoned US20110312803A1 (en) | 2010-06-17 | 2011-06-01 | System for variable loading of reagents into microfluidic device for genetic analysis |
US13/150,126 Abandoned US20110312541A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with primer-linked linear probes |
US13/149,970 Abandoned US20110312673A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device with multi-layer structure |
US13/149,956 Abandoned US20110312661A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with array of chambers and corresponding diffusion barriers for electrochemiluminescent detection of targets |
US13/149,929 Abandoned US20110311411A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic thermal bend actuated surface tension valve |
US13/150,261 Abandoned US20110312612A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences with probes between a working electrode and a photosensor |
US13/150,040 Abandoned US20110312705A1 (en) | 2010-06-17 | 2011-06-01 | Test module for pcr amplification using low pcr mixture volume |
US13/150,087 Abandoned US20110312615A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with parallel nucleic acid amplification section |
US13/149,953 Abandoned US20110312658A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for concentrating nucleated cells in a biological sample |
US13/150,061 Expired - Fee Related US8388910B2 (en) | 2010-06-17 | 2011-06-01 | Portable test module for excitation of electrochemiluminescent probes |
US13/150,266 Abandoned US20110312835A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with electrochemiluminescent probes and integrated photosensor for detection of target sequences |
US13/150,243 Abandoned US20110312814A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module with excitation source |
US13/149,991 Abandoned US20110312556A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with trigger photodiode in each hybridization chamber |
US13/150,163 Abandoned US20110312777A1 (en) | 2010-06-17 | 2011-06-01 | Test module with digital memory |
US13/150,202 Abandoned US20110312794A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates epidemiological databases with location data |
US13/150,093 Abandoned US20110312741A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for analysis of mitochondrial dna |
US13/150,055 Abandoned US20110312717A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, incubation and nucleic acid amplification |
US13/150,251 Abandoned US20110312822A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for electrochemiluminescent detection of target nucleic acid sequences |
US13/150,143 Abandoned US20110312842A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device |
US13/150,022 Abandoned US20110312699A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with on-chip semiconductor controlled pcr section |
US13/150,184 Expired - Fee Related US8425845B2 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis LOC with hybridization array with calibration chamber containing probe that lacks a reporter |
US13/150,020 Abandoned US20110312564A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and nucleic acid amplification |
US13/149,962 Abandoned US20110312666A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with triggered photodetection of fluorescing probe-target hybrid |
US13/150,218 Abandoned US20110312609A1 (en) | 2010-06-17 | 2011-06-01 | Test module for orientation-independent operation |
US13/150,044 Abandoned US20110312573A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and parallel nucleic acid amplification |
US13/149,952 Abandoned US20110312657A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a mobile telephone |
US13/150,271 Abandoned US20110312840A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sample inlet, electrochemiluminescent probes and integrated photosensor for detection of target sequences |
US13/149,966 Abandoned US20110312670A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with an ebook reader |
US13/150,270 Abandoned US20110312839A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for electrochemiluminescent detection of target sequences with working electrode in contact with photosensor |
US13/150,264 Abandoned US20110312833A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences using transparent electrodes |
US13/150,160 Abandoned US20110312845A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with laminar structure |
US13/150,075 Abandoned US20110312731A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with large angle of collection of emission light |
US13/150,253 Abandoned US20110312824A1 (en) | 2010-06-17 | 2011-06-01 | Test module with waste storage incorporating porous element |
US13/150,056 Abandoned US20110312718A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using recombinase polymerase amplification |
US13/150,272 Abandoned US20110308945A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with thick electrodes for electrochemiluminescent detection of target sequences |
Family Applications After (288)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/150,096 Abandoned US20110312616A1 (en) | 2010-06-17 | 2011-06-01 | Test module with parallel dna and rna amplification sections |
US13/150,255 Abandoned US20110312825A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection with electrode pairs having complementary and mutually interdigitated finger formations |
US13/150,000 Abandoned US20110312690A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section having two-dimensional control of input heat flux density |
US13/149,964 Abandoned US20110312668A1 (en) | 2010-06-17 | 2011-06-01 | Loc with dialysis section for retaining insoluble sample constituents after amplification and passing soluble constituents to a detection section |
US13/149,997 Abandoned US20110312558A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and tandem nucleic acid amplification |
US13/150,244 Abandoned US20110312815A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with humidity sensor |
