CN1997454B

CN1997454B - Thermal reaction device and method for using the same

Info

Publication number: CN1997454B
Application number: CN2005800225837A
Authority: CN
Inventors: 费德里科·顾特塞德; 马克·A·恩格; 黄江; 埃默森·全; 罗伯特·格罗斯曼; 菲里普·林; 周厚朴; 吉克·金保尔; 马丁·皮普瑞兹克; 杰弗里·费塞
Original assignee: Fluidigm Corp
Current assignee: Standard Biotools Corp
Priority date: 2004-05-02
Filing date: 2005-05-02
Publication date: 2010-06-16
Anticipated expiration: 2025-05-02
Also published as: SG166024A1; CN1997454A

Abstract

An MxN matrix microfluidic device for performing a matrix of reactions, the device having a plurality of reaction cells in communication with one of either a sample inlet or a reagent inlet through a via formed within an elastomeric block of the device. Methods provided include a method for forming vias in parallel in an elastomeric layer of an elastomeric block of a microfluidic device, the method comprising using patterned photoresist masks and etching reagents to etch away regions or portions of an elastomeric layer of the elastomeric block.

Description

Thermal reaction device and use its method

Prioity claim

The application requires each priority of applying for below: the U.S. Patent application No.11/058 that on February 14th, 2005 proposed, 106; The U.S. Patent application No.11/043 that on January 25th, 2005 proposed, 895; The No.10/876 that on June 23rd, 2004 proposed, 046; And the U.S. Patent application No.10/837 of proposition on May 2nd, 2004,885; Each application is combined with its full text that is used for all purposes by reference.

The cross reference of related application

The application also relates to the U.S. Patent application No.10/818 that on April 5th, 2004 proposed, 642; The U.S. Provisional Patent Application No.60/460 that on April 3rd, 2003 proposed, 634; The U.S. Patent application No.10/819 that on April 5th, 2004 proposed, 088; The U.S. Patent application No.10/818 that on April 5th, 2004 proposed, 642; The U.S. Patent application No.10/3063 that on November 27th, 2002 proposed, 798; The U.S. Provisional Patent Application No.60/391 that on June 24th, 2002 proposed, 529; And U.S. Provisional Application No.60/335,292; Each application is combined with its full text that is used for all purposes by reference.

Background

Recently, in order to implement to be provided for to prepare various chemistry and the biochemical analysis and synthetic with analytical applications, the trial of having developed and having made microfluid system.Because with respect to the analysis of implementing on grand scale and synthetic can realize a large amount of advantages according to microminiaturization, therefore the target of this equipment appears making.This advantage comprises utilizes this equipment to implement to analyze or synthesize the real minimizing in needed time, cost and space.In addition, microfluidic device has the possibility that is applicable to the use automatic system, thereby has the additional advantage that further cost reduces and reduce operator's error because of artificial disturbance reduces.Microfluidic device has been suggested and has been used for various application, comprises for example Capillary Electrophoresis, gas-chromatography and cell separation.

Yet the realization of these advantages various is intricately often hindered because of relevant with manufactured up to now microfluidic device.For example, many present microfluidic devices are by the substrate manufacturing based on silica; These materials are difficult to process and are complicated, and are frangible by the equipment of these material manufacturings.In addition, the fluid transport in many existing microfluidic devices requires with the adjusting to complicated electric field of the controllable way of using described equipment.

Therefore, except the present restriction of existing equipment, consider the aforementioned advantages of using microfluidic device to realize, still have needs the microfluidic device that is designed to implement various chemistry and biochemical analysis.Because its importance in modern biochemistry, specifically need to be utilized implement various nucleic acid amplification reactions equipment, this equipment also has simultaneously and is used for enough versatilities that all kinds are analyzed.

Having the equipment of implementing the nucleic acid amplification effect will serve many purposes.For example, this equipment may be used as analysis tool, to determine whether to exist or lack interested objectives nucleic acid in sample.Therefore, this equipment may be utilized the concrete pathogen of test (for example, virus, bacterium or fungi) existence, and identification purposes (for example, patriarchy (paternity) and court use).This equipment can also be utilized and detect or the previous special nucleic acid that is associated with disease specific or genetic disorder of characterization.As analysis tool the time, this equipment also may be used to implement gene type analysis and gene expression analysis (for example, different genes expression study).Replacedly, can use this equipment to be used for further analysis by preparation method with the enough nucleic acid that increases, such as amplified production, cell type, dna fingerprint etc.In the range gene engineering is used, also can use amplified production, for example be inserted in the vector that can be used then the production of the protein that is used to want with transition cell.

General introduction

The plurality of devices and the method that are used to implement microfluid analysis here are provided, have comprised to be utilized the equipment of putting back the thermal cycle reaction of answering with embodiment such as nucleic acid.This equipment is different from conventional microfluidic device and is: they comprise elastomeric element; In some example, many or armamentarium is made up of elastomeric material.

Concrete equipment is designed to implement thermal cycle reaction, and (for example, PCR), described equipment comprises one or more elastic valve, flows through the solution of equipment with adjustment.Therefore, also provide the equipment that uses this design to implement the method for amplified reaction.

Some equipment comprise that blind influx moves passage, and the moving passage of described blind influx comprises the zone of playing the reflecting point effect.Concrete this equipment comprises the flow channel that is formed in the elastomeric material, and the moving passage of the blind influx of a plurality of and flow channel fluid communication, the area limiting conversion zone of the moving passage of each blind influx.Described equipment comprises also and intersects at one or more control channels that each blind influx moves passage that wherein elastic diaphragm separates one or more control channels and the moving passage of blind influx in each crosspoint overlapping.At this equipment elastic diaphragm is provided with deflection and goes into the moving passage of blind influx or move the passage withdrawal, in response to incentive action power from blind influx.Described equipment can further comprise a plurality of protection passages ideally, its be formed in the elastomeric material and overlapping flow channel and/or reflecting point in one or more.The protection passage is designed to have perforation fluid stream therebetween, to reduce the flow channel from described equipment and the evaporation of reflecting point.In addition, described equipment can comprise the one or more reagent that are deposited in each reflecting point ideally.

In a certain equipment, flow channel is one of in a plurality of flow channels, and each flow channel is communicated with the moving passage phase fluid of multistage blind influx of branch therefrom.In the equipment of this design, in some instances, a plurality of flow channels are inner mutually to be connected, so that fluid can be imported into each reflecting point via single inlet.In miscellaneous equipment, yet a plurality of flow channels are isolated mutually, can not flow into another flow channel so that import the fluid of a flow channel, and each flow channel is included in the inlet at the place, one or both ends that fluid can be imported into.

Miscellaneous equipment comprises having at least 50 point/cm ²The reflecting point array of density, reflecting point typically is formed in the elastomeric material.Miscellaneous equipment has more high density, for example at least 250,500 or 1000 point/cm ²

Another miscellaneous equipment comprises the reflecting point that is formed in the elastic substrates, and the reagent that is used at the elastic substrates place implement to react is non-covalent fixing.Reagent can be one or more reagent, is used for implementing in fact any type reaction.In certain embodiments, reagent comprises a kind of reagent that is used to implement nucleic acid amplification reaction.Therefore, in some equipment, reagent comprises primer, polymerase and one or more nucleotides.In miscellaneous equipment, reagent is nucleic acid-templated.

Various matrixes are also provided or based on the equipment of array.In these equipment some comprises: (i) more than first flow channel; it is formed in the elastic substrates; (ii) more than second flow channel; it is formed in the elastic substrates; intersect with more than first flow channel; with the defined reaction lattice array; a plurality of separation valve doors that (iii) can be energized; it is set in more than first and second flow channel; with with the solution in each reflecting point with isolated at the solution at another reflecting point place; and (iv) a plurality of protection passages, its overlapping one or more flow channels and/or one or more reflecting point are to stop solution evaporation therefrom.

