WO2005002730A1 - Procede et dispositif pour la microfluidique - Google Patents

Procede et dispositif pour la microfluidique Download PDF

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Publication number
WO2005002730A1
WO2005002730A1 PCT/GB2004/002850 GB2004002850W WO2005002730A1 WO 2005002730 A1 WO2005002730 A1 WO 2005002730A1 GB 2004002850 W GB2004002850 W GB 2004002850W WO 2005002730 A1 WO2005002730 A1 WO 2005002730A1
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Prior art keywords
droplet
reaction mixture
carrier fluid
aqueous reaction
cells
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PCT/GB2004/002850
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English (en)
Inventor
Stephan Mohr
Philip John Royle Day
Nicholas J. Goddard
Peter Robert Fielden
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The University Of Manchester
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Publication of WO2005002730A1 publication Critical patent/WO2005002730A1/fr

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/30Injector mixers
    • B01F25/31Injector mixers in conduits or tubes through which the main component flows
    • B01F25/314Injector mixers in conduits or tubes through which the main component flows wherein additional components are introduced at the circumference of the conduit
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30Micromixers
    • B01F33/302Micromixers the materials to be mixed flowing in the form of droplets
    • B01F33/3021Micromixers the materials to be mixed flowing in the form of droplets the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30Micromixers
    • B01F33/3031Micromixers using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/502769Containers 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 multiphase flow arrangements
    • B01L3/502784Containers 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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0605Metering of fluids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0654Lenses; Optical fibres
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087Multiple sequential chambers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/02Drop detachment mechanisms of single droplets from nozzles or pins
    • B01L2400/027Drop detachment mechanisms of single droplets from nozzles or pins electrostatic forces between substrate and tip
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the present invention relates to microfluidic methods and devices for the accurate and sensitive, quantitative detection and analysis of chemical entities, including chemical and biological samples such as a minor population of cells within mixed populations of cells.
  • the invention relates to the amplification and subsequent detection of nucleic acid sequences from the minor population of cells of interest.
  • the analysis and manipulation of small quantities of chemicals may be carried out using conventional techniques.
  • nanoliter sized plugs in miniaturised chemical and biological reactions whereby each plug can act as a microreactor.
  • the content of the plug has to be compatible with the device in which it is created due to its contact with the device.
  • the plugs are also unsatisfactory for the analysis and manipulation of very small quantities of sample. It is desirable to provide a method and device that could allow extremely small known volumes of chemicals (pico or nanoliters) to be monitored and manipulated whilst being kept separate from any chemicals that may result in unwanted side reactions.
  • a first object of the present mvention is to provide improved microfluidic methods and devices for the monitoring and manipulation of chemical entities, including biological samples.
  • a further object of the present invention is to provide improved methods for detecting a minor population of cells within mixed populations of cells.
  • a first aspect of the present mvention provides a method of monitoring and/or manipulating at least one chemical entity, the method comprising: (i) obtaining an aqueous reaction mixture comprising at least one chemical entity; (ii) introducing a volume of the aqueous reaction mixture into a volume of a carrier fluid immiscible with the aqueous reaction mixture to form a droplet of the aqueous reaction mixture supported by the carrier fluid; and (iii) monitoring and/or manipulating the chemical entity within the droplet.
  • the method of the first aspect provides a general schema to provide enhanced monitoring and/or manipulation of a chemical entity on a microscale.
  • the chemical entity is kept within its own discrete environment and may be manipulated, for example by the addition of further reagents to the droplet and/or by movement ofthe droplet through area or zones of particular conditions, such as heat or light.
  • reagents may be added to the aqueous mixture prior to formation of the droplet or to the droplet itself.
  • Droplets containing different chemicals may be merged together.
  • the droplet is transfeired within the carrier fluid, the droplet being surrounded by the fluid except for when it is desired to merge the droplet with another droplet.
  • the sensitivity of the method is regulated by controlling the volume of aqueous mixture used to form the droplet.
  • aqueous reaction mixture In order to expose the aqueous reaction mixture to different conditions for the manipulation thereof, (such as zones at different temperatures) it is preferable to introduce the aqueous reaction mixture into an immiscible moving carrier fluid that is supported in a microfluidic flow manifold or chip.
  • a microfluidic flow manifold or chip Such devices represent an important aspect of the invention.
  • the device may be fabricated by direct machining of polymeric substrates or by injection moulding.
  • a second aspect of the present invention provides a microfluidic device comprising: a substrate with different zones that are subjectable to defined conditions, the substrate bearing a continuous conduit adapted to carry an immiscible carrier fluid that passes through the different zones; a reservoir which is comiected to the conduit with means to introduce droplets of an aqueous reaction mixture from within the reservoir into the carrier fluid within the conduit, the droplet being supported by the carrier fluid; and optionally a detector capable of identifying products within individual droplets which have travelled through the different zones within the conduit.
  • the method of the present invention is particularly applicable to the detection of minor population of cells within mixed population of cells.
