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WO2006005371A1 - Dispositif microfluidique destine a effectuer une pluralite de reactions et ses utilisations - Google Patents

Dispositif microfluidique destine a effectuer une pluralite de reactions et ses utilisations Download PDF

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Publication number
WO2006005371A1
WO2006005371A1 PCT/EP2004/009149 EP2004009149W WO2006005371A1 WO 2006005371 A1 WO2006005371 A1 WO 2006005371A1 EP 2004009149 W EP2004009149 W EP 2004009149W WO 2006005371 A1 WO2006005371 A1 WO 2006005371A1
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WO
WIPO (PCT)
Prior art keywords
capillary
primers
diffusion
reactions
reagents
Prior art date
Application number
PCT/EP2004/009149
Other languages
English (en)
Inventor
François Chatelain
Jean Berthier
Original Assignee
Commissariat A L'energie Atomique
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Filing date
Publication date
Application filed by Commissariat A L'energie Atomique filed Critical Commissariat A L'energie Atomique
Priority to PCT/EP2004/009149 priority Critical patent/WO2006005371A1/fr
Priority to JP2007520786A priority patent/JP2008506376A/ja
Priority to EP05775164A priority patent/EP1776473A1/fr
Priority to CA002571921A priority patent/CA2571921A1/fr
Priority to PCT/EP2005/008743 priority patent/WO2006005636A1/fr
Publication of WO2006005371A1 publication Critical patent/WO2006005371A1/fr

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00353—Pumps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00511—Walls of reactor vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00513—Essentially linear supports
    • B01J2219/0052—Essentially linear supports in the shape of elongated tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00657—One-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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

Definitions

  • the present invention relates to a microfluidic device for perforrning a plurality of reactions.
  • the present invention features a method and device for performing a plurality of chemical and/or biological reactions in parallel by providing an array of releasable reagents into the inner surface of a capillary.
  • the present invention is exemplified by performing a large number of polynucleotide amplification reactions using the capillary array.
  • the present invention features a method and device for coupling the amplification of polynucleotides and the detection and/or analysis of the amplified products.
  • the draft sequences currently available for a large number of genomes can serve as reference sequences for assays that are being developed to compare individual sample sequences to such a reference.
  • Such comparative analyses between a reference sequence and a sequence obtained from a person displaying a disorder, or having a predisposition to such a disorder or to a disease should allow the diagnosis, the prognosis and/or the provision of appropriate treatment of said human disorder or disease.
  • Analytical tools of nucleic acid sequences are thus required to study the genetic diversity especially based on single or polynucleotide polymorphisms as compared to the sequences generated by the sequencing of entire genomes.
  • PCR polymerase chain reaction
  • the first step of PCR consists in heating the reaction mixture containing the target DNA, a large excess of primers, the four nucleotide bases, and DNA polymerase, such that the paired strands of all of the DNA in the sample denature, or separate. The single strands are thus accessible for the primers.
  • the sample is cooled to allow do ⁇ ble-strands to form again. Because of the large excess of primers, the two strands of the denatured or separated strands of the target DNA templates bind to the complementary primers instead of with each other.
  • the temperature is adjusted to obtain maximum activity for the DNA polymerase enzyme. For each DNA sequence that is annealed to a short primer, the enzyme will extend the primer's sequence such that the original double helix for the DNA sequence is replicated.
  • Nucleic acid amplification technologies have been developed in various fields including, for example, the food industry or agro-industry for contaminant detection, or the medical field for disease diagnostic or genetic testing.
  • PCR is typically performed in disposable reaction tube such as small, plastic, microcentrifuge tubes or test tubes which are placed in an instrument containing a thermally controlled heat exchanger.
  • PCR reaction volume is generally comprised between 10 microliters and 1.5 milliliters in conventional heat block or liquid bath heat exchanger PCR instrument designs where the reaction mixture has been stored in microcentrifuge tubes.
  • the procedures should also be easy to carry out, i.e., requiring a minimum of manual steps to be performed by a technician.
  • Multiplex amplification consists in performing multiple PCR amplifications in the same reaction tube by mixing different pairs of primers, said pairs of primers being designed so that they do not interact each other.
  • the different amplified products mixed in the same reaction tube can be detected, for example, by size separation through capillary electrophoresis.
  • PCT patent application WO 01/27327 (Protogene, October 6, 2000) describes a device for performing multiplex amplification by bringing two arrays into close apposition and allowing reagents on the surfaces of the two arrays to come into contact.
  • array-based devices are hampered by their format: planar arrays are indeed difficult to handle and do not allow complex enzymatic reactions to take place.
  • a first object of the present invention is thus to provide methods and device to perform a plurality of polynucleotide amplifications in parallel in a single reaction volume.
  • one object of the present invention is to provide methods and device to perform a plurality of reactions in a single reaction volume, especially a plurality of chemical reactions.
  • the method and device of the invention are indeed capable of generating large amounts of products per unit time by carrying out large numbers of reactions using a single reaction volume. Furthermore the present invention is amenable to full automation.
  • single capillary can be advantageously used to perform different reactions in a single reaction volume, wherein corresponding reagents for each reaction are released in a discrete area of the capillary.