Front equipment can be utilized to implement a large amount of differential responses, to comprise to relate to those (for example, thermal cycles of foranalysis of nucleic acids) that temperature is adjusted.The method of using some sidewalk for visually impaired people property equipment to implement relates to providing and comprises the microfluidic device that is formed on the flow channel in the elastomeric material; And the moving passage of a plurality of blind influxs that are connected with flow channel, the stub area defined reaction point of the moving passage of each blind influx.At least a reagent is introduced into each reflecting point, and it is detected to be reflected at one or more reflecting points place then.Method can excellently selectively be included in the reflecting point at least a reagent heating.Therefore, for example, method can relate to introduces the parts that are used for nucleic acid amplification reaction, and then parts is carried out thermal cycle to form the product of amplification.

Other method relates to provides the microfluidic device that comprises one or more reflecting points, each reflecting point to comprise to be used to first reagent of implementing to analyze, and described reagent is deposited on the elastic substrates non-covalently.Then, second reagent is imported into one or more reflecting points, thereby first and second reagent mix are to form reactant mixture.Detected subsequently in the reaction that one or more reflecting points are between first and second reagent.

Another other method relates to provides microfluidic device, and described microfluidic device comprises and is formed on intrabasement reflecting point array and has at least 50 point/cm ²Density.At least one reagent is introduced into each reflecting point.Then, detection is in the reaction at one or more reflecting points place.

Another other method relates to provides microfluidic device, and described microfluidic device comprises and is formed at least one reflecting point in the elastic substrates and also has a plurality of protection passages to be formed in the elastic substrates.At least one reagent is introduced into each reflecting point, is heated in reflecting point then.Before the heating or during fluid flow through the protection passage, to reduce evaporation from least one reflecting point.Reaction at least one reflecting point is detected subsequently.

Also provide and be designed to reduce the other equipment of fluid from the equipment evaporation.Usually, this equipment comprises cavity, and described cavity is formed in the part of the microfluidic networks in the elastic substrates; And a plurality of protection passages, its overlapping cavity and be separated by barrier film and cavity.Protection passage at this equipment is made into suitable size, (i) allows flow of solution to cross wherein, and (ii) so that because of barrier film in of the effect deflection of incentive action power to the protection passage, flow into, flow out or the solution that flows through cavity does not reduce basically.Miscellaneous equipment comprises (i) one or more flow channels and/or one or more reflecting point; And (ii) a plurality of protection passages, its overlapping microfluidic device and be separated by elastomer and there wherein protects interval between the passage between 1 μ m to 1mm.In miscellaneous equipment, at interval between 5 μ m to 500 μ m, in miscellaneous equipment, between 10 μ m to 100 μ m, in another miscellaneous equipment, at interval between 40 μ m to 75 μ m.

The synthetic of implementing foranalysis of nucleic acids in the reflecting point of some microfluidic device also is provided.In below some these synthetic comprises one or more: reagent and the washing agent of retardance combined with protein on elastomeric material.Retarding agent is typically by selecting (for example gelatin or albumin, for example bovine serum albumin (BSA)) in the group that comprises protein.Washing agent for example can be SDS or Triton.

Brief Description Of Drawings

Figure 1A is schematically illustrating of example apparatus, has to intersect vertically and the matrix design of water flow channel.

Figure 1B-E shows the amplification view of the part of equipment shown in Figure 1A, and its operation is shown.

Fig. 1 F is schematically illustrating of another example matrix designing apparatus, and it utilizes the protection passage to reduce the sample evaporation.

Fig. 2 is the plane of example sidewalk for visually impaired people equipment.

Fig. 3 A is the plane of another example sidewalk for visually impaired people equipment.

Fig. 3 B is schematically illustrating of more complicated sidewalk for visually impaired people equipment, based on the unit of the universal design shown in Fig. 3 A.

Fig. 4 is the plane that utilizes the equipment of Mixed Design.

Fig. 5 shows the rising (ramp up) of enforcement thermal cycle reaction and the icon of fall time.

Fig. 6 is presented at the position of the position (spotted reagent) of the interior point sample reagent of reflecting point in the type equipment of sidewalk for visually impaired people, and reagent correct arrangement in equipment corner reflecting point is shown.

Fig. 7 A and 7B are respectively the cross-sectional view and the schematic diagrames of another mixed type microfluidic device, and expression is used to this equipment tested described in the embodiment 1-4.

Fig. 8 is a bar chart, draws the mean F AM/PRI/ control ratio that is used for six kinds of different beta actin TaqMan reactions therein.These be reflected in the microfluidic device (chip) and Macro (grand) TaqMan reaction in by thermal cycle.Described the first and the 4th collection of stripes that does not have DNA that is controlled to be.Error bars is the standard deviation of these ratios.

Fig. 9 is the figure of depicted example pin mark sampling technology.From source (for example microtiter plate (microtiterplate)), draw reagent, then by loading pin contact substrate is printed.Scrub step and be included in the vacuum drying stirring in deionized water afterwards.

Figure 10 is a bar chart, describes the FAM signal strength signal intensity that is used for microfluidic device (chip) described in the example 1 (seeing Fig. 7 B) based on test described in the example 2.Data are (FAM signal/PRI signal) form of being weighed by the FAM/PRI ratio of standard drawing lines.Error bar is the standard deviation in road along the line." 1.3X " and " 1X " refers to the concentration degree that the point sample utmost point and probe are relevant to its nominal value.

Figure 11 is a bar chart, is presented at the average VIC/PFI/ control ratio in the 9-10 hole that is used for Macro TaqMan (bar figure) and TaqMan reaction in the microfluidic device (solid bars).Two negative controls (control) and two samples with 100pg/nl genomic DNA are by thermal cycle, and aforesaid reaction part has the standard utmost point/probe of 4 times.Error bar is represented the standard deviation of average ratio.

Figure 12 is a bar chart, shows the FAM/PRI/ control ratio in each 10-1nl hole that is used for single current circulation passage (the seeing Fig. 7 B) branch from microfluidic device.The quantity of genomic DNA is 0.25pg/nl, and it is average that this target that produces each hole backs up.

Figure 13 is a bar chart, describes to use the average VIC/PRI/ control ratio that is used for CYP2D6SNP of the microfluidic device shown in Fig. 7 B.Allele 1 (Al-1) is the Position Control that is used for the VIC probe at benchmark or wild-type allele CYP2D6*1.Allele 2 (Al-2) is the Position Control that is used for the FAM probe at modification or mutation allele CYP2D6*3.Control does not have dna profiling.Use genomic DNA with 100pn/pl or 20pn/pl.Error bar is the standard deviation of these ratios.

Figure 14 is a bar chart, is presented at the mean F AM/PRI/ control ratio that is used for CYP2D6SNP in the microfluidic device shown in Fig. 7 B.Sample be relevant to identical described in Figure 13 and the example 3.

Figure 15 is the schematic diagram that is used for the microfluidic device of example 4 tests.

Figure 16 is the polyacrylamide gel that comprises from the PCR product of Macro PCR and PCR reaction in the microfluidic device shown in Fig. 7 B.The result illustrates the approximate drift of different DAN bases to length on the left side.Comprising the drawing lines that scatters band is that molecular weight indicates.The drawing lines that indicates " Macro " is the PCR product from the Macro reaction of different diluent.The drawing lines that indicates " In chip (in the chip) " is the PCR product that produces in chip.Comprise the non-specific background signal of many band drawing lines that connects gel.

Figure 17 a-17d is depicted in two decision design isolating microfluidic device in valve closing and the valve closes state.