  • a third aspect of the present invention provides a method of detecting analytes from a minor population of cells within mixed populations of cells, the method comprising: i) obtaining an aqueous reaction mixture comprising analytes from a mixed population of cells; ii) adding assay reagents suitable for detecting analytes from the minor population of cells of interest to the aqueous reaction mixture; iii) introducing a volume of the aqueous reaction mixture into a volume of a carrier fluid immiscible with the aqueous reaction mixture to form a droplet of the aqueous reaction mixture supported by the carrier fluid; iv) performing some or all of an assay, in the droplet, wherein an assay product is representative ofthe presence ofthe minor population of cells of interest; and v) analysing the droplet for the presence ofthe assay product.
  • the method of the third aspect of the invention represents a general schema to increase sensitivity (and also specificity) of biological sample detection. It is particularly useful for aiding the determination of disease thresholds.
  • the method may also be used to simplify current laboratory multi-step and contamination prone processes with savings relating to time, money and increased assay throughput, quality and reliability.
  • the method is also useful as a basis for improved clinical diagnosis and prognosis.
  • the method offers for the first time an ability to place accurate numbers to characterise the state or progression of disease, which could be used in a dynamic context if used in serial samples as a function of time.
  • the method ofthe third aspect ofthe invention represents an improved method for detection of analytes from minor populations of cells in a number of fields.
  • improvements include: (a) increasing the sensitivity of pre-existing assays; (b) potential application to the analysis of single cells which may be mandatory for stem cell analyses; (c) definitive assessment of thresholds for diseases and infectious agents, including the assessment of host response; (d) hastening of reaction and ease of interpretation; (e) reducing ambiguity of result assessment and implementing a new regime of assessment by sequential analysis; and (f) improved mass spectrometry delivery systems; (f) PCR in presence of inhibitory substances - crude extracts relying upon dilution to remove the effect ofthe inhibitory substance; and (g) general flow cytometric analyses.
  • the mixed population of cells may include cultured cells and biological tissue samples, such as biopsies or blood samples, and samples of other biological fluids such as lymph, sputum, cerebrospinal fluid, and the like. Additionally, non-clinically related applications in forensic sciences, genetic modified organisms, environmental and toxicological testing would further benefit from the invention.
  • the method of the third aspect of the invention is particularly applicable to analysis of biological samples of interest that comprise substantially single cells free in the aqueous reaction mixture.
  • this method of the invention may further comprise dispersing cells from a biological sample of interest to provide a population of substantially single cells.
  • droplets may be formed that contain whole cells (in which case subsequent lysis may be required to release assayable cell contents) or droplets may be formed from cells that have been previously ruptured in the aqueous reaction mixture.
  • the aqueous reaction mixture may advantageously comprise an agent capable of lysing biological cells, in order to better permit entry of the other components ofthe reaction mixture into cell nuclei present in the sample.
  • Simple alkali lysis followed by neutralisation, application of detergents, proteolytic enzymes or use of somcation or high voltage are procedures successfully applied to releasing the nucleic acids from whole cells. The procedure is also amenable to accepting nuclear material from fixed paraffin wax embedded tissues.
  • a droplet may be formed which contains a cell (or cell contents) and none, some or all of the assay reagents required to perform the assay.
  • the methods of the first and third aspects of the invention represent the application of discrete microfluidic technology to control the amount of a chemical or chemicals or number of cells, or amount of cell content, contained within each droplet. Since the aqueous reaction mixture is present in the form of discrete volumes supported in an immiscible carrier fluid each droplet provides reaction products that may be analysed independently from the products of other droplets. Alternatively the droplets may be coalesced for combined analysis (e.g. on a down-stream gene array).
  • each droplet only contains a few cells and most preferred that each droplet contains a single cell. Since the contents of each droplet remain separate during subsequent assay procedure each assay result will be representative of the characteristics of the single cell. Furthermore the reaction products in each droplet may be specific to a single cell from a minor population.
  • the minor population may be differentiated from the other cells types on a cell by cell basis. Accordingly the prior art failings of sensitivity (e.g. resulting from cross-contamination by other cells which reduces the capability to detect populations of cells present at low copy number within a tissue) is obviated.
  • each droplet contains a single cell
  • the third aspect of the invention provides considerable benefits over the prior art even in the case that the droplet contains up to ten cells, up to a hundred cells, or more. It will be appreciated that pre-testing for a specific assay set-up will confirm the detection limit of one test cell amongst a maximum excess of other non-target cells.
  • the cells When the method involves the generation of cell lysates that are subsequently made into droplets, the cells may be treated to release their contents within a reservoir containing the aqueous reaction mixture.
  • the dilution of cells into the solution in the reservoir will be known and hence the expected concentration of cell contents calculated.
  • the dilution will be controlled such that one copy (or at least a defined number) of a particular cell content will be present in a droplet. Hence alterations in copy number will be amenable to detection.
  • aqueous reaction mixtures comprising cell lysates, rather than whole cells, may be used to generate droplets according to the invention.
  • the analysis of the reaction mixture for the presence of the assay product may be performed at a site remote from the site at which the droplet is introduced into the carrier fluid.
  • Transport of the droplet may be achieved either by movement of the carrier fluid (such that it transports the droplet) or by independent movement of the reaction mixture with a static carrier fluid.