US13/149,897 Abandoned US20110312626A1 (en) | 2010-06-17 | 2011-06-01 | Test module incorporating spectrometer |
US13/149,920 Abandoned US20110312639A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating leukocytes and pathogens from blood |
US13/150,221 Abandoned US20120004145A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for wafer-scale spotting of locs |
US13/150,258 Abandoned US20110312828A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection using electrode pairs optically coupled to photodiode |
US13/150,154 Abandoned US20110312596A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface tension valve at reagent reservoir outlet |
US13/150,006 Abandoned US20110312538A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes for detecting targets in a fluid and a positive control probe for detecting a nucleic acid sequence known to be present |
US13/150,236 Abandoned US20110312808A1 (en) | 2010-06-17 | 2011-06-01 | Test module with controlled exposure of fluorophores to excitation light source |
US13/150,134 Abandoned US20110312770A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with nucleic acid amplification using primers covalently attached to linear probes |
US13/149,907 Abandoned US20110312632A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section and diffusion mixer |
US13/150,059 Abandoned US20110312575A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using a nicking enzyme and a dna polymerase |
US13/150,165 Abandoned US20110312778A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control chambers incorporating probes designed to be noncomplementary to nucleic acid sequences in the amplicon |
US13/150,071 Abandoned US20110312728A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with non-imaging optics |
US13/150,081 Abandoned US20110312527A1 (en) | 2010-06-17 | 2011-06-01 | Method of analysing the nucleic acid content of biological fluid |
US13/150,048 Abandoned US20110312711A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with controllable shunts peripheral to integrated photodiodes |
US13/150,199 Abandoned US20110312851A1 (en) | 2010-06-17 | 2011-06-01 | Device for high density spotting of oligonucleotides |
US13/150,057 Expired - Fee Related US8383065B2 (en) | 2010-06-17 | 2011-06-01 | Test module with integral photosensor for electrochemiluminescent detection of hybridization |
US13/150,153 Abandoned US20120028842A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating probes that hybridize for any amplicon |
US13/150,257 Abandoned US20110312827A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for detection of target sequences with electrochemiluminescent luminophore and functional moiety for quenching photon emissions |
US13/150,127 Abandoned US20120053088A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for biochemical processing and analysis |
US13/150,053 Expired - Fee Related US8394340B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low mass electrochemiluminescent probe spots |
US13/150,122 Abandoned US20110312587A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with primer-linked stem-and-loop probes |
US13/150,110 Abandoned US20110312754A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of mitochondrial dna via electrochemiluminescence modulated hybridization |
US13/150,083 Abandoned US20110312736A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow rate sensor |
US13/149,906 Abandoned US20110312631A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with non-specific nucleic acid amplification section and subsequent specific amplification of particular sequences in a separate section |
US13/150,150 Abandoned US20110312773A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with fault-tolerant multiple valve assembly |
US13/150,051 Abandoned US20110312714A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for amplification of nucleic acids using dna polymerases of thermophiles |
US13/150,117 Abandoned US20110312761A1 (en) | 2010-06-17 | 2011-06-01 | Test module for chemically and thermally lysing cells |
US13/149,890 Abandoned US20110312622A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume hybridization chambers for electrochemiluminescent detection of target sequences |
US13/150,262 Abandoned US20110312831A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequence with electrochemiluminescent metalorganic complex |
US13/149,978 Abandoned US20110312678A1 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having dialysis device, loc and interconnecting cap |
US13/150,180 Abandoned US20110312785A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device for spotting fixed array of locs |
US13/150,168 Abandoned US20110312779A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for operation under external microprocessor control |
US13/149,910 Abandoned US20110312549A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with multi-layer dialysis section |
US13/149,914 Abandoned US20110312636A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating leukocytes from blood |
US13/150,135 Expired - Fee Related US8383064B2 (en) | 2010-06-17 | 2011-06-01 | Genetic test module with low oligonucleotide probe mass and reagent volumes |
US13/150,109 Abandoned US20110312753A1 (en) | 2010-06-17 | 2011-06-01 | Loc with integral led driver for excitation led |
US13/150,152 Abandoned US20110312844A1 (en) | 2010-06-17 | 2011-06-01 | Biochemical deposition device |
US13/150,220 Abandoned US20110312610A1 (en) | 2010-06-17 | 2011-06-01 | Test module with long fluorescence lifetime probes |
US13/150,224 Abandoned US20110312855A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for high density spotting of oligonucleotides |
US13/150,193 Abandoned US20110312789A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with flash memory |
US13/150,124 Abandoned US20110312766A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with feedback controlled incubation section |