Figure 18 a and 18b are depicted in and carry out the image that thermal cycle reaction is isolated microfluidic device afterwards.Figure 18 a describes two coloured images, and Figure 18 b describes to reduce the residual signal afterwards of control danger signal.

Figure 19 describes the par that duplicates in every hole and positive polarity hole number chart relatively.

Figure 20 describes isothermal duplication and disposes a SCORPION.

Figure 21 depicted example matrix microfluidic device plane.

Figure 22 A describes the substrate according to the microfluidic device with integral pressure accumulation well (well) of the embodiment of the invention;

The expanded view of the microfluidic device shown in Figure 22 B depiction 8A further comprises elastomer block;

Figure 22 C is the overall diagram of the microfluidic device shown in Figure 22 B;

Figure 22 D is the plane of the microfluidic device shown in Figure 22 B;

Figure 22 E is the cross-sectional view of the microfluidic device shown in Figure 22 B;

Figure 23 is the cross-sectional view of the path (via) that uses among some embodiment in microfluidic device of the present invention.

Figure 24 is the perspective view of situation that is used to encourage microfluidic device according to the embodiment of the invention.

Figure 25 describes to be pushed to the cutaway view according to the platen of the upper surface of the microfluidic device of the embodiment of the invention;

Figure 26 A is the simplification overall diagram according to the system of the embodiment of the invention;

Figure 26 B is the perspective view of receiving platform in the system of Figure 26 A;

Figure 26 C is at the back plane figure of plate interface or platen inner fluid selection schemer (routing) according to another embodiment of the present invention;

Figure 27 A and 27B are the cross-sectional side views that shows according to the interface platen of the embodiment of the invention, shown in the interface platen be matched on the carrier;

Figure 28 A is the perspective view according to the integral carriers of the embodiment of the invention;

Figure 28 B be according to the present invention in the perspective view of integral carriers of use PCR of another embodiment;

Figure 29 A is the simplification cross-sectional view that is used for the system of constraints graph 28A carrier;

Figure 29 B is the simplification cross-sectional view that is used for the system of constraints graph 28B carrier;

Figure 29 C is the simplified plan view of carrier part among Figure 28 B;

Figure 29 D is to use the simplified plan view of the vacuum chuck (vacuum chuck) of system among Figure 28 B; And

Figure 30 is to use the simplification overall diagram of the vacuum chuck of system among Figure 28 B.

Describe in detail

I. Definition

If there is not specific qualification, all technology used herein and scientific terminology have the common implication of understanding of those skilled in the art in the invention.Offer the technical staff below with reference to general definition: Singleton etc., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed.1994) with numerous terms used in the present invention; THECAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger etc. (eds.), Springer Verlag (1991); And Hale﹠amp; Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY (1991). as used herein, below term return if there is not specific qualification then to belong to their implication.

" flow channel " is commonly referred to as solution by its flowable circulation path.

If do not have specific indication, term " valve " to refer to therein that interpolation is gone into circulation passage and control channel and by the separated structure of elastomeric diaphragm, described elastomeric diaphragm responsing excitation power can be deflected into flow channel or regain from flow channel.

" sidewalk for visually impaired people " or " blind alley passage " refers to flow channel, and it has inlet and does not singly have independent outlet.Therefore, the flow of solution of turnover sidewalk for visually impaired people appears at same position.The filling process of the one or more sidewalk for visually impaired people of filling is called " blindly filling (blind fill) " sometimes for short.

" isolation reflecting point " is not commonly referred to as not the reflecting point that is communicated with other reflecting point fluids of existing on the equipment.When being relevant to the sidewalk for visually impaired people and being used, isolating reflecting point is the zone of sidewalk for visually impaired people end, and described zone can be by the valve blockage that is associated with the sidewalk for visually impaired people.

" through hole (via) " refers to the passage that is formed in the elastomeric material equipment, with provide fluid at equipment outside port and one or more flow channel between pass in and out.Therefore, through hole can be used as sample and inputs or outputs, for example.

Term " elastic materials " and " elastomeric material " have the employed common implication in this area.Therefore, for example, Allcock etc. (Contemporary Polymer Chemistry, second generation Ed) have described the elastic materials that common conduct is present in the polymer at temperature place between its glass transition temperature and the condensing temperature.Elastomeric material shows and elastic performance to stretch because polymer chain is subjected to twist motion easily to make main chain in response to active force, presents previous shape there not being under the active force main chain twine again.Usually, elastomer distortion when applying active force, but when removing active force, return back to its original shape then.Can specialize by Young's modulus by the elasticity that elastomeric material is showed.The elastomeric material of disclosed here utilization in microfluidic device typically has the Young's modulus at about 1Pa-1Tpa, in other example between about 10Pa-100Gpa, in another other examples between about 20Pa-1Gpa, in another other examples between about 50Pa-10Mpa, and in the object lesson approximately between the 100Pa-1Mpa.The needs that depend on concrete application can also utilize the elastomeric material with the Young's modulus outside these scopes.

Some microfluidic device described here is by the elastomer polymer manufacturing, for example, and GE RTV 615 (prescription), ethylene-silane crosslinked (type) silicone elastomer (family).Yet this microfluid system is not limited to this a kind of prescription, model or this adoption compound; But almost any elastomer polymer all is to use.The otherness of given polymer chemistry, parent, synthetic method, reaction condition and possible additive, have a large amount of may elastomer system, it can be used makes little valve of monolithic elasticity and pump.The selection of material typically depended on to be implemented use desired concrete material parameter (for example, solvent resistance (solvent resistance), hardness, ventilative property and/or temperature stability).The disclosed here other details that is relevant to the elastomeric material type that can be used in the manufacturing of microfluidic device parts is set forth in (2000) Science 288:113-116 such as Unger, in open WO02/43615 of PCT and WO01/01025, it is combined with the full text that it is used for all purposes by reference at this.

Term " nucleic acid ", " polynucleotides " and " oligonucleotide " are used the condensate form of the nucleotides that is used to comprise random length here, include but not limited to ribonucleotide (ribonucleotide) or deoxyribonucleotide.The difference of between these terms, on length, not wanting.These belong to the primary structure that only refers to molecule in addition.Therefore, in specific embodiment, these terms can comprise three, two and single stranded DNA, and three, two and single stranded RNA.They also comprise change, for example by methylating and/or gland (capping), and the unchanged form of polynucleotides.Especially, term " nucleic acid ", " polynucleotides " and " oligonucleotide " comprise polydeoxyribonucleotide (comprising the 2-deoxy-D-ribose), polyribonucleotide (comprising D-ribose), be the N-of purine or pyrimidine base or any type polynucleotides of C-glucosides, and other polymer that comprise non-nucleotide main chain (nonnucleotidic backbone), for example polyamide (such as peptide nucleic acid (PNA)) and poly-morpholino (polymorpholino) (can from Corvallis Oregon (Kang Walisi city Oregon) Anti-Virals Co., Ltd buy the same) polymer and other composition sequence specific nucleic acid polymer with Neugene, suppose that polymer comprises nucleic acid base salt (nucleobases) in the structure that allows base pairing and base stacking, for example in the structure of DNA and RNA.