  • the droplets may be introduced into an immiscible moving carrier fluid, supported in a microfluidic flow manifold, by a number of means.
  • the simplest procedure is to induce outflow of the aqueous fluid from a channel branch, or jet, or other orifice that feeds into the carrier fluid.
  • Pressure, or positive volumetric displacement are well known means of inducing flow of the droplet into the carrier fluid.
  • both the volume of the droplet and the frequency of successive droplets may be achieved with a level of precision that ensures that the discrete droplets/droplets avoid cross-contamination, with either each other or through intermittent conduit wall contact.
  • the inventors have demonstrated that the droplets so formed will tend to travel at the centre of a microfluidic conduit filled with a carrier fluid, thus avoiding wall contact.
  • a preferred method by which such outflow may be achieved is through the application of pressure, or positive volumetric displacement, to the aqueous reaction mixture in a reservoir.
  • This may be acliieved by means such as displacement of a plunger in a barrel containing the aqueous reaction mixture.
  • the displacement may be achieved by changing the volume of a reservoir containing the aqueous reaction mixture, for example by applying an electric current to a piezo electric material in which a channel (suitable for holding the aqueous reaction mixture) has been formed.
  • means of transporting droplets in the static immiscible carrier fluid include physical phenomena such as electrostatics, dielectrophoresis, ultrasonic agitation and thermally-induced convection.
  • An alternative method of inducing the requisite generation of droplets is by electrical droplet generation.
  • the presence of buffer salts in the aqueous sample phase will mean that the sample has moderately high conductivity, in contrast with the carrier fluid which should have significantly lower conductivity than the sample, and preferably be a good insulator.
  • two or more side channels will be disposed opposite or adjacent to each other across the main conduit containing a flowing carrier fluid.
  • One will contain the aqueous sample, while the other will be filled with a solid conductor, which may be a metal, metal-filled polymer, conducting carbon or graphite or a conducting carbon-filled polymer.
  • the solid conductor cannot move, only the aqueous sample will move into the main channel, where it will be subject to shear forces from the flow ofthe carrier fluid. When enough sample has entered the main channel, it will shear off from the end ofthe side channel and be carried along as a charged droplet.
  • the force experienced by the sample at the end ofthe side channel is given by:
  • A is the cross-sectional area ofthe side channels
  • Fis the applied potential difference
  • d is the distance across the main conduit between the ends of the side channels.
  • P is the pressure. It can be seen from this equation that the pressure is independent of the cross-sectional area of the side channel. To generate a droplet, it is necessary that the pressure exerted on the aqueous sample exceeds the internal pressure of the carrier fluid. If we take a typical fluid pressure of 0.1 atmospheres (10,000 Pa) and a fluid with a dielectric constant of 2.5 and a side channel separation of 100 ⁇ m, we find that to balance the internal pressure will require an applied voltage of -2377 volts. Any higher voltage will result in the formation of droplets. The applied voltage can be applied continuously, which will result in a continual stream of droplets at a rate controlled by the separation of the aqueous sample from the end of the side channel (the maximum droplet generation rate), or can be pulsed to provide droplets on demand.
  • two power supplies be used.
  • One will provide the bias voltage, which will just balance the internal pressure and stop the carrier fluid from entering the side channel, and one that will be pulsed to generate droplets.
  • the pressure depends on the square ofthe applied voltage, so to double the pressure will require only a 41% increase in applied voltage.
  • An adjustable 2.5kN power supply may be used to provide bias and a pulsed lkN power supply to generate the droplets.
  • similar results may be achieved through the application of a suitable high voltage AC waveform.
  • the assay procedure according to the third aspect of the invention involves some sort of amplification ofthe analyte to be detected from the minor cell population.
  • polymerase chain reaction (PCR) amplification and detection ofthe amplified PCR products represent a preferred assay procedure according to the method ofthe third aspect ofthe mvention.
  • PCR quantitative measurement of minor nucleic acid populations within large heterogeneous nucleic acid populations cannot be accurately achieved using conventional PCR.
  • the inventors have demonstrated that, if too highly diluted, PCR cannot detect low copy number aberrant nucleic acid sequences, and therefore, for example, current minimal residual disease thresholds are related to and limited by assay sensitivity rather than being a true reflection ofthe threshold level above which a cancerous clonal population requires remedial action.
  • One of the great benefits of preferred, PCR based, methods according to the third aspect of the invention is that the methods allow true and reproducible quantitative measurement of nucleic acids from minor cell populations and thus reflects actual levels of minimal residual disease.
  • PCR primers may be selected that only amplify nuclear material (such as fusion gene transcripts) found in the minor cell population.
  • the primers may be designed to specifically lead to the amplification of a mutation in an oncogene that transforms a cell in cancer.
  • the primers may also be designed to allow amplification of a transcript for a protein that is only expressed in the cancerous state. It will be appreciated that methods of the third aspect of the invention that utilise PCR and such primers are particularly useful for making a prognosis for cancer patients or in the diagnosis of cancer. Such primers are particularly useful for making assessments of minimal residual disease (MRD) in cancer patients (e.g. clinical assessment of reoccurring clonal populations in leukaemia).
