WO2000058724A1 - Microscale total analysis system - Google Patents
Microscale total analysis system Download PDFInfo
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- WO2000058724A1 WO2000058724A1 PCT/EP2000/002887 EP0002887W WO0058724A1 WO 2000058724 A1 WO2000058724 A1 WO 2000058724A1 EP 0002887 W EP0002887 W EP 0002887W WO 0058724 A1 WO0058724 A1 WO 0058724A1
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- reaction chamber
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- inflow channel
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- fluid inflow
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0672—Integrated piercing tool
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
- B01L2300/165—Specific details about hydrophobic, oleophobic surfaces
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
Definitions
- This invention relates to apparatus for detecting the presence of a target species in an aqueous sample, and also to apparatus for determining the concentration and reaction kinetics of target species.
- the invention is applicable to the monitoring of many different molecular interactions, in particular molecular recognition between an immobilised affinity partner and a species in solution, such as immunoglobulin/antigen interaction, DNA hybridisation, hapta er-protein interaction, drug and virus detection and high throughput screening of synthetic molecules.
- the time needed to reach reaction equilibrium is directly dependent on the mass transport of the molecule.
- the diffusion time of a molecule in a solution is proportional to the square of the path length; typically a small molecule needs less than one second to diffuse through 10 ⁇ m while it needs two hours to traverse one millimetre.
- the chemical partners In order to decrease the equilibrium time of the reaction, the chemical partners must therefore be placed as close as possible to each other; by reducing the reactor size to microdimensions, immobilising one partner on the surface of the reactor and filling the reactor with the second partner, the equilibrium time can be dramatically decreased.
- the use of icroreactors not only enhances the speed of affinity assays, but also facilitates the obtaining of information concerning reaction kinetics, which is important in the understanding of the ther odynamic stability of complexes.
- the affinity constant K d is the ratio between the forward and reverse reaction rate constants k + and k_ , which represent the association and the dissociation constants respectively.
- a strong complexation is characterised by a very fast association and a very slow dissociation, which in the particular case of sorbent affinity assays are adsorption and desorption from the surface of the microreactor .
- thermodynamic properties can be used for the study of cress- reactivity between several antigens or of non-specific adsorption of a matrix element during an affinity assay.
- the complexation of higher affinity partners is favoured and the non-specific adsorption is then reduced to a minimum.
- This fact may be an important factor in the decrease of the detection limit in immunosorbent assays due to the minimisation of the background signal.
- This method can also be applied to monitor the adsorption of antigens of different molecular weight.
- the diffusion coefficient of a molecule is proportional to its mass, the diffusion time of the molecule through the reaction chamber is different for small and large molecules.
- the K d may be the same for all molecules, whereas the diffusion coefficient is different for each of them.
- microscale total analysis systems ( -TAS) 1 , and they have already been recognised as convenient means of manipulating and analysing small sample quantities 2"8 .
- ⁇ -TAS devices to date have been produced by photolithography, wet chemical etching or thin film deposition on substrates such as glass, quartz and silicon 9 ⁇ 10 .
- plastic substrates have also been micromachined using either silicone rubber casting 11_i4 injection moulding - 5 embossing 16, 17 or laser photoablation 1S .
- These structures are planar devices with channels of micrometre size that are often sealed by thermal or anodic bonding to a glass cover. Interconnected channels may be fabricated easily, which makes possible the rapid separation and reactions in volumes of few a picoliters.
- Other advantages of ⁇ -TAS are the reduction of sample and reagent consumption and the increase of precision and reproducibility relative to bench scale apparatus 21, 22 .
- Another type of immunoassay device has been developed for simultaneous analysis of multiple samples 6 .
- biotin-labelled antibodies are patterned onto an avidin- coated waveguide so as to form an array of six vertically oriented stripes of captured antibodies immobilised on the waveguide surface by avidin-biotin bridges .
- Samples are then analysed using a sandwich immunoassay format by patterning another array of six horizontally oriented lines containing the corresponding fluorescent-labelled antigen at various concentrations. Fluorescent complexes on the surface of the waveguide are then excited by a diode laser, and the fluorescence intensities of the 36 square dots is collected by a CCD camera.
- This immunosensor allows the analysis of multiple samples in parallel and simultaneous detection of more than one analyte per sample.