" probe " is a kind of nucleic acid, and it uses one or more chemical bonds to use complementary base right usually in conjunction with the target nucleic acid of complementary series, uses hydrogen bond to constitute usually, thereby forms duplex.Probe combination or hybridization " probe binding site ".Probe can indicate by detecting, and to allow the detection easily of probe, probe hybridization once is to its complementary target particularly.For example, the sign that is attached to probe can comprise various different sign the known in the art arbitrarily, and they can detect by chemistry or physical means.The suitable sign that can be attached to probe includes but not limited to that radioisotope, fluorogen, chromophore, quality indication, electron density particle, magnetic particle, spin indicate, molecule, electro-chemical activity molecule, enzyme, confactor and the enzyme substrate of emission chemiluminescence.Probe can obviously change in size.Some probes are suitable ends.Usually, probe is long at least 7 to 15 nucleotides.Other probe is long at least 20,30 or 40 nucleotides.Another probe is longer slightly, at least 50,60,70,80,90 nucleotides long.Another probe is longer, at least 100,150,200 or more a plurality of nucleotides long.Probe can also be long for any specific that falls in the scope of front.

" primer (primer) " is singly to revolve polynucleotide, it can play the synthetic starting point effect of DNA of template direction at the proper temperature place at suitable cushion base under appropraite condition (just, four kinds of different NTPs and the reagent that is used for polymerization for example or under the situation of DNA or RNA polymerase or counter-rotating transcriptase).The appropriate length of primer depends on the desired length of primer, but it is long typically to be at least 7 nucleotides, and more typically changes between 10 to 30 nucleotides are long.The short primer molecule generally requires lower temperature, to form sufficiently stable compound association with template.Primer needn't reaction template particular sequence, but must be enough complementary to hybridize with template.Term " primer point " or " primer binding site " refer to the sections of the target dna of primer hybridization." primer to " indicates and comprises an aligning primer of 5 ' " upstream primer " and 3 ' " downstream primer ", 5 ' " upstream primer " with the fill-in hybridization of 5 ' end of dna sequence dna that will be amplified, 3 ' " downstream primer " with the fill-in hybridization of 3 of the dna sequence dna that will be amplified ' end.

" perfect complementation " primer has the sequence of the complete complementation on the whole length of primer, and does not have off resonance.Primer is perfect typically complementary to the part (subsequence) of target sequence." off resonance " refers to the nucleotides in the primer and is not complementary point with nucleotides in the target nucleic acid that it aligns.Term when using with reference to primer " complementary basically " expression primer and its target sequence are not perfect complementary; Replace it, primer is only fully complementary, selectively to hybridize to spiral separately at desirable primer binding site place.

Nucleic acid of term " complementation " expression is consistent or selective cross with another nucleic acid molecules.Compare total specificity disappearance, when more the hybridization selectivity of multi-selection appears at the hybridization generation.Typically, in the stretching of 14-25 nucleotides at least, have about at least 55% when identical,, selective cross will take place, and preferably at least 60%, more preferably at least 75%, and then most preferably at least 90%.Preferably, a nucleic acid is hybridized specially to all the other nucleic acid.Referring to M.Kanehisa, Nucleic AcidsRes.12:203 (1984).

Term " sign " refers to molecule or the molecule aspect that can pass through physics, chemistry, electromagnetism and other correlation analysis technology for detection.The example of the detected sign that can be utilized includes but not limited to that radioisotope, fluorogen, chromophore, quality indication, electron density particle, magnetic particle, spin indicate, molecule, electro-chemical activity molecule, enzyme, confactor, the enzyme that links to nucleic acid probe and the enzyme substrate of emission chemiluminescence.Expression that term " can detect sign " and the reagent that indicates conjugated or have some inherent feature () reagent for example, size, shape or color, described feature allow it not have conjugated to indicate and detected to separating.

" polymorphic mark " or " polymorphic point " is the place of diffusion nidus.Preferred mark has at least two allele, and each allele occurs with 1% frequency greater than selected colony, more preferably with greater than 10% or 20%.Polymorphic place can with a base to the same little.Polymorphic mark comprises that restriction fragment length polymorphic, variable number are connected and repeat that (VNTR ' s), hypervariable zone, microsatellite (minisatellite), dinucleotides repeat, tetranucleotide repeats, simple sequence repeats and the insertion element of for example Alu.But the first recognition allele form is designed to reference to form arbitrarily, and other allelic form allele that be designed to substitute or variable.The allelic form of frequent appearance is called as the wild type form sometimes in the group of selecting.Two times of homotype combination or heterozygosis that organism can be an allelic form.The two pairs of allele is polymorphic to have two kinds of forms.The three pairs of allele is polymorphic to have three kinds of forms.

" mononucleotide is polymorphic " (SNP) appears at by the occupied polymorphic point of mononucleotide, and described polymorphic point is the position that changes between the allele sequence.Allelic highly conserved sequence (for example, changing in the member less than crowd 1/100 or 1/1000) is arranged before or after this position usually.Mononucleotide is polymorphic usually owing to replacing another to produce at nucleotides of polymorphic site.Conversion is that a purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine.Otherwise transversion is a pyrimidine replace purine or.Mononucleotide is polymorphic also can be by nucleotide deletion or nucleotides about the insertion of reference allele and produce.

" reagent " broadly refers to any medicament of using in reaction.Reagent can comprise self can be monitored single agents (for example, when it is heated monitored material) or the mixture of both or more reagent.Reagent can be lived (for example, cell) or abiotic.The reagent exemplary that is used for nucleic acid amplification reaction is including but not limited to buffer solution, metal ion, polymerase, primer, template nucleic acid, nucleotides, mark, dyestuff, nuclease and the like.The reagent that is used for enzyme reaction comprises, for example, and substrate, confactor, connection enzyme, buffer solution, metal ion, inhibitor and catalyst.Be used for based on the reagent of the reaction of cell including but not limited to cell, cell-specific dyestuff be bonded to the aglucon (for example, activator and antagonist) of cell receptor.

" aglucon " is meant any molecule, has another specificity or the non-specific binding molecule (that is, anti-aglucon) to this aglucon for this molecule, described combination since anti-aglucon to the identification of aglucon part.

II. General introduction

A large amount of different microfluidic devices (becoming chip sometimes) with unique flow channel structure here are provided, use these equipment to implement the method that various high-throughputs are analyzed in addition.These equipment are designed to need in the temperature controlled analysis, relate in particular in the analysis of thermal cycle (for example, nucleic acid amplification reaction).Microfluidic device is in conjunction with the specific design feature: supposition has the equipment of the footprint that is significantly less than many conventional microfluidic devices, this equipment will easily be combined with other instruments and is used for automatic analysis.

Some microfluidic device has used the design that typically is called as " sidewalk for visually impaired people " or " blind filling " here, its Partial Feature is to have a large amount of sidewalk for visually impaired people, as indicated in qualifying part, described sidewalk for visually impaired people is the flow channel with dead end or blind end, so that solution can only at one end pass in and out sidewalk for visually impaired people (just, not having the import and the outlet of the sidewalk for visually impaired people of separation).These equipment only need to be used for the single valve of each sidewalk for visually impaired people, form the enclosed areas point with zone, sealing sidewalk for visually impaired people.During the manufacturing of this equipment, one or more reagent depositions that will be used to implement to analyze are at reflecting point, thereby cause the obvious minimizing of input and output quantity.In addition, the sidewalk for visually impaired people is connected to interconnective channel network, so that can fill all reflecting points from the sample input of single or limited quantity.Because the single valve of the reduction of the complexity of input and output and fund is used to seal each reflecting point, increased the space that can be used for reflecting point.Therefore the characteristics of these equipment mean: each equipment can comprise a large amount of reflecting points (for example, until thousands of) and can realize that high reflecting point density (for example, surpasses 1000-4000 reflecting point/cm ²).Individually and jointly, than traditional microfluidic device, these characteristics also directly change obviously reducing of these instrument size into.