  • MRD minimal residual disease
  • the primers may be designed to amplify a microbial gene foimd in a pathogen but not in a host organism. It will be appreciated that methods ofthe third aspect of the invention employing such primers can be used to detect a pathogen in a multi- cellular orgamsm. Accordingly the method is useful in the diagnosis of microbial infections (whether protozoa, fungal, bacterial or even viral). For instance preferred methods ofthe third aspect ofthe invention may be used to diagnose malaria.
  • the aqueous reaction mixture may also contain nucleotides, primers and a DNA polymerase (e.g. Taq polymerase). Droplets comprising the aqueous reaction mixture may then be injected into the carrier fluid which then flows into a PCR reactor. Real-time optical detection may be performed on the outflow from the PCR reactor to enable the dynamics ofthe reaction to be recorded.
  • a DNA polymerase e.g. Taq polymerase
  • the temperature of the bulk carrier fluid may be varied thereby inducing PCR in the aqueous reaction mixture.
  • the droplets are introduced into a PCR reactor wherein they are transported through zones of different temperature in order to perform the polymerase chain reaction.
  • zones of different temperature may be formed within a "PCR chip".
  • a circuitous conduit, carrying the carrier fluid and aqueous reaction mixture passes through a solid substrate. Different zones ofthe substrate may be heated to different temperatures, and the temperature of the aqueous reaction mixture controlled by controlling the length of time that the reaction mixture spends in the different heated zones.
  • PCR chip that may be adapted for use according to the invention. It will be appreciated that the Kopp PCR chip should be adapted, as discussed in more detail below, such that there are: (a) means for introducing droplets of aqueous reaction mixture into the carrier fluid (the carrier fluid may then flow through the chip to allow PCR reactions to occur in each droplet); and (b) means for detecting PCR product in each droplet.
  • a PCR reactor comprising: a substrate with different zones that are heatable to defined temperatures and the substrate bears a circuitous conduit adapted to carry an aqueous-immiscible carrier fluid that passes through the different zones; a reservoir which is connected to the conduit with means to introduce droplets of an aqueous reaction mixture from within the reservoir into the carrier fluid within the conduit, the droplet being supported by the carrier fluid; and a detector capable of identifying PCR products within individual droplets which have travelled through the different zones within the conduit.
  • the PCR reactor according to the fourth aspect of the invention is particularly useful for carrying out the method according to the third aspect ofthe invention.
  • the PCR reactor may serve as either a standalone device or may be used for integrated applications in both routine and research fields.
  • the device may be used in the fields of prognostics, diagnostics, toxicology, security systems and forensic sciences.
  • the device is highly suited to rapid, sterile, reproducible, integrated and cost effective multi-parallel or serial sensitive quantitative analyses.
  • a prefened embodiment of the PCR reactor is adapted for application to a high throughput experimental scheme, to enable many discrete PCR amplifications and associated diagnostic assays.
  • This may be achieved by discrete microfluidics, where the PCR reaction, sample and detection components are contained within single droplets supported in the carrier fluid (e.g. an immiscible fluid phase, such as an oil).
  • the carrier fluid e.g. an immiscible fluid phase, such as an oil.
  • the droplets can be generated such that their diameter is significantly less than the cross-section ofthe conduit into which they are introduced, hi this way, the droplets may be transported along the channel structure defined on the substrate without touching the conduit walls and without being forced to merge with similar preceding or following droplets.
  • the PCR reactor according to the fourth aspect of the invention is adapted for use in miniaturised integrated analysis systems and particularly in Micro Total Analytical Systems ( TAS).
  • TAS encompasses miniaturised integrated analysis systems. These systems allow the production of meaningful data from raw biological samples without the requirement for manipulation by an operator.
  • Preferred PCR reactors according to the fourth aspect of the invention allow the introduction of a biological sample into the reactor according to the principles of ⁇ TAS.
  • Cell or nucleic acid dilution may be regulated in the reservoir and the volume of droplet introduced into the carrier fluid such that enhanced sensitivity of detection and increased assay quantitation is achieved according to the method ofthe third aspect ofthe invention.
  • the inventors believe that the conventional approach is incorrect because they have found that sensitivity increases with respect to detecting a minor nucleic acid population within an excess of other nucleic acids when the method of the third aspect of the invention is followed.
  • the procedure is able to also eliminate the averaging effect of PCR to permit the detection of cells possessing different levels of a nucleic acid that is common to all cells, for example a proto-oncogene that becomes chromosomally amplified, or disease- related de-regulated or de-no vo expression of a transcript.
  • RNA may be quantified (rather than DNA) and therefore the magnitude of change i.e. copy number of a particular transcript, will be amplified.
  • RNA may be quantified using the same methodology as used to quantify DNA (e.g. a modified Kopp Chip) but will also employ an upstream reverse transcriptase to convert mRNA into cDNA. For example Obeid et al.
  • the substrate may be comprised of messenger RNA or (mRNA) added to a reverse transcriptase enzyme, nucleotide triphosphates and an appropriate buffer.
  • mRNA messenger RNA
  • mRNA reverse transcriptase enzyme
  • nucleotide triphosphates an appropriate buffer.