US13/150,128 Abandoned US20110312767A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubation section having temperature feedback |
US13/149,981 Abandoned US20110312680A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting hybridization of target nucleic acid sequences with electrochemiluminescent resonant energy transfer, primer-linked, linear probes |
US13/149,975 Abandoned US20110312677A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of targets with electrochemiluminescent resonant energy transfer probes |
US13/150,213 Abandoned US20110312800A1 (en) | 2010-06-17 | 2011-06-01 | Test module for gravity-independent operation |
US13/150,235 Abandoned US20110312807A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with a membrane seal to prevent dehumidification of the mixture |
US13/150,179 Abandoned US20110312603A1 (en) | 2010-06-17 | 2011-06-01 | Test module with loc having on-chip electronics for module control |
US13/150,079 Abandoned US20110312733A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with nucleic acid amplification section |
US13/150,036 Abandoned US20110312571A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and parallel nucleic acid amplification |
US13/150,240 Abandoned US20110312812A1 (en) | 2010-06-17 | 2011-06-01 | Genetic test module with feedback-controlled humidifier |
US13/149,908 Abandoned US20110312548A1 (en) | 2010-06-17 | 2011-06-01 | Test module with diffusive mixing in small cross sectional area microchannel |
US13/150,097 Abandoned US20110312744A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for amplifying mitochondrial dna in a biological sample |
US13/149,984 Abandoned US20110312682A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for amplifying and detecting target nucleic acid sequences using electrochemiluminescent resonant energy transfer, stem-and-loop probes with covalently attached primers |
US13/150,267 Abandoned US20110312836A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for electrochemiluminescent detection of target sequences |
US13/150,178 Abandoned US20110312784A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting targets with probes, detection photodiodes and a calibration photodiode |
US13/150,035 Abandoned US20110312703A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for rapid pcr amplification |
US13/149,992 Abandoned US20110312557A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and parallel nucleic acid amplification |
US13/150,003 Abandoned US20110312693A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with feedback controlled pcr section |
US13/149,965 Abandoned US20110312669A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with electrochemiluminescent probes and photosensor with large angle of collection for probe emittted light |
US13/150,200 Abandoned US20110312793A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low mass of probes |
US13/150,068 Abandoned US20110312577A1 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume hybridization chambers and reagent reservoir for electrochemiluminescent detection of target nucleic acid sequences |
US13/149,895 Abandoned US20110312625A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and tandem nucleic acid amplification |
US13/150,070 Abandoned US20110312579A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel incubation and parallel nucleic acid amplification functionality |
US13/150,130 Abandoned US20110312768A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with pcr amplification using primers covalently attached to stem-and-loop probes |
US13/149,925 Abandoned US20120052562A1 (en) | 2010-06-17 | 2011-06-01 | Test module with microfluidic device having laminar structure and sample receptacle |
US13/150,046 Abandoned US20110312709A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences using electrochemiluminescent probes and calibration probes with detection photosensors and calibration photosensors |
US13/150,167 Abandoned US20110312599A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with a pcr section with single activation, outlet valve |
US13/150,085 Abandoned US20110312582A1 (en) | 2010-06-17 | 2011-06-01 | Test module with nucleic acid amplification section |
US13/150,234 Abandoned US20110312856A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for dispensing reagents, loading oligonucleotide spotting devices and spotting oligonucleotide probes |
US13/150,042 Abandoned US20110312707A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for pcr amplification of nucleic acids |
US13/150,249 Abandoned US20110312820A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and prisms for simultaneous excitation of oligonucleoutide probes |
US13/150,101 Abandoned US20110312747A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for biochemical processing and analysis |
US13/150,033 Abandoned US20110312077A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences in a fluid with calibration chamber containing probes designed to be non-complementary with any nucleic acid sequences in the fluid |
US13/150,195 Abandoned US20110312790A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low-volume hybridization chamber |
US13/150,151 Abandoned US20110312595A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with mixing section |
US13/150,209 Abandoned US20110309275A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly |
US13/150,194 Abandoned US20110312850A1 (en) | 2010-06-17 | 2011-06-01 | Biochemical deposition device with high deposition rate |
US13/150,142 Abandoned US20110312591A1 (en) | 2010-06-17 | 2011-06-01 | Loc with low-volume hybridization chamber and reagent reservoir for genetic analysis |
US13/149,995 Abandoned US20110312687A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with low volume hybridization chambers and reagent reservoirs for genetic analysis using electrochemiluminescent target detection |
US13/150,156 Abandoned US20110312617A1 (en) | 2010-06-17 | 2011-06-01 | Monolithic microsystems technology device for oligonucleotide spotting |