  • products present in a discrete region of a capillary have an axial controllable diffusion capacity which can be used to allow them to mix with other products present in the discrete region of the capillary and to prevent them from mixing with other products present in other distinct regions.
  • a microfluidic device comprising a PCR amplification reaction chamber coupled with capillary electrophoresis is described in US patent 6,372,484.
  • the capillary allows electrophoresis of the amplified product and further analysis.
  • a device for performing PCR amplifications in capillary, comprising a system to control temperature is also commercialised from Roche Diagnostics (the Lightcycler ⁇ , F. Hoffmann-La Roche Ltd, Basel,
  • capillaries are used either as a means for moving small PCR reaction volumes from different reaction chambers or as a means for optimising efficient heat transfer to small PCR reaction volumes, thereby limiting the time of the reaction.
  • a first object of the invention thus concerns a capillary suitable for performing a plurality of reactions, wherein said capillary comprises a plurality of functional rings (FR 1 -FR n ) designed in its inner surface, each functional ring having an inner surface which comprises at least a first area
  • each functional ring having a first releasable reagent releasably attached to said first area (R) within the inner surface of the functional ring of the capillary and a second releasable reagent releasably attached to said second area (F) within the inner surface of the functional ring of the capillary, and wherein in each determined ring, said area (F) is overlapping or is close enough to said area (R) for allowing their respective reagents to mix after their release and for performing a reaction involving at least the two released reagents as substrates.
  • the term "capillary” refers to a single tube or a single channel having a diameter less than 1 millimeter.
  • the inner surface of the functional ring is a part of the inner surface of the capillary, identical to or smaller than the whole surface of the functional ring, which comprises the areas R and F.
  • Any suitable material for capillary may be used in the present invention. These materials include glass, silicon, quartz, metal, among others, and more preferably glass, silicon or quartz.
  • the capillary is rigid, and is preferably made of glass, as illustrated in examples 1 and 2.
  • the capillary of the invention has a diameter of between 1 and 500 ⁇ m, preferably between 1 and 200 ⁇ m, and more preferably between 1 and 100 ⁇ m.
  • the term "functional ring” refers to a functional unit of the capillary which corresponds to the section of the inner surface of the capillary wherein, at least two releasable reagents are attached in a releasable manner, one first reagent being attached to a first area and one second reagent being attached to a second area, said second area overlapping or being close enough to the first area for allowing the reagents to mix especially by diffusion, after their release, and for performing at least one reaction involving at least the two released reagents as substrates.
  • capillary for performing reactions triggered by the mixing of two reagents released in each functional ring
  • many variations of said capillary are within the contemplation of the present invention.
  • more than two reagents can be attached to the same functional ring allowing to perform one reaction using more than two reagents or to perform multiple reactions in the same reaction volume, such as multiplex amplifications.
  • Each reagent occupies a definite area when attached to the inner surface of the capillary, which area may extend after the release of said reagent in such a manner that the reagents of one functional ring are allowed to react together after mixing as a result of the controlled diffusion.
  • molecular diffusion of released reagents and reaction products in a confined reaction region is based on the Einstein law depending upon the viscosity of the fluid, the temperature and hydrodynamic radius of the reagents and reaction products.
  • the reagents occupies the capillary section delimited by the initial functional ring and, surprisingly, the axial diffusion is weak in this first period of time.
  • Axial diffusion is becoming more important in broader range of time.
  • This result has two causes: geometrical and biochemical.
  • the diffusion coefficient is isotropic so that the speed of diffusion is the same in the radial and axial directions.
  • the characteristic time for diffusion in the radial direction on a distance equal to the capillary radius is smaller than that in the axial direction on a distance equal to the spacing between the functional rings.
  • the primers can be recaptured on the wall in the functional rings and this leads to a slower diffusion in the axial direction.
  • the one skilled in the Art will thus determine the structure of the capillary, the density of the attached reagent to the inner surface of the capillary, and the dimensions of each area for obtaining locally a concentration of both reagents after their release equal to or above the critical minimum concentration necessary for the reaction to take place, while avoiding mixing of the other reagents from distinct functional rings.
  • said definite area is a cylindrical section of said inner surface as shown in figure 1.
  • the two reagents of a functional ring can be mixed and attached to the inner surface in the same cylindrical section, the first area (R) and the second (F) being thus indistinguishable.
  • the two reagents can be attached to distinct areas which correspond to neighbour sections of the capillary as shown in figure 1.
  • capillary of the invention is that no specific mechanical methods such as microfabricated reaction wells or chemical methods such as chemical barrier including hydrophobic chemicals are necessary to prevent reagents of one functional ring from entering another functional ring after release.
  • each functional ring has an axial length of between 20 ⁇ m and 10 mm, preferably between
  • the number of functional rings in the capillary-for performing may vary between about 2 to 1000, preferably between about 2 to 100, and more preferably between 10 to 50.
  • the capillary can comprise a region forming a spacer (S) between two functional rings, wherein no reagent is attached to the inner surface of the capillary in said spacer region, wherein the spacer prevents the reagents released from two distinct functional rings to mix together, as shown in figure 1.