  • One temperature incubation typically 42°C and then straight into PCR (see Obeid et al supra).
  • Droplets within the conduit of the Reactor must be subjected to temperature cycling for the PCR process to achieve the desired amplification. This may be achieved through known means of bulk temperature cycling, or through transport of the droplet stream, along the conduit, through a meandering channel that re-enters the temperature cycle as many times as required (in this case, the temperature cycle is realised through differential heating of two, or three, zones within the supporting substrate structure).
  • a first zone is kept at about 95°C and a second zone kept at about 60°C.
  • the first zone is kept at about 95°C; the second zone kept at about 77°C; and the third zone kept at about 60°C.
  • the zones may be heated by any means that generates a stable temperature, but preferably through the electrical heating of a conductive element which incorporates a feedback mechanism to ensure maintenance of a stable and fixed pre-selected temperature.
  • the temperature zones may be supplied with the same PCR aqueous fluid with common sample.
  • the droplets may be continually generated for a fixed time period or for the generation of a pre-determined droplet number.
  • An alternative to this is to introduce a statistical criterion that forces the prolonged production of droplets until sufficient data have been collected to realise a preset data quality of the data ensemble.
  • This approach has the distinct advantage of economy of both PCR sample and assay time, and ensures the achievement of a pre-determined data quality.
  • the discrete droplets comprise different samples, thus providing a significantly high throughput of different samples.
  • the droplet outlet ofthe conduit manifold either in single or multiparallel embodiment, may be manipulated to waste, or may be collected, sorted, combined or stored in suitable array formats.
  • the detector detects fluorescence of PCR reaction products.
  • An epifluorescence configuration may be used, providing a set of detection zones (e.g. one per thermal cycle).
  • a set of holes may be provided for GRIN (gradient index) lenses. These lenses may be approximately 2mm in diameter.
  • GRIN lenses are convenient for this application in that they are cylindrical in shape and do not require complex machining to provide a secure mount. The lenses will be chosen to bring collimated light incident on the outside face of the GRDST lens to a focus just beyond the inner face ofthe lens, in the main conduit. As droplets pass through the focus, fluorescence will be excited.
  • any fluorescence emission falling on the end of the GRIN lens will be collimated on exit from the outer face of the lens.
  • a dichromic filter may be fitted to direct only the fluorescence emission onto a multi-channel photomultiplier. Up to 32 channels of fluorescence can then be monitored simultaneously. It should be noted that the PCR Reactor of this embodiment of the invention is limited to 32 thermal cycles. It may be useful to omit the first few thermal cycles from the detection system, as there will generally be too little PCR product to detect.
  • a most preferred PCR reactor according to the invention comprises a single channel device with on-chip droplet generation, off-chip carrier and sample reservoirs and pumps, two-sided heating (two-zone PCR) and GRIN lens array for detection.
  • a method of analysing for the presence of a nucleic acid sequence of interest within a tissue of interest comprising: i) obtaining an aqueous reaction mixture comprising nucleic acids from biological cells from the sample of interest; ii) adding polymerase chain reaction (PCR) reagents suitable for amplifying the nucleic acid sequence of interest; iii) introducing droplets of the aqueous reaction mixture into a carrier fluid immiscible with the aqueous reaction mixture such that a single copy of the nucleic acid of interest is contained within the droplet, the droplet being supported by the carrier fluid; iv) performing the polymerase chain reaction to produce, in the droplet, an amplification product representative of the presence of the nucleic acid sequence of interest; and analysing the aqueous reaction mixture for the presence ofthe amplification product.
  • PCR polymerase chain reaction
  • PCR based methods are particularly useful in analysing for the presence of cancer cells within a biological tissue.
  • cancer cells may represent only a small proportion of the total number of cells present in a tissue, the majority of cells being non-transformed and healthy.
  • tissue samples such as biopsies
  • PCR based methods are also particularly useful for helping a clinician to test a biopsy sample to evaluate whether or not the tissue is free of cancerous cells (i.e. minimal residual disease (MRD) analysis).
  • MRD minimal residual disease
  • a method of analysing for the presence of cancer cells in a biological sample comprising: i) obtaining an aqueous reaction mixture comprising the biological sample; ii) adding polymerase chain reaction (PCR) reagents suitable for amplifying a nucleic acid sequence indicative of cancer cells; iii) introducing a volume of the aqueous reaction mixture comprising a defined number of cells into a volume of a carrier fluid immiscible with the aqueous reaction mixture to form a droplet of the aqueous reaction mixture in the carrier fluid, the droplet being supported by the carrier fluid; iv) performing the polymerase chain reaction to produce an amplification product representative ofthe presence ofthe nucleic acid sequence indicative of cancer cells; and v) analysing the droplet for the presence of the amplification product; wherein the presence of the amplification product indicates that the biological sample contains
  • PCR based methods according to the third or fifth aspects ofthe invention are also able to detect the presence of pathogens, such protoza, fungi, bacteria or viruses, which may also be present at relatively low numbers in a biological sample.