US13/150,155 Abandoned US20110312774A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for diffusive mixing in small cross sectional area microchannel |
US13/150,245 Abandoned US20110312816A1 (en) | 2010-06-17 | 2011-06-01 | Test module with led for simultaneous excitation of oligonucleoutide probes |
US13/150,225 Abandoned US20110312801A1 (en) | 2010-06-17 | 2011-06-01 | Test module with lanthanide metal-ligand complex fluorophore |
US13/149,972 Abandoned US20110312675A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device with flow-channel structure for capillary-driven fluidic propulsion without trapped air bubbles |
US13/149,999 Abandoned US20110312689A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sensor-triggered photodetection of fluorescent probe-target hybrid |
US13/150,030 Abandoned US20110312701A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences with calibrated photodetection of probes in hybridization array |
US13/150,147 Abandoned US20110312593A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubator having two-dimensional control of input heat flux |
US13/150,211 Abandoned US20110312799A1 (en) | 2010-06-17 | 2011-06-01 | Usb-interfaceable portable test module for detection of hybridized probes |
US13/150,076 Abandoned US20110312581A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with nucleic acid amplification chamber heater bonded to chamber interior |
US13/150,131 Expired - Fee Related US8354074B2 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume reagent reservoir |
US13/150,161 Abandoned US20110312776A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating reporters |
US13/150,223 Abandoned US20110312611A1 (en) | 2010-06-17 | 2011-06-01 | Test module with transition metal-ligand complex fluorophore |
US13/149,894 Pending US20110312624A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure having active valve for capillary-driven fluidic propulsion without trapped air bubbles |
US13/150,141 Abandoned US20110312082A1 (en) | 2010-06-17 | 2011-06-01 | Dispensing apparatus for wafer-scale dispensing of reagents |
US13/149,947 Abandoned US20110312653A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume hybridization chambers |
US13/150,252 Abandoned US20110312823A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and mirrors for simultaneous excitation of oligonucleoutide probes |
US13/150,041 Abandoned US20110312706A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with hybridization chambers containing probes for electrochemiluminescent detection of target nucleic acid sequences in a fluid and calibration chamber containing probes sealed from the fluid |
US13/149,912 Abandoned US20110312635A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with flow-channel structure for capillary-driven fluidic propulsion without trapped air bubbles |
US13/150,174 Abandoned US20110312783A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control comprising empty chambers |
US13/149,893 Abandoned US20110312545A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and tandem nucleic acid amplification |
US13/149,902 Abandoned US20110312629A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section having stomata tapering counter to flow direction |
US13/149,900 Abandoned US20110312628A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with mst layer and overlying cap |
US13/150,177 Abandoned US20110312602A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal bend actuated surface tension valve |
US13/150,263 Abandoned US20110312832A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of target sequences with electrochemiluminescent probes in hybridization chambers |
US13/150,019 Abandoned US20110312698A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr section having short thermal cycle times |
US13/150,108 Abandoned US20110312752A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume reagent reservoir |
US13/150,091 Abandoned US20110312584A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module with driver for excitation of electrochemiluminescent luminophores |
US13/150,115 Abandoned US20110312759A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with reagent reservoir |
US13/150,139 Abandoned US20110312590A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with elongate incubation chamber |
US13/150,204 Abandoned US20110312795A1 (en) | 2010-06-17 | 2011-06-01 | Diagnostic test module with a loc with integral photosensor and excitation led for detection of hybridization assay results |
US13/149,935 Abandoned US20110312645A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled hybridization chambers for electrochemiluminescent detection of targets |
US13/149,903 Abandoned US20110312547A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by number of active outlet valves |
US13/150,121 Abandoned US20110312764A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubator |
US13/150,089 Abandoned US20110312740A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with capillary meniscus marching velocity sensor |
US13/150,146 Abandoned US20110311394A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal bend actuated surface tension valve |
US13/150,067 Abandoned US20110312727A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel nucleic acid amplification functionality |
US13/150,004 Abandoned US20110312694A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with delay-triggered photodetection of fluorescent probe-target hybrid |
US13/150,116 Abandoned US20110312760A1 (en) | 2010-06-17 | 2011-06-01 | Reagent microvial with authentication integrated circuit |
US13/150,182 Abandoned US20110312604A1 (en) | 2010-06-17 | 2011-06-01 | Loc having on-chip electronics for use in a test module to control module communications |
US13/150,259 Abandoned US20110312829A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis and electrochemiluminescent detection of target sequences |
US13/149,931 Abandoned US20110312641A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with sample inlet and probe hybridization section |