  • S spacer
  • the reactions which are compatible with the present device and method are very broad, encompassing all non-unimolecular reactions, preferably chemical reactions involving two or more reagents, provided that the reactions can be performed in the same reaction solution comprising optionally one or more reagent in common.
  • the reagents can thus be selected, but are not limited to, among the peptides, polysaccharides, carbohydrates, lipids, proteins, cells, viruses, and nucleic acids.
  • the reaction is triggered by releasing of the reagents attached to the inner surface of the capillary, at least for one functional ring and their mixing.
  • the reagents and the reaction products remain confined to a discrete region in the functional ring according to the limited axial diffusion in the capillary.
  • the releasable reagents can be synthesized and attached to the capillary in situ or can be synthesized in a first step and attached to the capillary in a second step.
  • the reagents are immobilized via a releasable site, for example, by tethering to an immobilized molecule with a cleavable moiety.
  • Methods for attaching releasable reagents at a specific area to the inner surface of the capillary are described in the Art, and in particular in US patent application
  • the capillary is suitable for performing one or several polynucleotide amplifications.
  • the capillary is suitable for performing a plurality of polynucleotide amplifications in parallel.
  • each functional ring has an inner surface which comprises at least a first area (R) and a second area (F), wherein a first releasable oligonucleotide as the forward primer of one polynucleotide amplification, is releasably attached to said first area (R) within the inner surface of the capillary, and a second releasable oligonucleotide suitable for use as the reverse primer is releasably attached to said second area (F) within the inner surface of the capillary.
  • a forward primer is a primer which can prime extension after hybridisation to the one strand (+) of a target DNA.
  • a reverse primer is a primer which can prime extension after hybridisation to the complementary strand (-) of said target DNA.
  • Each functional ring comprises at least one couple of reverse and forward primers for amplifying one specific nucleic acid sequence. Different primer sequences specific for different regions of the same target sequence can be attached to the same capillary, allowing multiple amplifications of different fragments of the same target sequence to perform.
  • Primers can be released using any usual known methods in the Art, including, enzymatic, chemical, thermal or photolytical treatment.
  • primers may be initially hybridised to immobilized polynucleotides and subsequently released by strand separation from the inner surface of the capillary.
  • one or more primers for polynucleotide amplification reactions may be covalently immobilized on an array via a cleavable site.
  • a cleavable site may be introduced in a moiety, bound to the inner surface of the capillary, during in situ synthesis.
  • the immobilized moieties containing releasable sites may be prepared before they are covalently or noncovalently immobilized on the capillary.
  • said cleavable moiety is cleavable by photolysis.
  • the density of oligonucleotides attached within the inner surface of the capillary in each functional ring is between about 1 and 1000 fentomoles per square mm, preferably between 10 and 100 fmoles/mm 2 .
  • each functional ring is divided into two cylindrical sections comprising respectively the reverse primer and the forward primer for one amplification reaction.
  • the number of functional rings on the capillary is between about 2 to 100, preferably between 10 and 50.
  • capillaries of the invention can be easily integrated in a microfluidic device.
  • the device should provide means for circulating the reaction solution, i.e., to move the reaction solution into the capillary (where the reaction takes place), after its introduction into the device, to immobilize the solution flow during the reaction period, and to remove the solution containing the reaction products from the capillary, before, during or after analysis of said reaction products.
  • solution flow in the capillary is pressure- induced
  • said means for circulating the reaction solution comprises pressure-driven syringe pump.
  • solution flow is, for instance, electro- kinetically induced, and/or induced by shear forces, gravimetrical forces, capillary action, and the like.
  • the device should also provide means for releasing the releasable reagents.
  • Such means can be based for example on thermal methods or photochemical methods initiated by irradiation.
  • said means may comprise irradiation sources which include, but are not limited to, laser light, CRTs, LEDs, Resonant Microcavity Anodes, photodiodes, broad wavelength lamps, and the like. Irradiation can be focused to discrete sites, for example, some specific functional rings along the capillary length using optical or physical methods.
  • the capillary is preferably made of optical clear material, such as glass or quartz, allowing the light to cross the capillary walls.
  • the device may also advantageously comprise means for producing collimated light beams, including for instance, individual lasers, masks, arrays of mirrors, and TV screens.
  • means for producing collimated light beams including for instance, individual lasers, masks, arrays of mirrors, and TV screens.
  • Detection of the reaction products in the capillary can be performed by methods including, without limitation, photochemical, electrochemical, electrophoretic, fluorescent, UV/VIS absorbance, MS, IR, and/or chromatographic methods.
  • Optical detection methods are usually used since they alleviate the problem posed by the necessity to detect the products at the capillary end. Suitable methods are for instance UV-VIS spectrophotometry, direct fluorescence, and time-resolved fluorescence.
  • the device comprises means for carrying out optical detection, including an excitation light source associated to a detector of fluorescent emission such as a PMT, a scanner or a linear charged coupled device (CCD) to generate a quantitative fluorescent image of the capillary as a means for quantitating the reaction products labelled with fluorescent material.
  • an excitation light source associated to a detector of fluorescent emission such as a PMT, a scanner or a linear charged coupled device (CCD) to generate a quantitative fluorescent image of the capillary as a means for quantitating the reaction products labelled with fluorescent material.