  • a method of analysing for the presence of a pathogen in a biological sample comprising: i) obtaining an aqueous reaction mixture comprising the biological sample; ii) adding polymerase chain reaction (PCR) reagents suitable for amplifying a nucleic acid sequence indicative ofthe pathogen; iii) introducing a volume of the aqueous reaction mixture comprising a defined number of cells into a volume of a carrier fluid immiscible with the aqueous reaction mixture to form a droplet of the aqueous reaction mixture in the carrier fluid, the droplet being supported by the carrier fluid; iv) performing the polymerase chain reaction to produce an amplification product representative of the presence of the nucleic acid sequence indicative of the pathogen; and v) analysing the droplet for the presence ofthe amplification product; wherein the presence of the amplification product indicates that the biological sample contains pathogens.
  • PCR polymerase chain reaction
  • Example 1 illustrates that a mixture of cells in a sample is detrimental to the sensitivity of conventional assays for detecting a minor population of cells in the sample
  • Example 2 illustrates droplet formation by volumetric displacement of fluid at a microfluidic junction
  • Example 3 investigates droplet formation on demand using a high voltage pulse
  • Example 4 illustrates a microfluidic device according to one embodiment ofthe present invention, its incorporation into a PCR microreactor and investigates the flow behavior of droplets through the device
  • Example 5 investigates droplet merging
  • Example 6 illustrates a PCR microreactor according to another embodiment ofthe invention and studies fluorescent detection ofthe contents ofthe droplets
  • Figure 26 is a schematic diagram ofthe optical set up for each channel ofthe PCR chip;
  • Figure 27 is a plot of fluorescence intensity (a.u) versus time (10 "4 sec) for fluorescin containing droplets at the end of cycle 7 and 8;
  • Figure 28 is a schematic illustrating the steps ofthe polymerase chain reaction.
  • EXAMPLE 1 Experiments were conducted to demonstrate that a mixture of cells in a sample is detrimental to the sensitivity of conventional assays for detecting a minor population of cells in the sample. This illustrates that the usefulness of the methods and PCR reactor according to the invention for increasing the sensitivity of detection of a minor population of cells within a heterogeneous mixture of cells.
  • Biological material typically comprises several differentiated cell types, and many cell types may be present in such low numbers that they become difficult to detect.
  • Fig 1 shows schematically how different mixed populations of cells might be represented with respect to a specific oncogene.
  • Fig 1 also indicates how PCR tends to provide an averaging effect. Whilst this phenomenon is satisfactory for correctly ascertaining gene copy number from within homogeneous cell populations, current applications of PCR are unable to detect minor cell populations within heterogeneous cell populations and tend to deliver a result resembling the average gene content per cell. Even if a unique sequence is to be analysed it is highly likely that reaction sensitivity and sample error will produce a false low or even negative reaction result.
  • Fig 2 further demonstrates the concept.
  • the upper section of the figure represents how present working practice attempts to determine the presence of a sub- population of cells (via a marker gene) from an unknown total population of mixed cell types. This situation contrasts markedly with the one portrayed in the lower part of figure 2 where the total mixed cell population entering the reaction is known, leaving only the amount of cells comprising the sub-population to be determined by PCR experimentation.
  • the invention suggests that a dilution of target sequences will effectively increase assay sensitivity.
  • the invention is therefore able to alter assay sensitivity by both a change in concentration of nucleic acid offered to the reaction and the volume used per reaction, and the whole assembly is in dynamic flow and is amenable to parallelisation.
  • Suspensions ofthe neuroblastoma tumour cell line LAN 1 which possess some 80 copies of the oncogene MYCN DNA were used either directly from cell culture or following storage at -80°C in 10% DMSO. Cells were prepared for FACS using
  • MYCN and ⁇ globin are shown in Table 1, and were designed using Primer ExpressTM software (PE/ABI, Foster City, CA).
  • flanking forward and reverse amplimers were selected by applying the criteria suggested in the TaqMan ® Universal 2 x PCR Master Mix handbook (Perkin Elmer Applied Biosystems P/N 4304437).
  • MYCN probes used FAM as the 5' reporter dye and the ⁇ globin probe used NIC as the 5' reporter dye, the 3' quenching dye was TAMRA or QSY-7 (Molecular Probes, Germany), ⁇ globin was chosen as a reference gene as it is present on chromosome 11 which is highly stable within cells of neuroblastoma tissues.
  • the recommended rapid reaction optimisation procedure was adopted for all real-time PCRs which used 2x master mix.
  • SK LAN 1 cells have approx 80 DNA copies of MYCN per cell
  • FIG. 4 is a schematic illustrating various preferred means for vesicle or droplet generation, namely pressure driven, pressure, electrically driven and electrically driven droplet merging.
  • the formation of droplets by volumetric displacement at a microfluidic junction was investigated.
  • Kloehn N6 syringe pumps were used to inject a dispersed and continuous phase flow of water C into an oil flow A B, as shown in Figure 5 of the accompanying drawings.
  • the pumps had a resolution of 48000 steps and a minimum pump speed of 40 steps per second.
  • a 100 ⁇ l syringe was used for the water and a 250 ⁇ l for the oil.