- Luminescence is the generic term referring to the emission of an electromagnetic radiation (UV, visible or IR) by an excited molecule that relaxes to its ground state, can be induced by photoexcitation (photoluminescence) or by a chemical reaction (chemiluminescence and electrochemiluminescence) .
- Chemifluorescence (CF) is another class of luminescent reactions which combines the reaction mechanisms of both PL and CL. In this case, a fluorogenic substrate A is converted to a fluorescent product C by chemical reaction, and luminescence is generated by excitation of this product:
- one of the reactants of the assay system that is capable of generating luminescence can be attached to a molecule in order to "label" it specifically.
- the presence or absence of an observable label attached to one or more of the binding materials is then used as an indicator of the existence of an analyte of interest.
- a large body of experiments has been developed to detect and quantitate trace amounts of pharmaceuticals, microorganisms, hormones, viruses, antibodies, nucleic acids and other proteins by such methods.
- competitive and sandwich immunoassays using luminescence detection are now used on a routine basis 27, 2B .
- a molecule is labelled with an enzyme that catalyzes the luminescence reaction.
- Typical examples are the detection of immunoreagents labelled with Horse Radish Peroxidase (HRP) or Alkaline Phosphatase (ALP) which, in the presence of hydrogen peroxide and hydroxide ions, respectively facilitate the oxidation of luminol and dioxetanes and the hydrolysis of phosphate-containing reagents.
- HRP Horse Radish Peroxidase
- ALP Alkaline Phosphatase
- ALP has been used in CF assays to cleave a phosphate group from a fluorogenic substrate to yield a highly fluorescent product 9 .
- Luminescence assay methods are widely used in the analysis of peptides, proteins, and nucleic acids.
- CL has been shown to be a highly sensitive detection method in both flow injection analysis (FIA) and high-performance liquid chromatography 30 ⁇ 32 , and it has also been employed in capillary electrophoresis (CE) 33, 34 for the detection of amino acids neurotransmitters 3S , rare-earth metal ions 3 ⁇ or labelled proteins 37 .
- FIA flow injection analysis
- CE capillary electrophoresis
- luminescence is the most commonly used detection method 27
- US 4,621,059 discloses a method in which the light emitted by a luminescent substance flowing through a capillary column and reacting with an immobilised enzyme is collected through a plurality of optical fibers that are arranged along the longitudinal direction of the column in order to determine the enzyme activity or the quantity of analyte of interest from the distribution of the luminescence intensity.
- US 5,624,850 describes a method for performing immunoassays in capillaries in which fluorescence is used to detect an analyte of interest in translucent capillaries having an inner diameter from -0.1 ⁇ m to 1.0mm.
- homogeneous chemiluminescence immunoassays can be carried out, for example as described in US 5,017,473, in which a light absorbing material and a luminescent labelled tracer are incubated with the analyte/anti-analyte complex, so that all the emitted light is absorbed by the light-absorbing material except that associated to the bound tracer.
- a method is disclosed in US 5,585,069 in which two or more samples are processed in parallel in a system comprising a plurality of wells that are connected by one or more channels to move a sample from one well to the other using mechanical or electrokinetic pumping.
- the channels are simply used as connections between two wells, and are not used as reaction or detection chambers .
- the present application therefore provides, in one aspect, apparatus comprising: at least one reaction chamber; at least one fluid inflow channel communicating with the or each reaction chamber; and gate means adapted to prevent passage of aqueous fluid through the fluid inflow channel (s) into the reaction chamber (s) , until such fluid is acted upon by a fluid entry force; wherein the gate means comprises at least a portion of the or each fluid inflow channel having a hydrophobic inner surface.
- the apparatus has a plurality of reaction chambers, which take the form of microchannels , each having an associated fluid inflow channel.
- a plurality of microchannels may be served by a single inflow channel, feeding into a common conduit communicating with the microchannels.
- the fluid entry force is provided by aspiration means connected to a common conduit communicating with each microchannel at its end distal the inflow channel.
- the apparatus comprises a rotatable support member and the fluid entry force is provided by centrifugal force upon rotation of the substrate.
- the support member may form the substrate of the microchannel apparatus, with the microchannels being arranged generally radially.
- the rotatable support member may serve as a support for one or more devices having parallel microchannels .
- the advantage of a common source of fluid entry force for all of the microchannels is that simultaneous filling may be ensured, the fluid samples being prevented from entering the microchannels by the hydrophobic gate means until the fluid entry force is applied. Furthermore, the degree of fluid entry force may also readily be controlled, to ensure rapid filling of the microchannels, and adequate mixing.