US13/150,125 Abandoned US20110312069A1 (en) | 2010-06-17 | 2011-06-01 | Microvial with digital memory for storage of oligonucleotide specification data |
US13/149,892 Abandoned US20110312623A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis, nucleic acid amplification and prehybridization filtering |
US13/150,183 Abandoned US20110312848A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for wafer-scale spotting of locs |
US13/150,228 Abandoned US20110312802A1 (en) | 2010-06-17 | 2011-06-01 | Test module with probes suspended in fluid |
US13/150,054 Abandoned US20110312716A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using an isothermal reaction |
US13/149,958 Abandoned US20110312663A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with time delayed detection of fluorescence from hybridized probes |
US13/150,028 Abandoned US20110312569A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with small cross sectional area microchannel |
US13/150,077 Abandoned US20110312732A1 (en) | 2010-06-17 | 2011-06-01 | Test module using lanthanide metal-ligand complex, electrochemiluminescent luminophores |
US13/150,066 Abandoned US20110312576A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device for multi-stage amplification of nucleic acid sequences |
US13/149,990 Abandoned US20110312685A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pcr using adaptor primers and target detection using electrochemiluminescent resonant energy transfer probes |
US13/149,891 Abandoned US20110312841A1 (en) | 2010-06-17 | 2011-06-01 | Fabrication system for lab-on-a-chip (loc) devices with differing application specific functionality |
US13/150,172 Abandoned US20110312782A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with digital memory |
US13/150,049 Abandoned US20110312712A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for pcr amplification of nucleic acids from whole blood |
US13/150,192 Abandoned US20110312607A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration photosensor output subtracted in a differential circuit from the output of hybridization photosensors |
US13/149,985 Abandoned US20110312683A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with tablet computer |
US13/150,074 Abandoned US20110312730A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with parallel dna and rna amplification functionality |
US13/149,941 Abandoned US20110312649A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with optically transparent hybridization chambers |
US13/150,227 Abandoned US20110309276A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal boiling-initiated valve |
US13/150,164 Abandoned US20110312846A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with fluidics on both sides of supporting substrate |
US13/150,260 Abandoned US20110312830A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc device with electrochemiluminescent probes having a functional moiety for quenching photon emissions configured to change proximity to a luminophore upon forming a probe-target hybrid |
US13/150,208 Abandoned US20110312852A1 (en) | 2010-06-17 | 2011-06-01 | Robotic system for loading oligonucleotides into spotting devices |
US13/150,238 Abandoned US20110312810A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for detection of hybridization of targets with oligonucleotide probes |
US13/150,222 Abandoned US20110311415A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal bend-actuator surface tension valve |
US13/150,078 Abandoned US20110312526A1 (en) | 2010-06-17 | 2011-06-01 | Method of analysing the nucleic acid content of a blood sample |
US13/149,937 Abandoned US20110312647A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with temperature feedback controlled hybridization chambers |
US13/150,189 Abandoned US20110312618A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device with high spotting rate |
US13/149,917 Abandoned US20110312637A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for separating pathogens from a biological sample |
US13/149,942 Abandoned US20110312650A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with optically transparent hybridization chambers for electrochemiluminescent detection of targets |
US13/149,968 Abandoned US20110312671A1 (en) | 2010-06-17 | 2011-06-01 | Single use microfluidic device with photosensor for electrochemiluminescent detection of targets |
US13/149,944 Abandoned US20110312651A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low mass probe spots |
US13/150,032 Abandoned US20110312702A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis and nucleic acid amplification |
US13/150,084 Abandoned US20110312737A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for electrochemiluminescent detection of targets |
US13/150,118 Abandoned US20110312762A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with fluorescence resonance energy transfer (fret) probes |
US13/149,955 Abandoned US20110312660A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a laptop computer |
US13/150,007 Abandoned US20110312559A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and parallel nucleic acid amplification |
US13/150,137 Abandoned US20110312081A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus for array of microfluidic devices |
US13/150,230 Abandoned US20110312804A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with aperture with geometry to promote unpinned flow-through of fluid |
US13/150,148 Abandoned US20110312843A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device for complete assay spotting of locs |
US13/149,960 Abandoned US20110312553A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with non-imaging optics for electrochemiluminescent detection of targets |
US13/150,268 Abandoned US20110312837A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis and electrochemiluminescent detection of target sequences |