  • CCD linear charged coupled device
  • the microfluidic device according to the invention can further comprise means for controlling the temperature in the capillary.
  • the device temperature is usually cycled between 40 and 95 0 C.
  • Systems were developed and are commercially available to enable PCR to be performed in capillaries. Such a system, for instance, the Lightcycler from Roche Diagnostics (F. Hoffmann-Laroche Ltd, Basel, Switzerland) (PCR in capillaries), the P/ACETM Series system from Beckman Coulter, Inc.
  • the device may comprise a plurality of capillaries.
  • Another aspect of the invention relates to a method for performing a plurality of reactions, comprising the use of a capillary according to the invention as above-defined or the microfluidic device of the invention comprising such capillary as above defined.
  • the invention concerns a method for performing a plurality of reactions, comprising the steps of: a) providing a capillary according to the invention as above-defined or the microfluidic device of the invention comprising such capillary as above defined; b) filling the capillary with a reaction solution comprising one or more reagent(s) in common to all reactions and an appropriate buffer; and, c) releasing reagents attached to the inner surface of the capillary; thereby allowing one or several reactions to perform in said capillary, each reaction occurring in the discrete region where appropriate reagents are released and mix.
  • the reagents from distinct functional rings of the capillary are released simultaneously, thereby allowing multiple reactions to perform in parallel in a single reaction volume.
  • the invention thus also concerns a method for performing a plurality of polynucleotide amplifications, comprising the use of a capillary appropriate for performing multiple polynucleotide amplifications according to the invention as above-defined or the microfluidic device of the invention comprising such capillary as above defined.
  • the invention concerns a method for performing a plurality of polynucleotide amplifications, comprising the steps of: a) providing a capillary appropriate for performing multiple polynucleotide amplifications according to the invention as above-defined or the microfluidic device of the invention comprising such capillary as above defined; b) filling said capillary with a reaction solution appropriate for polynucleotide amplifications and an appropriate buffer; and, c) releasing primers attached to the inner surface of the capillary; thereby performing one or several polynucleotide amplifications in said capillary, each amplification reactions occurring in the discrete region where appropriate reverse and forward primers are released and mix.
  • the primers from distinct functional rings of the capillary are released simultaneously, thereby allowing multiple polynucleotide amplifications to perform in parallel in a single reaction volume.
  • a reaction solution appropriate for polynucleotide amplifications may comprise at least one of the following components:
  • the target DNA in particular, genomic DNA or cDNA
  • - salts such as KCI or NaCI solution and/or other appropriate PCR buffers, typically a mixture of NaCI and MgCI 2 ; - dATP, dCTP, dTTP, dGTP;
  • the target might be mRNA as well, the first step is then a reverse transcription to generate cDNA.
  • the solution can also comprise DNA binding dye such as the SyBR green dye, labelled probes containing fluorophores, allowing detection of the amplicons in real time.
  • the reaction solution might include fluorescent dXTP for use in appropriate detection methods.
  • the reaction solution may also further comprise fluorescent-quencher probes type TaqMan, or fluorescent intercalating agents such as SyBR Green.
  • the reaction solution may comprise DNA extracted from a biological sample.
  • the DNA may be extracted from a biopsy, from biological fluid including blood, urine, saliva ...
  • the DNA may be extracted from a biopsy or a biological fluid of an animal, a non-human mammal or a human. It can also be extracted from a cell culture, eucaryotic cells or prokaryotic cells, bacteria, fungi or yeasts.
  • polynucleotide amplification reactions known to those skilled in the Art may be suitable for the instant invention.
  • the most common form of polynucleotide amplification reaction, known as the PCR reaction is well known in the Art.
  • the polymerase used to direct the nucleotide synthesis may include, for example, E. coli DNA polymerase I 1 Klenow fragment of E. coli DNA polymerase, polymerase muteins, heat- stable enzymes, such as Taq polymerase, Vent polymerase and the like.
  • amplification of either or both strand of the target nucleic acid comprises the use of one or more nucleic acid-modifying enzymes, such as DNA polymerase, a ligase, an RNA polymerase, or an RNA-dependent reverse transcriptase.
  • nucleic acid-modifying enzymes such as DNA polymerase, a ligase, an RNA polymerase, or an RNA-dependent reverse transcriptase.
  • polynucleotide amplification reactions may include self-sustained sequence replication (S3R), nucleic acid sequence-based amplification (NASBA), strand displacement activation (SDA), ligase chain reaction (LCR), and Qp replicase system, among others.
  • S3R self-sustained sequence replication
  • NASBA nucleic acid sequence-based amplification
  • SDA strand displacement activation
  • LCR ligase chain reaction
  • Qp replicase system among others.
  • the present invention may be used to couple the amplification and detection procedures of the amplified products, thus providing an environment for simultaneously carrying out a plurality of amplification reactions followed by simultaneous detection of the amplified products.
  • the method further comprises the step (d) of capturing the amplified products by a plurality of immobilized polynucleotide probes on the inner surface of the capillary through hybridization.
  • the invention is thus directed to a method of detecting and analysing nucleic acid in a sample, comprising the use of a capillary suitable for performing multiple polynucleotide amplifications according to the invention as above-defined or the microfluidic device of the invention comprising such capillary as above defined.