- the microchannels may also be emptied in an efficient and rapid manner, by application of an increased force to the fluid in the channels, for example by increasing the degree of aspiration, or by increasing the rate of rotation of the rotation support member. An exact end point of an assay may thereby be achieved. In many instances it is advantageous for the sample to be expelled before monitoring for bound target species.
- a liquid reagent or a washing fluid may be supplied in a sealed cavity forming a reservoir, there preferably being one such reservoir per microchannel.
- the reservoirs may be arranged to communicate with their respective microchannels via normally closed valves, and may be caused to expel their contents through such valves when acted upon by respective pistons.
- there may be a single reservoir, communicating via a normally closed valve with a common conduit feeding all microchannles .
- Detection of target species with the microchannels may be achieved by conventional means.
- preferred embodiments of apparatus are constructed so that at least a portion of the surface of the microchannel is formed of an electrically conductive material.
- This may for example be a conductive polymer material or an electrode.
- at least a portion of the microchannel walls may be formed of a semi-conductor material such as indium oxide.
- the semi-conductor material is transparent.
- detection may be achieved by luminesce or fluorescence means, n which case an electromagnetic radiation detector, such as a photodiode or a photomultiplier, is provided.
- One particular advantage of the invention is that chemical reagents may be immobilised on the inner surfaces of the microchannels, thus providing the possibility of ELISA - type assays in a ⁇ -TAS-type system.
- a number of different types of reagent may be attached to the microchannel walls, for example oligonucleotides, polypeptides, proteins (such as enzymes), or other natural or synthetic molecules. Conveniently, these may be adsorbed onto the surface of the microchannel walls, or covalently linked thereto, (for example by means of amide bond formation with succinimide) , or electrostatically linked thereto (for example by means of a crosslinker such as polylysine) .
- the inner surface of the microchannel and/or of the fluid inflow channel may also be provided with chemically functional groups formed by chemical or physical treatment .
- the invention also extends to a method of manufacturing an apparatus as defined above, comprising the following steps which may be performed in either order or simultaneously: forming at least one reaction chamber; and forming at least one fluid inflow channel communicating with the reaction chamber (s), at least a portion of the or each fluid inflow channel having a hydrophobic inner surface adapted to act as gate means to prevent passage of fluid through the fluid inflow channel into the reaction chamber (s) until such fluid is acted upon by a fluid entry force.
- the apparatus is preferably formed in two main parts: a substrate in which the microchannels (and possibly also the inflow channels) are formed as depressions (for example by injection moulding, hot embossing, photoablation, casting or polymerisation on a mould) ; and an overlying layer applied over the substrate and over the depressions, to form the microchannels (and optionally also the inflow channels) .
- the inflow channels are not produced in the substrate they may, for example, be produced by drilling through a laminated overlying layer using a laser, or by depositing above the inlet of the reaction chamber a joint made of a hydrophobic material such as polydimethylsiloxane (PDMS) .
- PDMS polydimethylsiloxane
- the apparatus may be formed from any suitable material, for example, ceramics, glass, semiconductors, polymers, or combinations thereof.
- both the substrate and lamination layer are formed of polymer material, which not only permits ready formation of the microchannels (for example by photoablation) , but also allows the two components to be fused together by a thermal lamination technique.
- at least one of the polymers is of a material which has a relatively low melting point, for example polyethylene with a melting point of under
- the lamination layer may with advantage be of an elastomeric material, such as polydimethyl siloxane (PDMS) .
- PDMS polydimethyl siloxane
- the lamination layer be formed of a substantially transparent material, and the substrate of a substantially opaque material (such as a ceramics material or a carbon-filled polymer) .
- the invention extends to a method of operating an apparatus as defined, comprising the steps of: placing at least one sample of an aqueous solution under test at the end of at least one fluid inflow channel distal at least one reaction chamber; causing the sample to enter the reaction chamber (s) via the fluid inflow channel (s) by applying a fluid entry force; and monitoring the sample in the reaction chamber (s) for the presence or concentration of a target substance.
- the fluid entry force is preferably applied by activating the aspiration means to apply reduced pressure to the microchannels for a period of time in the range 0.1 to 100s.
- the aspiration means may then be activated to provide an even lower pressure to the microchannels, optionally in conjunction with the supply of washing fluid from a reservoir.