Disclosed here other microfluidic devices use matrix design.Usually, such microfluidic device uses a large amount of cross one another levels and perpendicular flow passage, to locate the defined reaction lattice array in the crosspoint.Therefore, the equipment of this design also has the reflecting point array; Yet, use this design, great amount of samples input and corresponding output are arranged in order to regulate great amount of samples.The valve system that is called as convertible mobile array structure makes solution can optionally only flow through the bottom horizontal flow sheet passage, thereby allows the convertible sealing of the various flow channels in the matrix.Therefore, although sidewalk for visually impaired people equipment is designed to use under different situations the limited quantity sample to implement macromethod, yet matrix device is configured under the limited quantity situation great amount of samples be analyzed.Another other equipment make the combination of kind of design usually in this.

Its other characteristic of the microfluidic device that is described is, uses various parts, for example flow channel, control channel, valve and/or the pump of being made by elastomeric material.In some instances, in fact, entire equipment is by the elastomeric material manufacturing.Therefore, this equipment obviously is different from the main feature by the conventional microfluidic device of making based on the material of silicon on form and function.

The design of described equipment makes them can be combined and be utilized with a large amount of different heating system.Therefore, described equipment is useful implementing to require in the temperature controlled various analysis.In addition, these microfluidic devices that use in heating is used can be in conjunction with other design feature, being that sample evaporation from reflecting point minimizes.Such equipment generally includes a large amount of protection passages that are formed in the elastic devices, and water can flow into the water vapour pressure in the elastomeric material that increases the described equipment of formation by described protection passage, thereby reduces the sample evaporation from reflecting point.

In another embodiment, can the serviceability temperature recycle unit with the temperature of control microfluid.Preferably, microfluidic device may be suitable for realizing thermo-contact with microfluidic device.Wherein microfluidic device is by the base material of for example slide or the carrier board bottom supporting of plastic carrier for example, can in the zone of carrier or slide glass, form window, so that microfluidic device preferably has heating/cold-making block that the equipment of elastomer block can directly contact temperature cycles.In a preferred embodiment, heating/cold-making block has groove therein, to be connected the preferred part that reacts therein with the vacuum source of supplying with microfluidic device suction.Replacedly, firm thermal transfer plate can be bonded to microfluidic device, described microfluidic device is complementary with heating and cold-making block then, is used to implement heat-conduction effect.

The array format of some equipment means that this equipment can realize high-throughput.Jointly, high-throughput and temperature control capability make these equipment (for example, polymerase chain reaction-PCR) are useful to carrying out a large amount of nucleic acid amplifications.This indication that will at large be described as described equipment effectiveness here, especially their purposes in the temperature controlled any reaction of needs of being reflected at.Yet, should be understood that described equipment is not limited to these concrete application.Described equipment can be used in multiple other types analysis or the reaction.A plurality of examples comprise interactional analysis between the interaction of protein ligand and a plurality of unit and all cpds.Other example is provided hereinafter.

III. The general structure of microfluidic device

A. Pump and valve

Microfluidic device disclosed herein usually to small part by elastomeric material structure and use single or the soft lithographic printing of multilayer (MSL) technology or sacrifice layer method for packing (referring to, (2000) Science 288:113-116 such as Unger for example, in the open WO01/01025 of PCT, its both combined with the full text that it is used for all purposes by reference at this) structure.Make in this way, microfluidic device can be designed, and the solution of described equipment flow channel is flow through in control therein, uses one or more control channels of being separated with flow channel by elastic diaphragm or segment at least in part.This barrier film or segment can be deflected and enter flow channel or withdraw from flow channel, by exciting force being affacted control channel control channel are associated with flow channel.Be deflected the degree that enters flow channel or withdraw by the control barrier film from flow channel, flow of solution can be slowed down or complete blocked flow channel.Use the combination of such control channel and flow channel, people can prepare various dissimilar valves and pump, being used for regulator solution flows, as at (2000) Science288:113-116 such as Unger, with at length described the same among the open WO/02/43615 of PCT and the WO01/01025.

Equipment provided here combines this pump valve, selectively to isolate the reflecting point that allows reagent to react and locate.Reflecting point can be positioned in any diverse location in the equipment.For example, in some matrix type equipment, reflecting point is positioned in the infall of a cover flow channel.In the equipment of sidewalk for visually impaired people, reflecting point is positioned in the end of sidewalk for visually impaired people.

If use this equipment in temperature control reaction (for example, thermal cycle reaction), then, as described in more detail hereinafter, elastomeric device typically is fixed to supporting and goes up (for example, slide).Then, the structure that obtains can be placed on the temperature control panel, for example, to be controlled at various reflecting points place temperature.In the situation of thermal cycle reaction, this equipment can be placed on the thermal cycle plate of any amount.

Because this equipment by the elastomeric material manufacturing that is relative optical transparency, uses various different detection systems can easily monitor the reaction of any position on microfluidic device in fact.Yet great majority detect and typically to occur in reflecting point from being in (for example, in crosspoint that comprises flow channel or the zone at the cecum of flow channel).This equipment is also meaned by the fact of substantial transparent material manufacturing: do not have the current device of purposes can use concrete detection system to routine based on the microfluidic device of silicon.Use be incorporated into described equipment or with described device separates but with detector with the region alignment of detected equipment, can implement to detect.

B. The protection passage

For the evaporation from the elasticity microfluidic device that provides of sample and reagent is provided, protect passage can be formed in the described equipment in a large number here.Protection passage and the similar part of control channel are that typically they are formed in the elastomer layer, and described elastomer layer covers on flow channel and/or the reflecting point.Therefore, the same with control channel, protection passage and below flow channel and/or reflecting point are separated by the diaphragm or the segment of elastomeric material.Different with control channel, the cross-sectional area of protection passage is quite little.Usually, have diaphragm ratio than small size have the larger area diaphragm under same applied pressure with littler deflection.The design protection passage is protected passage being compressed so that solution (water typically) flows into.Come from the spreadable elastomer aperture of going into adjacent flow channel or reflecting point of the steam of protection passage, thereby increase the water-vapour density of adjacent flow channel or reflecting point, and reduce solution evaporation therefrom.

Usually, the protection passage is enough little so that compression separate the protection passage with below during the diaphragm of flow channel or reflecting point, can not limit solution basically and flow into, flow out or flow through flow channel or protect the overlapping reflecting point of passage.Term " restriction " basically or other similar terms mean when being used in this context: with under the same conditions into and out of or compare by the flow of solution of flow channel or reflecting point; into and out of or flow of solution by flow channel or reflecting point do not reduce more than 40%; typically be less than 30%; usually be less than 20%; and be less than 10% in some instances, be not compressed when obtaining the flow of solution by wherein at the protection passage.Usually this means that the protection passage has at 100 μ m ²With 50,000 μ m ²Between cross-sectional area, perhaps any integral body betwixt or non-integral cross-sectional area.Therefore, for example, in some example, cross-sectional area is less than 50,000 μ m ², in other example less than 10,000 μ m ², in another example less than 1000 μ m ², and in another example less than 100 μ m ², the protection passage can have any different shape, includes but not limited to circle, ellipse, square, rectangle, hexagon and octahedra shape.

The protection passage be designed to it adopt to implement thermal cycle reaction during and reduce under the condition from the sample of described equipment and evaporation of reagents to less than 50%, in other example less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%.Therefore, for example, the typical PCR reaction that comprises 40 circulations can be implemented in 120 minutes.The protection channel system is designed to during approximate this time frame evaporation is reduced to aforementioned limitations.In order to reach the level that this evaporation reduces, the protection passage typically is presented at least 10 lines/cm ²To 1000 lines/cm ²Density, perhaps arbitrary integer density value betwixt.Especially, the protection passage is generally at least 25 lines/cm ², at least 50 lines in other examples/cm ², at least 100 lines in another example/cm ², and in another example at least 500 lines/cm ²In order to reach this level that evaporation reduces, the protection passage typically present as from the outer edge of a line to the measured interval between 1mm to 1 μ m of the nearest outer edge of adjacent lines, perhaps arbitrary integer level of density betwixt.Especially, the protection passage is usually at interval between 500 μ m to the 5 μ m, in other examples between 100 μ m to 10 μ m, in example in addition between 75 μ m to 40 μ m.Therefore, typically be μ ml at least at interval, and less than 1mm, in other examples less than 500 μ m, in other example less than 400 μ m, in another example less than 300 μ m, in other examples less than 200 μ m, in another example less than 100 μ m, 50 μ m or 25 μ m.