  • a fraction of immobilized polynucleotides may contain non- releasable polynucleotides designed to probe (capture) amplification products.
  • the non-releasable probes can be attached to an area of the inner surface of the capillary, located in the corresponding functional ring containing the releasable primers for amplifying the products to be captured by said probes.
  • the non-releasable probe is attached to the inner surface of the capillary located between the (F) area containing the forward primer and the (R) area containing the reverse primer.
  • the probes can be attached to a different area than the functional ring containing the corresponding primers for amplifying the sequence to be captured.
  • the amplified products of a target nucleic acid may also be directly or indirectly analysed by detecting polynucleotide sequence variations.
  • Strategies for identification and detection of polynucleotide sequence variations in a capillary are described in the Art and in particular in US patent application 2003-0032035 (Chatelain et a/., CEA, 2002) or in WO 02/18949 (University of California, 2001).
  • said method further comprises the step of detecting polynucleotide sequence variations by detecting hybridisation complexes obtained at step (c).
  • the invention also concerns the use of a capillary as above-defined or a microfluidic device comprising such capillary, for protein detection and/or analysis.
  • FIG. 1 illustrates the body structure of a capillary according to the invention.
  • the capillary tube comprises functional rings (FR1,
  • each functional ring having an inner surface which comprises at least a first area (F 1 or F2) and a second area
  • R1 and R2 comprising releasable reagents, and wherein F area is closer to R area for allowing the respective reagents of a functional ring to mix together.
  • a spacer is placed between FR1 and FR2 in order to prevent mixing of reagents F1 and R1 with F2 and R2 by diffusion.
  • Figure 2 illustrates the release of reacting moieties and the approximate diffusion of the moieties in the capillary.
  • the upper diagram represents relative concentration of primers F and R in the capillary. PCR reactions occur at the boundaries between the zones F1 and R1, F2 and R2, etc; an optimal design is when the rings are sufficiently spaced so that PCR products do not mix together but not to much spaced in order to satisfy a criteria of minimal length of the system.
  • the lower diagram represents the relative concentration of the reaction product in the capillary.
  • a capillary tube comprising functional rings with forward (F) and reverse (R) primers on its inner surface (fig.1).
  • a capillary tube comprising functional rings was prepared according to the invention.
  • Each functional ring contains forward or reverse primers according to figure 1.
  • the carrier fluid containing the DNA to be amplified is injected inside the capillary. After it has been injected inside the capillary, the carrier fluid is at rest and the primers are released from the different rings and diffuse in the capillary.
  • the PCR reactions PCR1, PCR2, etc. are performed by a series of about 30 cycles of an adequate temperature transient, each transient being of the order of 30 seconds.
  • the couple of rings - containing forward and reverse primers - may be spaced by a spacer, as it is shown in the figure 2.
  • the goal of the modelling is to add to the feasibility of the method in numerically calculating the physical characteristics that allow for a satisfactory functioning of the device.
  • the first step consists in modelling only the molecular diffusion inside the capillary; the second step includes the effect of the association/dissociation on the walls.
  • the first step it is assumed that the primers are instantaneously released from the wall and that they have no adherence to the walls.
  • the second step it is allowed for the primers to temporarily associate on the walls, this phenomenon is supposed to follow a langmuiran kinetics.
  • the first case over-estimates the speed of diffusion along the capillary axis, because there are no delays due to adherence on the walls.
  • the second case it is assumed that the different primers do not interact, which is translated in the model by a unique diffusion coefficient for each of the components.
  • a first step we analyze the diffusion of the primers inside the capillary of the invention, assuming that these primers do not adhere to the walls.
  • This model gives an over-estimate of the speed of diffusion because there are no delays due to the association/dissociation of the primers with complementary sequences at the walls.
  • the primers are considered as particles bouncing elastically on the walls.
  • the equation for diffusion in an axisymmetric, cylindrical coordinate system (r,z) is
  • the dimensions of the capillary are such that the ratio between the inner radius and the total length of the capillary R/L is very small (this ratio is smaller than 5. 10 '3 ): from this ratio, a difficulty in the meshing of the system stems : a good numeric solution requires an almost equal size of the meshes in the axial and radial directions r and z. If one chooses 20 meshes along the r direction, meaning a mesh size of about 2.5 ⁇ m in this direction, it is necessary to have more than 8000 meshes in the axial z direction.
  • anisotropy ratio ⁇ - is equal to ⁇ - .
  • a reference concentration Co is defined by the number of primers initially immobilized on a ring divided by the corresponding annular volume:
  • the functionalized length is 1 mm and no spacing has been introduced between the rings.
  • the computational method is the Monte Carlo method where each primer executes a random walk in the prescribed domain. Statistically, diffusion is determined by the calculation of a great number of random walks. In each time step, each particle follows a straight line, and then at the next time step, its direction is randomly changed. When impacting a wall, the particle is supposed to bounce back.
  • the first parameter is constant and is given by the relation
  • the second parameter is the result of a random choice of uniform probability between 0 and 360.
  • the starting point is that of a great number of primers initially immobilized at the wall and randomly distributed along the wall in the functional ring.