- the fluid entry force is preferably applied by rotating the substrate or support at an angular velocity in the range 1 to 1,000 revolutions per minute for a period of time in the range 1 to 100s.
- the microchannels may then be evacuated by rotating the substrate or support at an increased angular velocity, in the range 10 to 100,000 revolutions per minute, for a period of time in the range 1 to 100s.
- Fig 1 is a schematic cross section of an embodiment of apparatus according to the invention illustrated a) after deposition of an aqueous sample drop on the hydrophobic gate, and b) after sample loading;
- Fig 2 is a schematic plan view of an embodiment of apparatus according to the invention, illustrating the steps of parallel sampling, loading and washing, achieved by aspiration;
- Fig 3 is a schematic plan view of an alternative embodiment of apparatus according to the invention, in which parallel filling and washing step are achieved by centrifugal force;
- Fig 4 is a schematic plan view of a further embodiment of apparatus according to the invention, in which the solution is loaded by slight aspiration and washed by strong aspiration,-
- Fig 5a is a partial plan view of an embodiment of apparatus according to the invention, incorporating a fluid reservoir adjacent the fluid inflow channel;
- Fig 5b consists of two vertical cross sections of the
- FIG. 5a apparatus together with an associated piston, illustrating the action of the piston in penetrating the sealed reservoir and expelling its contents through a valve into the reaction chamber;
- Fig 6 is a top plan representation of an embodiment of apparatus according to the invention manufactured by UV-
- Fig 7 is a calibration curve obtained from a fluorescence imager using ALP-DDi solution in the microchannel of an embodiment of apparatus according to the invention.
- Fig 8 is a graph illustrating fluorescence results indicating the level of binding between DDi-ALP in a test involving use of two microchannels of apparatus according to the invention, one incubated solely with BSA, and the other with BSA and anti-DDi (noted as BSA + Ab in the figure) ;
- Fig 9 is a fluorescence image obtained using apparatus of the type illustrated in Fig 6, in which different microchannels were incubated with different concentrations Of DDi-ALP;
- Fig 10 is a graph illustrating the intensity of fluorescence obtained from different concentrations of ALP- DDi in an apparatus of the type illustrated in Fig 6, having antibody-coated microchannels;
- Fig 11 is a graph illustrating variation of fluorescence intensity with time from an incubation of
- ALP-DDi in an embodiment of apparatus according to the invention having at least one antibody coated microchannel;
- Fig 12 is a graph illustrating fluorescence intensity
- D-Dimer utilising an embodiment of apparatus according to the invention, having at least one antibody-coated microchannel.
- microchannel devices of Figs 1 to 6 are produced by UV-Laser photoablation of commercially available polymers such as PET or polycarbonate.
- the photoablation procedure is performed in known fashion, for example as described previously by the present applicants 44 . Briefly, a polymer sheet is rinsed with distilled water and ethanol and then mounted on an X, Y machining stage (Microcontrol , France) .
- UV-Laser pulses (193 nm) (Lambda Physik LPX 205 i, Germany) are then fired at the polymer substrate target through a photomask and a 10:1 lense with a frequency of 50 Hz at 200 mJ/pulse, corresponding to a fluence per pulse of 1 J/cm 2 on the surface.
- the polymer substrate is moved horizontally with a X, Y stepping motor
- the microchannels are typically between 1 and l,000 ⁇ m in width, and in this example are approximately lOO ⁇ m wide.
- the depth of the channels was fixed at 40 ⁇ m, by controlling the number of laser pulses used (each pulse photoablates approximately 150 nm) .
- the channels are then sealed by thermal lamination of a layer of polyethylene over the base polymer sheet, the channels then exhibiting a trapezoidal shape in which three walls are composed of the substrate polymer (PET or Polycarbonate) and the top is composed of the lamination (Polyethylene) .
- Fluid inflow channels are opened either by firing enough laser pulses or are mechanically drilled through the hydrophobic lamination layer.
- the gates which may have a diameter between lO ⁇ m and 10mm, have hydrophobic inner surfaces due to the nature of the polymer, and therefore inhibit passage of aqueous fluids.
- microchannels are arranged parallel to each other, conveniently on a generally rectangular substrate.
- the inflow channel "gates" of the various microchannels are aligned with each other, to permit rapid and efficient loading with test solutions from a linear multiple pipette device (see Fig 2) .