  • a standard CCD video camera fitted with a 4x microscope objective was used to image the droplets fonned.
  • the camera had a frame rate of 30 fps and the frame size was 320 x 240 pixels.
  • the droplet volume was estimated by counting the number of droplets produced in 5 seconds and calculating the average droplet volume based on the flow rate ofthe water.
  • EXAMPLE 3 Whilst the method of Example 2 did generate droplets of controllable size, it only produced them as a constant stream. A method that could produce droplets into the continuous phase on demand is desirable as it would offer more confrol over the manipulation of the droplets. The droplets could be produced at different points in a microchannel network in a time fashion so that they collide and coalesce, before being transported to a different area of a microchip to be reacted further. For this reason a method was investigated to produce droplets on demand by using a high voltage pulse to push a controlled volume of water into the channel containing the flowing oil.
  • a chip was fabricated as in Example 2 but with an extra channel, filled with conducting epoxy, directly opposite the smaller water inlet.
  • the fluidic structures were fabricated on a 75 x 75 x 6mm sheets of polmethyl methacrylate (PMMA) using a precision CNC machine (Datron CAT3D), Datron GmbH, Germany) with 100 and 200 um diameter end mills.
  • the cross section of the channels were rectangular and between 100 and 200 um deep and between 100 and 500 um wide.
  • Figures 12a and 12b shows a two-phase flow microfluidic chip.
  • the chip consists of an oil inlet 10, a water inlet 12 and an added electrode 13. All inlets are comiected via a 100 um wide and 100 um deep channel to the main channel that is 30mm long and 200 um deep.
  • the electrode 13 consists of a thermosetting silver loaded epoxy (RS Ltd, UK) that is filled into a milled channel opposite the water inlet. This channel was milled and filled with silver epoxy before the main channel was milled to produce a smooth transition between the electrode and the main channel. Further downstream two more inlets 14 and 15 intersect the main channel for creating droplets of different reagent.
  • the fluid connectors were made by 1mm access holes drilled through the chip. Threads were cut to fit tube connectors (062 Minstan tube fitting, The Lee Company, USA) for easy connection to the pumps.
  • Figures 13 and 14 show a fluidic chip with a tapered main channel.
  • the main channel which is 200 um deep, is narrowed down from 300 um to 150 um and is then intersected by the 100 um deep and 100 um wide water inlet and the silver epoxy electrode.
  • the chip was sealed with a second sheet of Perspex in that a mirror image of the main channel is milled but which is only 100 um deep forming a total of 300 um. This method ensured that the droplets injected from the water inlet (100 um deep) flowed into the centre ofthe main channel to form spheres and were kept away from the channel walls.
  • the silver electrode was attached to a Brandenburg High Voltage Power supply and the water inlet was attached to ground via a piece of metal HPLC tubing which was inserted into the water inlet tube. It was hoped that applying a suitably high voltage to the silver electrode would overcome the dielectric forces of the oil and cause the water to be pushed into the oil channel.
  • a standard CCD video camera fitted with a 4x microscope objective was used to image the voltage effects.
  • the camera had a frame rate of 30 ⁇ s and the frame size was 320 x 240 pixels.
  • Figure 16 illustrates the effect of a voltage of +500N on the water inlet.
  • the water was drawn into the oil channel when voltages of 300N or greater were applied to the silver electrode.
  • EXAMPLE 4 A microfluidic device for enabling droplets to meander through different temperature zones was developed and the flow behaviour of droplets therethrough was studied.
  • a flow chip 38 was fabricated by milling into polycarbonate sheets of 4mm thickness and an overall dimension of 75 by 75 mm.
  • curable epoxy shows very good adhesion and solvent resistance and withstands temperatures from -150°C to +125°C.
  • Casting epoxy between two sheets of acetate 46 and separating them at dry ice temperatures results in a good surface quality important for subsequent scattering and fluorescent measurements.
  • Thermal experiments were conducted to test the behaviour ofthe seal at high temperatures (up to 100°C).
  • the aqueous sample is injected from a 100 x 100 ⁇ m injector into the oil
  • this main channel opens up to 400 x 500 ⁇ m via a tapered
  • the droplets were subjected to temperature cycling achieved through the meandering channel 40 between the heated top 42 and bottom surfaces 44 ofthe flow chip, see Figure 19.
  • the droplets oscillate between the two heated zones, whereby the number of cycles is determined by the number of complete oscillations between the two temperature zones.
  • Fig.20 shows one meander in a sequence of video frames. The top surface is heated to 60 °C and the bottom surface heated to 90 °C. In this sequence black droplets (flowing from left to right) turn into white ones on the hot surface and turn back to black subjected to the colder surface.
  • TMC thermal ink Chromazone
  • EXAMPLE 6 An alternative prototype PCR chip 50 was made and tested. It comprises a 16 thermo-cycle channel system and was fabricated as described in Example 4. The main channel meanders 52 between two different temperature zones provided by a heater block 54 at 95°C at the bottom and a heater block 56 at 60°C at the top ofthe chip and has a total length of almost 380 mm (see Figures 24a and 24b). This channel is made using a 500 um end mill tool creating a width of 500um and a depth of 300um. Injecting the sample from a 100 x 100 um channel into the oil filled channel produces the droplets. The droplet volume could be varied between lnl and 10 nl. Droplet speeds were measured from approx. 5mm/sec to several cm/sec.