  • the Brownian model is in agreement with the numerical « continuous » (4).
  • the function of the device for multiplex amplification requires areas where « forward » and « reverse » primers are present in a sufficient amount (the threshold is 25 nM - or 25 10 '6 mole/m 3 ).
  • the threshold is 25 nM - or 25 10 '6 mole/m 3 .
  • 2 criteria approximately equivalent to quantify the joint concentration i.e. a concentration of « forward » and « reverse » primers mixed in an elementary fluid volume.
  • the 2 PCR zones have a non-void intersection after 900 seconds.
  • a more detailed picture of the intersection of the intersection is obtained, where the intersection of the 2 "joint concentration" is defined by any of the 2 following formuJas
  • c 1 2 mm(min(c Fl ,c m ),min(c F2 ,c R2 ))
  • the design of the device is satisfactory until 500 seconds for functional rings of 1 mm, diffusion coefficients of 1. 10 "10 m 2 /s and initial surface concentrations of 50 femtomoles/mm 2 ). But after 500 seconds, overlapping of PCR areas occurs. If the diffusion coefficients are closer to 2.10 "11 m 2 /s, the design is satisfactory until 2700 seconds. In such situation, it is possible to reduce the size of the rings to less than 500 ⁇ m and not to have overlapping before 900 seconds.
  • Hibbert [5] who have solved numerically the coupled system diffusion in a volume/Langmuiran reaction at the walls.
  • there is no fluid advection unlike the 2 compartments model of Mason [6], which renders the conservation equation simpler.
  • it will not require much work to incorporate advection in our model.
  • the present model can handle the capture of antigenes on immobilized antibodies [7], [8].
  • Coupled system (reduced coordinates)
  • F 0 is the initial concentration in available association sites on the walls
  • J is the mass flux at the wall in mole/m 2 /s.
  • Equation (13) can be cast under the form
  • the ratio km T ° L is the Dammk ⁇ lher number for association
  • Equation (4) is described under the form « coefficient » equation (13) is a boundary condition for the mass flux and equation (15) is a differential equation coupled to the pde and described under the « weak » form. Values of the reference data are given in table 5
  • association and dissociation constants are not well known.
  • the association constant is taken from the work from the published work of Ligler [7]; thus the primers can be successively captured at the walls and released.
  • the diffusion distance at 1000 seconds was evaluated as a function of
  • Axial diffusion is slowed down when the adsorption Dammkohler increases. Physically, it is explained by temporary immobilization of the primers on the walls. This phenomenon is confirmed by the decrease of free primers in the capillary.
  • association coefficient k on is large, equal to 500 m 3 /moles/s and the dissociation coefficient k Off is also relatively large, equal to 4. 10 "2 1/s.
  • the data for the calculation are given in table 7:
  • the axial diffusion is very much reduced by the choice of the 2 Dammkh ⁇ ler numbers (a very strong association constant and a somewhat less strong dissociation constant, the ratio that characterizes the relative importance of the two mechanisms is equal to 2).
  • Axial propagation is nearly stopped because primers successively associate, then dissociate at the wall.
  • the free concentration is relatively small but still enough for the PCR reactions to take place.
  • the rings have an axial extent of 0.5 mm and they are separated by spacers of 0.5 mm.
  • Axial speed of diffusion is a key factor for the feasibility of the capillary PCR device. Transport of primers inside the capillary has to be slow enough if we want to incorporate many functional rings inside the capillary and still have a compact device.
  • Dc 0 capillary radius on the axial speed of propagation of the primers is weak as long as the ratio L f /R of the functionalized length to the radius is larger than 5.
  • a adsorption Dammk ⁇ lher number large enough to slow down the advancing front of diffusing primers, but a very important adsorption will result in a complete depletion of the primers.
  • a spacing between the couples of rings (F1 , R1), (F2, R2), etc., to constitute a diffusion « barrier could be functionalized with oligonucleotide complementary to the R1 and F2 type of oligonucleotides.
  • Example 1 Preparation of a capillary for performing a plurality of nucleotide amplifications in parallel
  • Capillaries are purchased from SGE, Milton Kaynes UK. The inside diameter is 100 ⁇ m and the diameter including the inner wall but excluding the Teflon jacket is 300 ⁇ m. These capillaries are made of fused silica and are UV transparent.
  • benzophenone (Bz) was used to create a photo-reactive surface.
  • the benzophenone was chosen because its photo chemical properties are well- known. In particular, it reacts with C-H under UV irradiation in a wide range of different chemical environments. Furthermore, benzophenone is chemically inert in the absence of light and stable in aqueous solutions. Capillaries were cleaned using a 3.6N NaOH solution in 50% ethanol and then washed with bi-distilled water. They were dried using a stream of air, and then treated with a 5% solution of aminopropylsilane in pure ethanol. The reaction was performed overnight.
  • Capillaries were rinsed, first with 95 % ethanol, then with bi-distilled water, and again with 95% ethanol.
  • the silane layer was reticulated by baking the capillaries at 115°C during 3 hours. The resulting amino-surface was then reacted with 20OmM Bz- isothiocyanate in DMF overnight. The capillaries are washed twice with DMF and twice with 95% ethanol.