US13/150,029 Abandoned US20110312570A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting target nucleic acid sequences with probes having long fluorescence lifetime fluorophores |
US13/150,105 Abandoned US20110312750A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with total reagent storage |
US13/150,196 Abandoned US20110312791A1 (en) | 2010-06-17 | 2011-06-01 | Test module with fault-tolerant multiple valve assembly |
US13/150,050 Expired - Fee Related US8398939B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with low-volume hybridization chambers for electrochemiluminescent detection of target nucleic acid sequences in a fluid |
US13/149,913 Abandoned US20110312071A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with large channels for cell transport and small channels suitable for biochemical processes |
US13/150,073 Abandoned US20110312729A1 (en) | 2010-06-17 | 2011-06-01 | Test module using transition metal-ligand complex, electrochemiluminescent luminophores |
US13/149,986 Abandoned US20110312537A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for amplifying and detecting target nucleic acid sequences using electrochemiluminescent resonant energy transfer, linear probes with covalently attached primers |
US13/150,111 Abandoned US20110312755A1 (en) | 2010-06-17 | 2011-06-01 | Test module with chemical lysis section |
US13/149,930 Abandoned US20110312552A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with conductivity sensor |
US13/150,002 Abandoned US20110312692A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and nucleic acid amplification |
US13/150,205 Abandoned US20110311407A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic boiling-initiated valve |
US13/150,149 Abandoned US20110312594A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization probes including positive and negative control probes |
US13/150,024 Abandoned US20110312567A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target nucleic acid sequences using hybridization chamber array and negative control chamber without probes |
US13/150,144 Abandoned US20110312592A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with incubation chamber between supporting substrate and heater |
US13/150,072 Abandoned US20110312580A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with nucleic acid amplification section and thermal insulation trench |
US13/150,207 Abandoned US20110312797A1 (en) | 2010-06-17 | 2011-06-01 | Portable test module for fluorescence excitation of probe nucleic acid sequences |
US13/149,922 Abandoned US20110312074A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with sample receptacle |
US13/149,971 Abandoned US20110312674A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral photosensor for electrochemiluminescence based detection of targets |
US13/149,946 Abandoned US20110312652A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with low-volume electrochemiluminescence-based probe spots |
US13/150,201 Expired - Fee Related US8398938B2 (en) | 2010-06-17 | 2011-06-01 | Microfluidic thermal bend actuated pressure pulse valve |
US13/149,957 Abandoned US20110312662A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for removing cell debris from a biological sample |
US13/150,047 Abandoned US20110312710A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and nucleic acid amplification |
US13/150,063 Abandoned US20110312724A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with incubation, nucleic acid amplification and prehybridization filtering |
US13/150,016 Abandoned US20110312562A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and parallel nucleic acid amplification |
US13/150,197 Abandoned US20110312792A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates epidemiological databases |
US13/149,976 Abandoned US20110312068A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device for separating nucleated cells in a biological sample from other constituents |
US13/150,176 Abandoned US20110312847A1 (en) | 2010-06-17 | 2011-06-01 | Spotting device with stored oligonucleotide specification data |
US13/150,062 Abandoned US20110312723A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for nucleic acid amplification using nucleic acid sequence based amplification |
US13/150,045 Abandoned US20110312708A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for isothermal amplification of nucleic acids |
US13/150,206 Abandoned US20110312796A1 (en) | 2010-06-17 | 2011-06-01 | Test module that updates medical databases |
US13/150,188 Abandoned US20110312542A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration chamber containing chamber with a blocked inlet spotted with reporter |
US13/150,060 Abandoned US20110312721A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, incubation, and nucleic acid amplification |
US13/150,031 Abandoned US20110312070A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr chamber for high rate of temperature change |
US13/149,967 Abandoned US20110312554A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis device, loc and interconnecting cap |
US13/150,095 Abandoned US20110312743A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detection of target nucleic acid sequences using electrodes configured for electrochemiluminescence of luminophores without a coreactant |
US13/150,246 Abandoned US20110312817A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with humidity sensor |
US13/150,191 Abandoned US20110312606A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with digital memory |
US13/149,954 Abandoned US20110312659A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with hybridization chambers and corresponding diffusion barriers |
US13/150,129 Abandoned US20110311408A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus |
US13/150,166 Abandoned US20110312079A1 (en) | 2010-06-17 | 2011-06-01 | Loc with digital memory to store epidemiological updates |
US13/150,138 Abandoned US20110312772A1 (en) | 2010-06-17 | 2011-06-01 | Loc for detection of hybridization of nucleic acid sequences with pcr amplification using linker primers |