The protection passage can be formed the channel network of separation, perhaps the smaller channels of branch's control channel.The protection described equipment of the extensible mistake of passage or concrete zone or a plurality of zone of described equipment are only arranged.Typically, the protection passage is close to and is provided with above flow channel and reflecting point, is main positions places that evaporation relates generally to these.The example location of protection passage on concrete matrix equipment is illustrated among Fig. 1 C, and protects the example location of passage on the equipment of concrete sidewalk for visually impaired people to be illustrated among Fig. 3 B and the 3C, and is described in detail hereinafter.

The solution that flows through the protection passage comprises the arbitrary substance that can reduce the water evaporation.Described material increases water vapor concentration a kind of of contiguous streamline and/or reflecting point, although or not increasing water vapor concentration hinders a kind of (blocking agent) that evaporates from the water of streamline and/or reflecting point.Therefore, a kind of selection is to utilize any aqueous solution in fact, and suitable in this case solution includes but not limited to water and buffering solution (for example, TaqMan cushioning liquid and PBS).Suitable blocking agent comprises for example mineral oil.

The protection passage typically is formed in the elastomer MSL technology that utilization is quoted as proof above and/or sacrifice layer method for packing.

Lower part is described a large amount of concrete structures in more detail, and it can be utilized implements various analyses, comprises the temperature controlled analysis of needs (for example, nucleic acid amplification reaction).Should be understood that, yet these structures are exemplary and will will be conspicuous for those skilled in the art to the remodeling of these systems.

IV. Matrix diagram

A. General introduction

Utilize the equipment of matrix diagram generally to have a plurality of vertical and bottom horizontal flow sheet passages, described a plurality of vertical and bottom horizontal flow sheet passage intersects to form the tie point array mutually, because different samples and reagent (or reagent set) can be imported into each flow channel, so great amount of samples can be tested with the high-throughput form under considerable reaction condition.Therefore, for example, if different sample is imported in M the different vertical flow channel each, and different reagent (or reagent set) is imported in N the varying level flow channel each, can implement M * N differential responses simultaneously then.Typically, matrix device comprises the valve of the convertible isolation that is used for vertical and bottom horizontal flow sheet passage.Differently described, make the valve location only pass the perpendicular flow passage or only pass flowing of bottom horizontal flow sheet passage to allow selection.Because such equipment allows to be relevant to the elasticity of the selection of the type of sample and quantity and amount of reagent and type, so these equipment very well are suitable for implementing to analyze, people attempt to screen great amount of samples under considerable reaction condition therein.Matrix device preferably the join protection passage to help to stop sample and evaporation of reagents.

The invention provides and be used for high-density matrix design, described design utilizes inter-layer fluid communication through hole in the microfluidic device, with the control line and the fluid line of structure perforation equipment.For example, be arranged in each layer that two layers of elastomer is blocked by making fluid line, more the high density reaction member arranges it is possible.Figure 21 has described the example matrix design, and wherein each first elastic layer 2110 (ground floor) and second elastic layer 2120 (second layer) with fluid passage is formed on therebetween.For example, reagent fluid passage in the ground floor 2110 is connected to reagent fluid passage in the second layer 2120 by through hole 2130, although the second layer 2120 also has sample channel therein, sample channel and reagent passage terminate in respectively in sample and the reagent chamber 2180.Sample and reagent chamber 2180 interconnect by interface channel 2150, and described interface channel 2150 has the interface valve 2140 that is associated with it with fluid flow between each chamber 2180 in the control reaction member 2160.In the use, interface is at first closed, and then reagent is imported reagent passage from the reagent inlet, and sample is imported sample channel by the sample inlet, and container valve 2170 is closed then, so that each reaction member 2160 and other reaction member 2160 are isolated.In case reaction member 2160 is isolated, then open interface valve 2140 so that sample chamber and reagent chamber be in interconnect among so that the reaction that can want.

Therefore, preferred aspect of the present invention is provided for the microfluidic device of the different samples of M quantity and the different reagent reactings of N quantity, described microfluidic device comprises: a plurality of reaction members, each reaction member comprise sample chamber and reagent chamber, sample chamber and reagent chamber are in the fluid connection by the interface channel, described interface channel has related betwixt interface valve, is used to control sample connection between sample chamber and the reagent chamber; A plurality of reagent inlet, each is in during the fluid of reagent chamber is communicated with; Wherein be in respectively with one of one of sample chamber or reagent chamber phase fluid by through hole one of in sample inlet or the reagent inlet and be communicated with.Specific embodiment comprises is formed in the elastomer block reaction member, and described elastomer block is formed by the multilayer that combines, but and interface valve be the deflection barrier film; The sample inlet is connected with sample chamber by sample channel, and the reagent inlet is connected with reagent chamber by reagent passage, sample channel part and reagent passage part are located approximately in parallel with each other, and each has the container value of being mutually related, and are used to control the fluid connection of perforation; Valve that is associated with sample channel and the valve that is associated with reagent passage operationally are communicated with, by public easy control channel mutually; The container public control channel is positioned along the line of one of approximately vertical sample channel or reagent passage.

Another aspect of the present invention is provided for realizing the method for feature in the elastic material block, and described method comprises step: first elastomeric layer is provided; On the first elastomeric material laminar surface, apply the photoresist layer; The photoresist layer is applied light pattern to form the pattern of the photo anti-corrosion agent material that reacts; Remove unreacted photo anti-corrosion agent material, the remaining photoresist pattern that has reacted on the first elastomeric material laminar surface; To first elastomeric layer apply etching reagent with etching not by the first elastomeric material laminar surface of the pattern covers of reacting photo anti-corrosion agent material, thereby remove the first elastomeric material layer region that does not have by the pattern covers of reacting photo anti-corrosion agent material, and remaining corresponding to the elastomeric material layer pattern that reacts the photo anti-corrosion agent material pattern.In the concrete preferred embodiment of this method, described method comprises that removal reacted the step of photo anti-corrosion agent material pattern; By splicing tape being applied to elastomeric layer and having reacted the surface of photo anti-corrosion agent material pattern, cause and implement to remove, separate splicing tape from elastomeric layer then, some or all have reacted the photo anti-corrosion agent material pattern by the surface removal from elastomeric material simultaneously; Making photoresist is SU8; Make etchant comprise tetrabutylammonium fluoride-trihydrate; Make and be characterized as through hole; The a plurality of elastomeric layers that elastic material block comprised bond together, wherein two or more elastomeric layers have the import and export that are formed on wherein, and import and export in elastomeric layer are connected with import and export in another elastomeric layer by through hole.

Microfluidic device of the present invention can further be integrated into vehicle equipment, described vehicle equipment is described in pending trial and the total US patent application serial number 60/557 that is proposed on March 29th, 2004 by Unger, in 715, it is used for all purposes here and is combined.The Unger carrier provides continuous fluid pressure on the plate, so that remain closed away from the valve of fluid pressure source, and family's air pressure for example.Unger is provided for automatic system in addition, is used for valve is as the present invention described herein feeded (charge) and encouraged.Another preferred embodiment, be used for adopting equipment with platen to the accumulator charging with to the automatic system that valve encourages, described platen coupling is on one or more surfaces of microfluidic device, wherein platen has in controlling two or more at least mouths that vacuum or pressure source are communicated with to fluid, and can comprise the mechanical part that is used to handle the microfluidic device part, for example, but be not limited to check-valves.