  • the Bz-modif ⁇ ed capillary was filled with the first oligonucleotide solution and a section of the capillary was UV365nm irradiated. Non-immobilized oligonucleotides were washed out of the capillary. The array was formed by repeating these steps for each oligonucleotide.
  • the light generator was an inverted microscope with a mercury lamp. The irradiation was performed orthogonally to the capillary through a 160 ⁇ m slot with 4 mW/mm 2 intensity. The capillary was moved under the device to create an oligonucleotide array.
  • the capillaries were prepared as described in example 1. After immobilization of the complementary oligonucleotides, the capillaries were hybridized with Cyanine 3 labelled primers following the above protocol: After washes, the hybridized labelled primers are detected through the wall of the capillary using a standard DNA chip scanner. The fluorescence intensities for each primer were recorded.
  • the capillaries were filled with the hybridization buffer and the temperature increased to 65°C for 1 hour.
  • the capillaries were flushed with fresh hybridization buffer and scanned.
  • the fluorescence intensities corresponding to each primer were recorded and showed a decrease to less than 30% of the intensities recorded before the release. We conclude that 70% of the hybridized primers were released in the capillary, suitable for PCR amplification.
  • Example 3 Amplification of the NAT2 gene
  • NAT2 N- acetyltransferase 2
  • a capillary is created to generate a PCR product for the NAT2 gene from human genomic DNA using a forward (5'- GTCACACGAGGAAATCAAATGC-3', sequence ID no. 1 ) and reverse (5'-
  • GTTTTCTAGCATGAATCACTCTGC-3' sequence ID no. 2 primers.
  • primers can be pre-synthesized and attached via a cleavable linker to the surface of the capillary or in situ synthesized in the capillary on a cleavable linker.
  • linkers can be used depending on the release mechanism.
  • the cleavage can be achieved upon light irradiation (photocleavable linker), with a reagent (chemical cleavage) or using a restriction enzyme (DNA sequence linker).
  • the primers complementary sequences can be pre- synthesized and covalently and permanently attached to the surface of the capillary or in situ synthesized in the capillary.
  • the capillary is then "loaded” by hybridization with a large excess of primers in solution.
  • the primers are released by a denaturation step at 94°C.
  • the capillary Before the PCR amplification reaction, the capillary is washed in 1x PCR buffer (5OmM KCI, 1.5mM MgCI2, 1OmM Tris-HCI pH 8.0). Then, for PCR amplification, PCR reagents containing 10ng human genomic DNA, PCR buffer, 200 ⁇ M deoxynucleotide triphosphates, and 1 unit DNA polymerase are added to the capillary. Primers are released (as described earlier) and allowed to mix with the PCR reagents. The capillary is then subjected to temperature cycling using a temperature cycling apparatus dedicated to accommodate capillaries. The resulting amplification product (amplicon) has a length of approx.
  • 1x PCR buffer 5OmM KCI, 1.5mM MgCI2, 1OmM Tris-HCI pH 8.0.
  • PCR product is analyzed using standard technologies known to the skilled in the art, e.g. using agarose gel electrophoresis.
  • the coding region of interest of the human N-acetyltransferase 2 (NAT2) gene spans 872 basepairs (GenBank accession number NM_000015). Numerous alleles are found in the population which can be associated with decreased function, determining slow, intermediate, and fast metabolism. The enzyme has been shown to play a role in the inactivation of aromatic and heterocyclic compounds including carcinogens. Carriers of certain alleles are likely to be more susceptible to certain cancers, i.e. colorectal and bladder cancer. A list of the alleles can be found in table 11.
  • Ph phenotype
  • R rapid
  • S slow
  • Fr frequency
  • AA amino acid
  • the low complexity, well understood genetics, and clinical relevance makes the NAT2 gene a good candidate to develop a proof-of-principle assay.
  • the assay described below is adapted from F. Chatelain and F.- Frueh, PCT/EP02/05459, US No. 60/288,526. Homozygous and heterozygous genotypes are determined at each polymorphic site and allele assignments are made according to the genotypes identified which ensures and improves the assay as each allele is defined by more than one SNP.
  • a capillary is created to generate a PCR product from human genomic DNA using a forward (5'-GTCACACGAGGAAATCAAATGC-S', sequence
  • primers can be presynthesized and attached via a cleavable linker to the surface of the capillary or in situ synthesized
  • probes are attached to the surface of the capillary, or in situ synthesized, to be used for the identification of the sequence of the NAT gene at the positions described in table 11.
  • probes are allele-specific containing allele- specific nucleotides (representing the positions of the nucleotides outlined in table 11) at their 3' end. These ends are exposed and can be elongated by (performing an enzymatic reaction known to the skilled in the art as
  • PCR buffer 5OmM KCI, 1.5mM MgCI2, 1OmM Tris-HCI pH 8.0.
  • PCR reagents containing 10ng human genomic DNA, PCR buffer, 200 ⁇ M dATP, 200 ⁇ M dGTP, 175 ⁇ M dTTP, 25 ⁇ M labeled dUTP (i.e. Cy3-dUTP), 175 ⁇ M dCTP, 25 ⁇ M labelled dCTP (i.e. Cy3-dCTP), and 1 unit DNA polymerase are added to the capillary. Primers are released (as described in example 2) and allowed to mix with the PCR reagents.