US13/149,993 Abandoned US20110312686A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with elongate pcr chambers |
US13/150,170 Abandoned US20110312781A1 (en) | 2010-06-17 | 2011-06-01 | Loc with digital memory to store genetic data updates |
US13/150,247 Abandoned US20110312818A1 (en) | 2010-06-17 | 2011-06-01 | Test module with excitation light and lens for simultaneous excitation of oligonucleoutide probes |
US13/150,094 Abandoned US20110312742A1 (en) | 2010-06-17 | 2011-06-01 | Single-use microfluidic device |
US13/150,086 Abandoned US20110312738A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with liquid sensor |
US13/150,136 Abandoned US20110312771A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pwm controlled incubation section |
US13/150,133 Abandoned US20110311409A1 (en) | 2010-06-17 | 2011-06-01 | Reagent dispensing apparatus with automatic collection and storage of reagent data |
US13/149,983 Abandoned US20110312681A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with dialysis section for removing erythrocytes from blood |
US13/149,904 Abandoned US20110312630A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,226 Abandoned US20110312620A1 (en) | 2010-06-17 | 2011-06-01 | System for variable microarray spotting and genetic analysis |
US13/149,980 Abandoned US20110312679A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface-micromachined dialysis section |
US13/150,080 Abandoned US20110312734A1 (en) | 2010-06-17 | 2011-06-01 | Test module with suspended electrochemiluminescent probes |
US13/150,025 Abandoned US20110312700A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pwm controlled pcr heater |
US13/150,181 Abandoned US20110312786A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with calibration chamber containing probe designed to be noncomplementary to nucleic acid sequences in the amplicon |
US13/150,090 Abandoned US20110312583A1 (en) | 2010-06-17 | 2011-06-01 | Test module with parallel nucleic acid amplification sections |
US13/150,241 Abandoned US20110312813A1 (en) | 2010-06-17 | 2011-06-01 | Single-use genetic test module |
US13/150,098 Abandoned US20110312745A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module with photosensor |
US13/150,102 Abandoned US20110312748A1 (en) | 2010-06-17 | 2011-06-01 | Loc with integral photosensor for detection of hybridization assay results |
US13/149,899 Abandoned US20110312546A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and tandem nucleic acid amplification |
US13/150,232 Abandoned US20110312621A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for dispensing reagents and loading oligonucleotide spotting devices |
US13/149,996 Abandoned US20110312688A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with pcr chamber between supporting substrate and heater |
US13/150,214 Abandoned US20110311413A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with liquid detector sensor feedback |
US13/150,219 Abandoned US20110311414A1 (en) | 2010-06-17 | 2011-06-01 | Fault-tolerant multiple valve assembly with thermal bend-actuator pressure pulse valve |
US13/150,009 Abandoned US20110312560A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, thermal lysis and tandem nucleic acid amplification |
US13/150,173 Abandoned US20110312600A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal bend actuated pressure pulse valve |
US13/149,961 Abandoned US20110312665A1 (en) | 2010-06-17 | 2011-06-01 | Loc with dialysis section for removing insoluble sample constituents from a nucleic acid mixture |
US13/150,239 Abandoned US20110312811A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for electrochemiluminescent detection of target sequences with electrodes profiled for greater peripheral edge length |
US13/150,023 Abandoned US20110312566A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis, chemical lysis, incubation and parallel nucleic acid amplification |
US13/150,123 Abandoned US20110312765A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with low oligonucleotide probe mass and low reagent volume |
US13/150,186 Abandoned US20110312849A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting device for ejecting low volume droplets |
US13/150,250 Abandoned US20110312821A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with waste storage |
US13/149,951 Abandoned US20110312656A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for pcr and probe hybridization |
US13/150,058 Abandoned US20110312720A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for genetic analysis with dialysis and nucleic acid amplification |
US13/149,918 Abandoned US20110312638A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with dialysis and nucleic acid amplification |
US13/150,169 Abandoned US20110312780A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with negative control chambers incorporating probes with no reporters |
US13/150,217 Abandoned US20110312854A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot for spotting arrays of locs |
US13/150,162 Abandoned US20110312598A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with reagent mixing proportions determined by outlet valve numbers |
US13/150,092 Abandoned US20110312585A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with parallel dna and rna amplification section |
US13/149,909 Abandoned US20110312633A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with dialysis section |
US13/150,158 Abandoned US20110311395A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with active valve at reagent reservoir outlet |
US13/150,008 Abandoned US20110312539A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes for detecting targets in a fluid and a positive control probe without a quencher for luminophore emissions |
US13/149,974 Abandoned US20110312067A1 (en) | 2010-06-17 | 2011-06-01 | Dialysis device for separating pathogens from a biological sample |