  • FIG. 25 shows droplets flowing through the PCR chip, at the bottom and the top ofthe chip.
  • the flow of droplets proved to be very stable, tested in a continuous run for more than 2 hours.
  • the droplets stay centred in the channel all along the way tlirough the chip, a very important fact that enables us the fluorescence of every individual droplet to be monitored at the end of each thermo cycle.
  • the optical set up for performing the fluorescence measurement was as follows:
  • the lower temperature block 54 was equipped with 16 GRIN (Gradient Refractive Index, Newport, UK) lenses 58 to focus excitation light into the flow channel (see Figure 26).
  • the correct working distance ofthe lenses have been calculated to take the refractive index steps ofthe air/seal and the seal/oil/sample into account.
  • the laser beam itself is split into 16 individual beams 60 of almost equal intensity (in line) via an acrylic holographic beam splitter 62 (Photonics & Analytical Marketing, Leeds, UK).
  • the beams are collimated to match the GRIN lens arcay with a pitch of 2 mm.
  • the setup Apart from the Ar+ -Laser (6mW @ 488nm, LaserGraphics, Germany) and the dual syringe pump (Harvard, US), the setup is housed in a box with all the crucial optics parts mounted onto an optical rail. Initial fluorescence measurements were performed in order to test and calibrate the system. Sample solutions of fluorescin were prepared to create droplets of well-defined amounts of a known flourophore.
  • Figure 27 shows an example of detected fluorescence of two neighboring detector elements for cycle 7 and 8.
  • the sampling rate during recording was 10kHz for all 16 individual channels.
  • This plot shows a scan of 4.5 sec, the droplets are produced with approx. 6 Hz.
  • Droplet size was estimated to 4nl and the concentration ofthe sample fluorescin solution was lxl 0 "3 M.
  • the system may be calibrated and its sensitivity enhanced to allow detecting fluorescence ofthe PCR reaction products per thermal cycle at real time.
  • EXAMPLE 7 A prefened application for the device and method of the present invention is the amplification of a DNA segment from a complex DNA mixture using the polymerase chain reaction which employs heat to produce the cyclic denaturation of double-stranded DNA into single-strands at high temperature, and the subsequent re-association of complementary sequences at lower temperature into double strands.
  • An aqueous sample containing the DNA to be amplified is injected into an oil- filled channel to produce a droplet that is fully supported by the oil.
  • a further aqueous sample containing the PCR reagents, namely oligonucleotides (forward or reverse amplimers or primers), DNA polymerase and deoxynucleotides is then introduced into the oil-filled channel to merge with the droplet containing the DNA sample.
  • the merged droplet is then passed through the different heat zones of the chip of allow denaturation, annealing and extension. Each of these steps is governed by temperature with denaturation being highest, annealing lowest and extension mid-way, although often the annealing and extension phases are combined, as illustrated in Figure 28.
  • the process starts with the target DNA sample that is typically double-stranded and possesses the sequence of the gene of interest.
  • Two synthetic oligonucleotides (termed forward and reverse amplimers or primers) are selected using software to ensure that they bind with appropriate specificity. They are chosen to each have complementary to opposite strands ofthe target gene, but also their orientation is such that uni-directional 5' to 3' DNA synthesis generates the DNA that exists between the two primers.
  • the primer extension product from one primer serves as a template for the other primer to hybridise and initiate DNA synthesis.
  • the main product from the PCR is thus the exponential production of a gene fragment flanked at both ends by the 5' sequence ofthe forward and reverse primers.
  • a predetermined number of cells By dividing a sample of known concentration into smaller aliquots a predetermined number of cells can thus be made to enter a droplet.
  • this droplet is mixed with appropriate PCR reagents by for example droplet fusion, effective quantitative in-vitro gene amplification can be monitored in real-time. Therefore a system of PCR based around droplet generation offers a means to integrate raw sample processing through to quantitative real-time PCR detection.
  • the process has the advantage of continuous flow high throughput, is highly reproducible, quantitative, greatly reduces the risk of contamination in addition to time and cost savings.

Abstract

L'invention concerne un dispositif et un procédé pour la microfluidique, permettant de surveiller, de manipuler des échantillons chimiques et biologiques et/ou d'effectuer des détections dans ces échantillons. Selon l'invention, un mélange réactionnel aqueux comprenant un échantillon est introduit dans un volume de fluide porteur qui est non miscible avec ce mélange réactionnel aqueux, cela de façon à former une gouttelette de mélange aqueux supportée par le fluide porteur. La sensibilité du procédé est ajustée par adaptation du volume de mélange aqueux utilisé pour former la gouttelette et ce procédé convient particulièrement pour la détection d'analytes à partir d'une population mineure de cellules se trouvant dans des populations mixtes de cellules.
PCT/GB2004/002850 2003-07-02 2004-07-01 Procede et dispositif pour la microfluidique WO2005002730A1 (fr)

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