- the microchannels are arranged radially on a generally circular substrate, either with the inflow channel gates towards the centre of the circle and the opposite (outflow) ends of the microchannels towards the circumference (Figs 3 and 6) , or vice versa (Fig 4) .
- a number of different means may be employed to provide the fluid entry force, of which the preferred means are aspiration and centrifugal force.
- a common conduit (3) is supplied at the outflow ends of the microchannels (2) , to which a reduced pressure is applied during operation of the device, to draw fluid into the microchannels through the fluid inflow gates.
- the aspiration means may also be utilised to supply a stronger aspirating force in order to expel the microchannel contents to a drain, optionally in conjunction with the supply of a washing fluid.
- the apparatus illustrated in Fig 4 operates in a similar fashion, with aspiration being applied to the common outflow drain (6) .
- fluid is compelled to pass through the fluid inflow gates and into the microchannels by spinning the substrate to produce centrifugal force.
- each microchannel has its own drain (7) .
- an aspiration driven device (as in Figs 2 and 4) a 2 ⁇ l sample is placed with a pipette on each gate (1) .
- the solution is then loaded into the microchannel by a brief aspiration from the common conduit (3; 6) .
- This technique ensures homogeneity of the solution over the whole microchannel.
- the microchannel is aspirated and rinsed three times with 2 ⁇ l . It is worth noting that the washing solution volume is much larger than that of the microchannel (about 100 nl) thus ensuring efficient washing.
- the filling and washing procedures may be achieved by placing 2 ⁇ l of solution over each gate (1) . Slow rotation results in loading of the sample into the microchannel (s) , and faster rotation is subsequently used to expel the sample from the microchannel (s) .
- Fig 5 illustrates an optional modification of apparatus according to the invention, in which each microchannel has an associated fluid reservoir (10) formed by a sealed cavity situated adjacent the fluid inflow gate (1) .
- the reservoir communicates with microchannels (2) by means of a normally closed valve (12) , which comprises valve member (13) which may be deformed under pressure into depression (14) .
- Reservoir (10) is capped by seal (15) , which may be broken by downward pressure applied by piston (11) , which is profiled to be a close fit within reservoir (10) .
- Downward movement of piston (11) within reservoir (10) increases the fluid pressure within the reservoir, thus opening valve (12) and allowing fluid from the reservoir to enter the microchannel.
- the reservoir may either be filled wiuh a reagent or with wash fluid.
- D-Dimer is used as a diagnostic indicator in thromboembolic events: deep vein thrombosis and pulmonary embolism can be diagnosed by monitoring D-Dimer concentration in blood.
- ELISA techniques for example the "Asserachro D-Di" of Diagnostica Stago.
- standard ELISA techniques are not suited for emergency situations, and alternative membrane-based techniques have been developed which use colour based detection systems 48 .
- these suffer from the disadvantage that the detection mechanism is too subjective.
- the detection of the enzyme was effected by a chemifluorescent substrate solution (VCR, Amersham) .
- VCR chemifluorescent substrate solution
- This system is based on the fluorescent detection of the AttoPhos substrate hydrolysed by ALP.
- the microchannels were then exposed to a Fluorescence Imager screen (MP840, Molecular Dynamics) and every channel was read for l minute. The image was then quantified using Image Quant software (Molecular Dynamics) .
- the calibration of the enzyme in the microchannel was achieved by mixing the substrate solution with different concentrations of enzyme and incubating for 5 minutes. The microchannels were then filled with the mixtures and analysed with the fluorescence imager.
- the enzyme was immobilised on the surface of the microchannels, and the VCR solution was added to the channels with fluorescence being measured 5 minutes later. Immobilisation of the proteins was achieved by physisorption for 1 hour at room temperature.
- the mouse IgG antibody (Serbio, France) was immobilised by placing either 10 or 100 ⁇ g/ml in the microchannel, followed by incubation for i hour in a wet chamber. The surface was then washed with PBS and 20 % Tween (Tween/Water :0.2 ml/L, Fischer Germany) , and blocked for 1 hour with a solution of 50 ⁇ g/ml of heat shocked BSA (Sigma, USA) in the washing buffer solution.
- the channels were individually filled with the antigen solution. After five minutes (except for the kinetic experiment, where other periods are specified below) , the microchannels were rinsed and a solution of 10 ⁇ g/ml of alkaline phosphatase labelled antigen (ALP-DDi) was introduced and rinsed again after five minutes .