  • the capillary then is subjected to temperature cycling using a temperature cycling apparatus dedicated to accommodate capillaries. After completion of the temperature cycling, the capillary is washed in a solution of triethylamine and alcohol (1 :1) for 15 minutes and rinsed several times with water, and analyzed during the PCR reaction, the template DNA is initially amplified by the primers (sequence ID nos. ⁇ and
  • the allele-specific probes containing allele-specific nucleotides at their 3' end are being extended. These probes remain attached to the surface of the capillary at known locations, allowing the identification of allele-specific amplification products based on their location on the surface of the capillary.
  • non allele-specific probes can be used. These probes are ending precisely one base short (upstream) of the polymorphic site.
  • ddNTPs di-deoxy nucleotide terminators
  • the four different ddNTPs are labelled in a different colour which can be distinguished by a colour detection device.
  • the use of such terminators allows extending the allele-specific probe by only one base.
  • the label which is characteristic for each of the four possible bases identifies attached nucleotide at each of the probe locations and, hence, identifies the sequence at the polymorphic site.

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Abstract

L'invention concerne un dispositif microfluidique destiné à effectuer une pluralité de réactions. L'invention concerne en particulier un procédé et un dispositif pour effectuer une pluralité de réactions chimiques et/ou biologiques en parallèle, grâce à la création d'un réseau de réactifs pouvant être libérés sur la surface interne d'un capillaire. Un grand nombre de réactions d'amplification de polynucléotides peuvent être effectuées au moyen du réseau capillaire. L'invention concerne également un procédé et un dispositif destinés à coupler l'amplification des polynucléotides et la détection et/ou l'analyse des produits amplifiés.
PCT/EP2004/009149 2004-07-13 2004-07-13 Dispositif microfluidique destine a effectuer une pluralite de reactions et ses utilisations WO2006005371A1 (fr)

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JP2007520786A JP2008506376A (ja) 2004-07-13 2005-07-13 複数の反応を実施するためのマイクロ流体装置及びその使用
EP05775164A EP1776473A1 (fr) 2004-07-13 2005-07-13 Dispositif microfluidique pour conduire plusieurs reactions et ses utilisations
CA002571921A CA2571921A1 (fr) 2004-07-13 2005-07-13 Dispositif microfluidique pour conduire plusieurs reactions et ses utilisations
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WO2009065620A3 (fr) * 2007-11-23 2009-07-30 Febit Holding Gmbh Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence
US10889851B2 (en) 2013-03-14 2021-01-12 Gen-Probe Incorporated Method for moving a processing vial between locations of an instrument

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US8358405B2 (en) * 2008-01-22 2013-01-22 Shimadzu Corporation Measuring apparatus, and liquid collecting and measuring system having the same
CN103620434B (zh) * 2011-05-17 2016-02-03 日立金属株式会社 磁力特性计算方法和磁力特性计算装置

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US20020013457A1 (en) * 1997-08-29 2002-01-31 Olympus Optical Co., Ltd. DNA capillary
US20020090641A1 (en) * 1998-06-11 2002-07-11 Hitachi, Ltd. Polynucleotide separation method and apparatus therefor
US20030044855A1 (en) * 1999-07-30 2003-03-06 Anderson N. Leigh Microarrays and their manufacture by slicing
WO2002089972A1 (fr) * 2001-05-03 2002-11-14 Commissariat A L'energie Atomique Dispositif microfluidique destine a l'analyse d'acides nucleiques et/ou de proteines, ses procedes de preparation et son utilisation
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WO2009065620A3 (fr) * 2007-11-23 2009-07-30 Febit Holding Gmbh Procédé d'extraction flexible permettant la production de bibliothèques de molécules spécifiques d'une séquence
US10889851B2 (en) 2013-03-14 2021-01-12 Gen-Probe Incorporated Method for moving a processing vial between locations of an instrument
US11279967B2 (en) 2013-03-14 2022-03-22 Gen-Probe Incorporated System and method for conducting an assay
US11434521B2 (en) 2013-03-14 2022-09-06 Gen-Probe Incorporated Method for conducting an assay
US11732289B2 (en) 2013-03-14 2023-08-22 Gen-Probe Incorporated Receptacle distribution system
US11732288B2 (en) 2013-03-14 2023-08-22 Gen-Probe Incorporated Assembly having reagent pack loading station
US11761027B2 (en) 2013-03-14 2023-09-19 Gen-Probe Incorporated System and method for receiving and storing reagent packs in an instrument
US11761026B2 (en) 2013-03-14 2023-09-19 Gen-Probe Incorporated Diagnostic system and method
US11834701B2 (en) 2013-03-14 2023-12-05 Gen-Probe Incorporated Reagent pack changer
US12110535B2 (en) 2013-03-14 2024-10-08 Gen-Probe Incorporated Method for reconstituting a reagent
US12173352B2 (en) 2013-03-14 2024-12-24 Gen-Probe Incorporated Method for receiving and storing reagent packs in an instrument

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