Another aspect of the present invention is provided for using substrate to be used to make elastic material block, carrier to stablize, preferably have one or more below features: well (well) or accumulating tank, its with elastic material block by being formed in the carrier or at least one passage phase fluid of use carrier is communicated with; Accumulator and elastic material block are by being formed in the carrier or using at least one passage phase fluid of carrier to be communicated with, and fluid flow port is communicated with the elastic material block phase fluid, wherein fluid flow port is preferably addressable for automatic vacuum or pressure source, such as above-mentioned automatic system, wherein automatic source further comprises the platen of port, described port and fluid flow port are complementary, and sealing fluid connects between the automatic system to form, and is used for fluid pressure or vacuum are imposed on elastic material block.In specific embodiment, the source also can realize being communicated with the fluid of the one or more drivers that are associated in carrier automatically, is used to be full of and discharge the pressure that remains on the integrator.In specific embodiment, carrier can further comprise the zone in the carrier zone that is positioned at the contact microfluidic device, wherein there is the material manufacturing that is different from carrier another part in the zone, is selected for other parts that the region material of improving the heat conduction and scattering parameter is different from carrier.The preferred material that is used to improve the heat conduction and scatters parameter includes, but are not limited to silicon, and preferably silicon is by high polish, available silicon-type in semiconductcor field for example, and as from wafer cutting and polishing wafer or part, chip (chip) for example.

Another aspect of the present invention is provided for using thermal source, for example, but be not limited to the PCR thermo cycler, it can be changed from its original manufacture state, thermal source have can matching vector the thermal conditioning part of part, preferably, conduction of the heat of carrier and distribution part are used for providing the thermal control of conducting and scattering part by heat to the elastomer block carrier.In a preferred embodiment, by vacuum source being applied to one or more passages in the thermal conditioning part that is formed on thermal source, improve thermo-contact, wherein passage is formed with the heat conduction of contact carrier and the surface of scattering part, attracts and the heat conduction of maintenance carrier and the position of scattering part to apply.In a preferred embodiment, the heat conduction and the distribution part of carrier do not have reality to contact with carrier, but be associated with carrier and elastic material block, by only heat conduction and distribution part being bonded to elastic material block, and remaining gap of conducting and scattering edge in the part around heat, with the parasitic heat effect that reduces to cause by carrier.Should be understood that, in described here many aspects of the present invention, the preferred elastomeric material piece may be substituted, use any known microfluidic device of the prior art not have description here, for example by the Affymetrix (R) of California, USA Santa Clara or by the equipment of the Caliper manufacturing of California, USA Mountain View GeneChip (R) (genetic chip) for example.Assign and describe microfluid or medium-scale fluid device to the United States Patent (USP) of Soane, Parce, Fodor, Wilding, Ekstrom, Quake or Unger, described equipment can be substituted elastic material block of the present invention to utilize hot advantage and improvement, for example attract the location, be reduced to other regional parasitic heat transmission of fluid device, this uses in the above in the context of elastic material block and is described.

B. Example design and purposes

Figure 1A provides a kind of diagram of example matrix equipment.This equipment 100 comprises seven perpendicular flow path 10s 2 and seven straight horizontal flow channels 104, and they intersect to form the array of 49 different intersections or reflecting point 106.Therefore this concrete equipment makes seven kinds of samples can be with wherein seven kinds of different reagent or cover reagent react.The row valve 110 of regulator solution flow can be by control channel 118 controls in vertical direction, and described control channel 118 can all be encouraged at single inlet 114 places.

Similarly, going valve 108 regulates liquid inventory in the horizontal directions; These are controlled by control channel 116, and described control channel 116 is by 112 excitations of single control inlet.As shown in Figure 1A, the control channel 116 of regulating row valve 108 depends on that the position changes on width.When control channel 116 was intersected perpendicular flow path 10 2, control channel 116 was enough narrow, so that it can not be deflected into perpendicular flow path 10 2 when it is energized, thereby reduced the liquid inventory that runs through wherein basically.Yet the width of control channel 116 is increased when its overlapping bottom horizontal flow sheet path 10 4; The flow of solution that this makes the diaphragm of control channel enough pass bottom horizontal flow sheet path 10 4 with obstruction greatly.

In operation, reagent R1-R7 be introduced into they separately bottom horizontal flow sheet path 10 4 and sample S1-S7 is injected goes into their perpendicular flow path 10s 2 separately.Therefore with at each perpendicular flow path 10 2 samples reagent in each bottom horizontal flow sheet path 10 4 mix intersecting 106 places, and described intersection 106 is the shape in well or chamber in concrete equipment.Therefore, in the concrete condition of nucleic acid amplification reaction, for example, the essential reagent of amplified reaction is imported into each bottom horizontal flow sheet path 10 4.The different IPs acid template is imported into perpendicular flow path 10 2.In some was analyzed, being imported into may be different between flow channel as the primer (primer) of reagent mixture part, and described reagent mixture is imported in each bottom horizontal flow sheet path 10 4.This makes nucleic acid-templatedly has a large amount of different primers with each that reacts.

Figure 1B-E shows the amplification view of the contiguous reflecting point in the equipment described in Figure 1A, how to operate in analysis to be described more specifically equipment.Be used for simple and clear purpose, do not show the intersection 106 of reacting hole form, and control channel 116,118 is omitted, only row and row valve 110,108 are shown (rectangular box).As shown in Figure 1B, by closed row valve 110 with open capable valve 108 and begin to analyze, cross bottom horizontal flow sheet path 10 4 to allow flow of solution, block simultaneously and flow through perpendicular flow path 10 2.Reagent R1 is imported into bottom horizontal flow sheet path 10 4 then and then flows through the length of bottom horizontal flow sheet path 10 4 fully, so that all reflecting points 106 are filled.Flow of solution by external pump can obtain passing bottom horizontal flow sheet path 10 4 still more typically obtains by peristaltic pump being incorporated into the elastomeric material equipment self.As described in detail in (2000) Science 288:113-116 such as for example Unger and the open WO01/01025 of PCT.

In case R1 is imported into, then closes capable valve 108 and open row valve 2 (seeing Fig. 1 C).This makes sample S1 and S2 be imported into perpendicular flow path 10 2 and flows through its flow channel separately.When sample flow was crossed perpendicular flow path 10 2, they discharged R1 from reflecting point 106, thereby at reflecting point 106 remaining samples.Then, as shown in Fig. 1 D, open capable valve 108 and allow S1 and S2 to scatter and mix with R1.Therefore, in each crosspoint or reflecting point 106 zones, obtain the mixture of sample and reagent (R1S1 and R1S2).Make after S1 and S2 and R1 scatter to continue the enough time, all row and row valve 108,110 are closed S1 and S2 are isolated in it separately within the reflecting point 106 and stop the mutual mixing (referring to Fig. 1 E) of S1 and S2.Allow mixture to react then, and by monitoring cross part 106 or comprise the cross shaped head zone detection reaction of cross part 106.Be used for the analysis (for example, in the thermal cycle of amplified reaction) of needs heating, equipment is placed on the heater, and is isolated in heating sample maintenance simultaneously.