- ALP-DDi alkaline phosphatase labelled antigen
- Figure 9 shows the fluorescence of the substrate in the channels after adsorption of different concentrations of ALP-DDi on the 10 ⁇ g.ml "1 adsorbed antibodies.
- the fluorescence intensity of the microchannel lines clearly shows the gradient of concentration in the different microchannels.
- the relative intensity of every microchannel is shown graphically in Figure 10. Saturation of channels is reached at about 30 ⁇ g/ml.
- Figure 11 shows the fluorescence intensity of microchannels that have been incubated for different periods of time. For short incubation times ( ⁇ 5 min) , the intensity grows linearly, showing that the antigens are very quickly captured by the antibodies. It is thought that all the antigens have still not reached the surface by diffusion. This first slope approximately follows the diffusion of the molecules to the walls. The molecules then react rapidly and the reaction becomes quasi diffusion-controlled. After 5 minutes of incubation, the reaction is controlled by slower kinetics driven by two different phenomena. Firstly, large molecules diffuse much more slowly and therefore reach the surface after a long time. In this case, the molecules can be partially degraded fibrin products, of which the molecular weight can be larger that 1000 kD .
- Figure 12 shows the fluorescence dependence of the D- Dimer concentration after a competitive immunoassay.
- the immobilised antibody sites are not occupied by the DDi, allowing the DDi-ALP to be present in a large amount and therefore to hydrolyse more fluorescent substrates.
- concentrations higher than 1000 ng.ml "1 D-Dimer molecules are present on most of the 5 antibody sites and therefore only a few sites are available for DDi-ALP.
- the central part of the concentration range (100-1000 ng.ml *1 ) shows the strong concentration dependence of the system, the two orders of magnitude detection range being in the range of interest for diagnostics applications
- 15 calibration may be reduced to less than 10 minutes, compared to a typical time of 3 hours for an ELISA in a microtiter plate.
- Hundreds of microchannels may be provided on a substrate, if desired, and the ability to ensure simultaneous filling provides the possibility of highly
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000608171A JP2002540425A (en) | 1999-03-29 | 2000-03-28 | Microscale total analysis system |
EP00922589A EP1166103B1 (en) | 1999-03-29 | 2000-03-28 | Microscale total analysis system |
AT00922589T ATE309534T1 (en) | 1999-03-29 | 2000-03-28 | NIKROM SCALE APPARATUS FOR CHEMICAL ANALYSIS |
AU42927/00A AU4292700A (en) | 1999-03-29 | 2000-03-28 | Microscale total analysis system |
DE60023862T DE60023862T2 (en) | 1999-03-29 | 2000-03-28 | MICROMASSETTE DEVICE FOR CHEMICAL ANALYSIS |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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GBGB9907249.8A GB9907249D0 (en) | 1999-03-29 | 1999-03-29 | Chemical assay apparatus |
GB9907249.8 | 1999-03-29 |
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PCT/EP2000/002887 WO2000058724A1 (en) | 1999-03-29 | 2000-03-28 | Microscale total analysis system |
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EP (1) | EP1166103B1 (en) |
JP (1) | JP2002540425A (en) |
AT (1) | ATE309534T1 (en) |
AU (1) | AU4292700A (en) |
DE (1) | DE60023862T2 (en) |
GB (1) | GB9907249D0 (en) |
WO (1) | WO2000058724A1 (en) |
Cited By (10)
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WO2003001192A1 (en) * | 2001-06-20 | 2003-01-03 | Cytonome, Inc. | Microfabricated separation device employing a virtual wall for interfacing fluids |
EP1314479A2 (en) * | 2001-11-24 | 2003-05-28 | GeSIM Gesellschaft für Silizium-Mikrosysteme mbH | Device for the transfer of liquid samples |
EP1412729A2 (en) * | 2001-06-20 | 2004-04-28 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
EP1424559A1 (en) * | 2001-08-09 | 2004-06-02 | Olympus Corporation | Micro flow passage device, connection device, and method of using the devices |