US13/150,140 Abandoned US20110311393A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal bend actuated pressure pulse valve |
US13/150,114 Abandoned US20110312758A1 (en) | 2010-06-17 | 2011-06-01 | Test module with thermal lysis section |
US13/149,973 Abandoned US20110312676A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral driver for excitation of electrochemiluminescent luminophores |
US13/150,157 Abandoned US20110312597A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with hybridization array with positive control chambers incorporating probes with no quenchers |
US13/150,216 Abandoned US20110312608A1 (en) | 2010-06-17 | 2011-06-01 | Test module with low-volume hybridization chamber and low-volume reagent reservoir |
US13/150,011 Abandoned US20110312695A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with hybridization chamber array with positive control chamber containing electrochemiluminescent reporter |
US13/150,132 Expired - Fee Related US8394339B2 (en) | 2010-06-17 | 2011-06-01 | LOC device with on-chip semiconductor controlled incubation section |
US13/149,916 Abandoned US20110312072A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with surface micro-machined chips and interconnecting cap |
US13/150,256 Abandoned US20110312826A1 (en) | 2010-06-17 | 2011-06-01 | Test module with laser for excitation of oligonucleoutide probes |
US13/150,052 Abandoned US20110312574A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection, genetic analysis and proteomic analysis with dialysis, chemical lysis, incubation and parallel nucleic acid amplification |
US13/150,187 Abandoned US20110312605A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with integral controller |
US13/150,104 Abandoned US20110312749A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with thermal lysis section |
US13/150,190 Abandoned US20110312788A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc with thermal boiling-initiated valve |
US13/150,237 Abandoned US20110312809A1 (en) | 2010-06-17 | 2011-06-01 | Test module with humidifier |
US13/150,203 Abandoned US20110312619A1 (en) | 2010-06-17 | 2011-06-01 | Device for high-density deposition of biochemicals |
US13/149,963 Abandoned US20110312667A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module for interfacing with a desktop computer |
US13/150,069 Abandoned US20110312578A1 (en) | 2010-06-17 | 2011-06-01 | Genetic analysis loc for non-specific nucleic acid amplification prior to specific amplification of particular sequences |
US13/149,933 Abandoned US20110312643A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of hybridization of nucleic acid targets |
US13/150,088 Abandoned US20110312739A1 (en) | 2010-06-17 | 2011-06-01 | Single-use test module for pcr amplification of targets and electrochemiluminescent detection of targets |
US13/150,001 Abandoned US20110312691A1 (en) | 2010-06-17 | 2011-06-01 | Loc device with electrochemiluminescent probes including positive and negative control probes |
US13/150,212 Abandoned US20110312853A1 (en) | 2010-06-17 | 2011-06-01 | Oligonucleotide spotting robot |
US13/150,039 Abandoned US20110312572A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection and genetic analysis with chemical lysis, incubation and nucleic acid amplification |
US13/150,106 Abandoned US20110312751A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of mitochondrial dna via fluorescence modulated by hybridization |
US13/150,014 Abandoned US20110312561A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with photodiodes with controllable shunts to detect fluorescing hybridized probes |
US13/150,210 Abandoned US20110312798A1 (en) | 2010-06-17 | 2011-06-01 | Test module with inbuilt lancet |
US13/149,928 Abandoned US20110312640A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with photosensor |
US13/150,231 Abandoned US20110312805A1 (en) | 2010-06-17 | 2011-06-01 | Test module with time delayed detection of fluorescence from hybridized probe |
US13/149,911 Abandoned US20110312634A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device with laminar structure |
US13/150,012 Abandoned US20110312696A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, chemical lysis and nucleic acid amplification |
US13/149,989 Abandoned US20110312684A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for pathogen detection with dialysis, lysis and nucleic acid amplification |
US13/150,185 Abandoned US20110312787A1 (en) | 2010-06-17 | 2011-06-01 | Loc having usb device driver for use in a test module to control usb connection |
US13/150,017 Abandoned US20110312563A1 (en) | 2010-06-17 | 2011-06-01 | Loc device for detecting target nucleic acid sequences in a fluid using hybridization chamber array and negative control chamber containing electrochemiluminescent probe designed to be non-complementary to any sequence in the fluid |
US13/150,064 Expired - Fee Related US8398940B2 (en) | 2010-06-17 | 2011-06-01 | USB-interfaceable portable test module for electrochemiluminescent detection of targets |
US13/149,932 Abandoned US20110312642A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detection of nucleic acid targets with electrochemiluminescent probes |
US13/150,100 Abandoned US20110312078A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic device for detecting target nucleic acid sequences in mitochondrial dna |
US13/149,948 Abandoned US20110312654A1 (en) | 2010-06-17 | 2011-06-01 | Apparatus for loading oligonucleotide spotting devices and spotting oligonucleotide probes |
US13/149,919 Abandoned US20110312073A1 (en) | 2010-06-17 | 2011-06-01 | Microfluidic test module incorporating surface micro-machined chips and interconnecting cap |
US13/685,105 Abandoned US20130079254A1 (en) | 2010-06-17 | 2012-11-26 | Microfluidic dialysis device |
Country Status (3)
Country | Link |
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US (355) | US20110312763A1 (en) |
TW (1) | TW201213798A (en) |
WO (23) | WO2011156844A1 (en) |
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