WO2005089944A3 (en) * | 2004-03-17 | 2005-12-08 | Reiner Goetzen | Microfluidic chip |
US7179423B2 (en) | 2001-06-20 | 2007-02-20 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
US7211442B2 (en) | 2001-06-20 | 2007-05-01 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
US7265348B2 (en) | 2002-11-08 | 2007-09-04 | Diagnoswiss S.A. | Apparatus for dispensing a sample in electrospray mass spectrometers |
US7700371B2 (en) | 2001-06-01 | 2010-04-20 | Arrowhead Research Corporation | Method for determining an analyte |
WO2011160015A2 (en) | 2010-06-17 | 2011-12-22 | Abaxis, Inc. | Rotors for immunoassays |
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JP2006226752A (en) * | 2005-02-16 | 2006-08-31 | Matsushita Electric Ind Co Ltd | Plate for bio-sample discrimination device |
JP5175778B2 (en) * | 2009-03-11 | 2013-04-03 | 株式会社東芝 | Liquid feeding device |
DE102011001550A1 (en) * | 2011-03-25 | 2012-09-27 | Friz Biochem Gesellschaft Für Bioanalytik Mbh | Device useful e.g. for promoting and blending reagents, comprises at least one reaction cell with at least a cavity exhibiting an aperture for carrying reagents, at least one reagent reservoir and at least one piston |
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- 2000-03-28 EP EP00922589A patent/EP1166103B1/en not_active Expired - Lifetime
- 2000-03-28 AT AT00922589T patent/ATE309534T1/en not_active IP Right Cessation
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US7700371B2 (en) | 2001-06-01 | 2010-04-20 | Arrowhead Research Corporation | Method for determining an analyte |
US7179423B2 (en) | 2001-06-20 | 2007-02-20 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
US7211442B2 (en) | 2001-06-20 | 2007-05-01 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
EP1412729A2 (en) * | 2001-06-20 | 2004-04-28 | Cytonome, Inc. | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
WO2003001192A1 (en) * | 2001-06-20 | 2003-01-03 | Cytonome, Inc. | Microfabricated separation device employing a virtual wall for interfacing fluids |
EP1412729A4 (en) * | 2001-06-20 | 2005-03-09 | Cytonome Inc | Microfluidic system including a virtual wall fluid interface port for interfacing fluids with the microfluidic system |
EP1424559A1 (en) * | 2001-08-09 | 2004-06-02 | Olympus Corporation | Micro flow passage device, connection device, and method of using the devices |
EP1424559A4 (en) * | 2001-08-09 | 2006-03-01 | Olympus Corp | Micro flow passage device, connection device, and method of using the devices |
EP1314479A3 (en) * | 2001-11-24 | 2004-03-24 | GeSIM Gesellschaft für Silizium-Mikrosysteme mbH | Device for the transfer of liquid samples |
EP1314479A2 (en) * | 2001-11-24 | 2003-05-28 | GeSIM Gesellschaft für Silizium-Mikrosysteme mbH | Device for the transfer of liquid samples |
US7265348B2 (en) | 2002-11-08 | 2007-09-04 | Diagnoswiss S.A. | Apparatus for dispensing a sample in electrospray mass spectrometers |
WO2005089944A3 (en) * | 2004-03-17 | 2005-12-08 | Reiner Goetzen | Microfluidic chip |
US7718127B2 (en) | 2004-03-17 | 2010-05-18 | microTec Gesellschaft für Mikrotechnologie mbH | Microfluidic chip |
WO2011160015A2 (en) | 2010-06-17 | 2011-12-22 | Abaxis, Inc. | Rotors for immunoassays |
EP2583100A2 (en) * | 2010-06-17 | 2013-04-24 | Abaxis, Inc. | Rotors for immunoassays |
EP2583100A4 (en) * | 2010-06-17 | 2013-11-06 | Abay Sa | Rotors for immunoassays |
US9816987B2 (en) | 2010-06-17 | 2017-11-14 | Abaxis, Inc. | Rotors for immunoassays |
US10371701B2 (en) | 2010-06-17 | 2019-08-06 | Abaxis, Inc. | Rotors for immunoassays |
US10969385B2 (en) | 2010-06-17 | 2021-04-06 | Zoetis Services Llc | Rotors for immunoassays |
Also Published As
Publication number | Publication date |
---|---|
DE60023862D1 (en) | 2005-12-15 |
EP1166103A1 (en) | 2002-01-02 |
DE60023862T2 (en) | 2006-07-27 |
EP1166103B1 (en) | 2005-11-09 |
GB9907249D0 (en) | 1999-05-26 |
AU4292700A (en) | 2000-10-16 |
ATE309534T1 (en) | 2005-11-15 |
JP2002540425A (en) | 2002-11-26 |
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