The revision of equipment shown in Figure 1A is presented among Fig. 1 F.Usually structure has and the many similar part described in Figure 1A, and the shared same reference numbers of the common ground among two figure.Equipment 150 differences shown in Fig. 1 F are that 4 pairs of bottom horizontal flow sheet path 10s are connected to public inlet 124.This uses the unique single injection port that enters inlet 124 in fact, makes two cover reagent be imported into two adjacent flow channels.The use of public inlet further is expanded with respect to perpendicular flow path 10 2.In this object lesson, use the single injection port that enters sample inlet 120, each sample is imported into five perpendicular flow path 10s 2.Therefore, use this concrete equipment, be useful on ten reaction repeated of each concrete combination of sample and reagent in fact.Certainly, the quantity of the reaction repeated quantity vertical and/or bottom horizontal flow sheet path 10 2,104 that can be ideally be connected to public inlet 120,124 by change is changed.

Equipment shown in Fig. 1 F also comprises the separation control channel inlet 128 of regulating control channel 130 and regulates another control channel inlet 132 of control channel 134, described control channel 130 can be used to arrange the flow of solution that flows to outlet 132, and described control channel 134 is adjusted to the flow of solution of outlet 136.Additionally, equipment 150 join protection passages 138.In specific design, protection passage 138 is formed the part of control channel 116.As previously mentioned, protection passage 138 is less than row valve 108; Therefore, the barrier film of protection passage 138 is not entered by walking around in the following bottom horizontal flow sheet path 10 4, so that flow of solution is interrupted.

At last, the difference of design shown in Fig. 1 F is not react in the hole of level and vertical streamline infall, but in intersection self.

V. The sidewalk for visually impaired people design

A. General introduction

Utilize the equipment of sidewalk for visually impaired people design to have concrete characteristics.At first, equipment comprises one or more flow channels, comes out from described one or more flow channel branch in one or more sidewalk for visually impaired people.As mentioned above, the stub area of this passage can be used as reflecting point.The valve that is formed by overlapping flow channel can be energized to isolate the reflecting point of end, sidewalk for visually impaired people.Valve is provided for isolating the mechanism of reflecting point convertiblely.

The second, the flow channel network that is connected with the sidewalk for visually impaired people is configured, so that all of reflecting point or great majority can use single or limited quantity inlet (for example, less than 5 or less than 10) is filled.The ability of filling the moving passage of blind influx is in the cards, because described equipment is by the elastomeric material manufacturing.Elastomeric material is enough porous, so that the air in flow channel and the sidewalk for visually impaired people can be escaped by these holes when solution is imported into passage.The hole of the material that utilizes in other microfluidic devices lacks the use of having got rid of the sidewalk for visually impaired people, if because do not provide through hole in somewhere, longshore current body path then air in the sidewalk for visually impaired people no longer overflows holding when also being injected into.

The 3rd characteristics are that during making, one or more reagent are deposited on (the other details about manufacturing process that sees below) on the elastomeric stratum non-covalently in reflecting point.Reagent is adhered to non-covalently, and is dissolved because reagent is designed to when sample is imported into reflecting point.Analyze the quantity maximization in order to make, different reagent or reagent set are deposited over each differential responses point place.

Design concrete sidewalk for visually impaired people equipment, so that reflecting point is arranged with array format.

Therefore, implement to adjust in those sidewalk for visually impaired people equipment of amplified reaction being designed to, for example, implement the needed one or more reagent of expansion reaction and during this equipment is manufactured, be deposited over each reflecting point place.Below this reagent for example comprises some or all: the dyestuff of primer, polymerase, nucleosides, confactor, metal ion, buffer, interpolation etc.In order to make the high-throughput maximization, the different primers of zones of different are deposited over each reflecting point in the selected DNA amplification.Therefore, when being imported into reflecting point via inlet, a large amount of expansion reactions can be performed at the different sections place of template when nucleic acid-templated.By equipment being placed on the thermal cycle plate and, can finishing the thermal cycle that must be used for amplified reaction at the various recycle units between the temperature that require.

Can be with the whole bag of tricks stable reagent.For example in some instances, one or more reagent are deposited on reflecting point non-covalently, and in other example, one or more reagent are covalently adhered to substrate at reflecting point.If bonding by covalency, reagent can be linked to substrate via link thing (1inker).Various link thing models can be utilized, for example photochemistry/to the non-persistent link thing of light, to hot unstability attachment and can be turned into the link thing that splits by enzyme.Some link thing is bifunctional (just, the link thing is included in the functional group of each end, and it links thing group on the element that is attached is responded with being positioned in); Functional group at each end can be identical or different.The example of the suitable link thing that can be used in some is measured comprises that the straight or branched carbochain connects thing, heterocycle link thing and peptide linkage thing.Various model link things are can be from Rockford, and the Pierce Chemical Company of Illinois (Illinois Rockford) obtains, and is described in EPA188256; US patent No.4671958,4659839,4414148,4669784,4680338,4569789 and 4589071, and by Eggenweiler, H.M, 1998,3,552 descriptions of Pharmaceutical AgentDiscovery Today.NVOC (6 nitro black false hellebore oxygen base carbonic acyl radical) link thing and other NOVC peer link thing are the examples (seeing, for example WO90/15070 and WO92/10092) that suitable photochemistry links thing.Peptide with protease split-point for example has been discussed in US5382513.

Fig. 2 is a kind of simplified plan view of utilizing the example apparatus of sidewalk for visually impaired people design.Equipment 200 comprises the flow channel 204 that is formed in the elastomeric material substrate 202 and a cover diverted flow passage 206 of branch therefrom.Each diverted flow passage 206 ends at reflecting point 208, thereby forms the reflecting point array.Overlapping diverted flow passage 206 be control channel 210, it is separated by barrier film 212 and diverted flow passage 206.Excitation to control channel 210 makes barrier film 212 be deflected into diverted flow passage 206 (just playing the effect of valve), thereby each reflecting point 208 is separated with the remaining reaction point.

The operation of this equipment comprises injects flow channel 204 with test sample book, and solution flows into each branched bottom 206 then.In case sample is filled each branched bottom 206, then encourage control channel 210 to cause the people having a common goal 206 that valve/barrier film 212 starts to be deflected into branch, thereby seal each reflecting point 208.When sample flowed into and remains on reflecting point 208, it dissolved in advance by the reagent of point sample at each reflecting point 208.In case dissolved, then reagent can react with sample.Valve 212 prevents to mix mutually at the reagent of each reflecting point 208 dissolvings by diffusion.Between sample and reagent, react and be detected then, typically in reflecting point 208.Reaction can preferably be heated, described in temperature control part hereinafter.

Fig. 3 A shows the example of more complicated to a certain degree blind influx circulation passage design.In this specific design 300, each bottom horizontal flow sheet passage 304 is enclosed within its end and is connected straight two perpendicular flow passages 302.A plurality of diverted flow passages 306 stretch away from each bottom horizontal flow sheet passage 304.Diverted flow passage 304 in this specific design is interlaced alternatives, be positioned between two branched bottoms 306 that are directly connected to adjacent level flow channel 304 so that be attached to the diverted flow passage 306 of any given level flow channel 304, perhaps be positioned between the diverted flow passage 306 that is directly connected to adjacent flow channels 304 and the perpendicular flow passage 302.When using the described design of Fig. 3 A, each diverted flow passage 306 ends at reflecting point 308.Also consistent with design shown in Fig. 3 A, control channel 310 overlapping each branched bottoms and isolated by barrier film 312 with following branched bottom.At port 316 place's start-up control passages.Vertical and bottom horizontal flow sheet passage 302,304 intersects so that sample to enter the mouth 314 cross level and perpendicular flow channel network such as the permission flow of solution, and finally enter each reflecting point 308 via diverted flow passage 306.

Therefore, in operation, sample is injected into inlet solution is imported each reflecting point.In case reflecting point is filled, then by start in port extruding control channel valve/barrier film with solution capture in reflecting point.The reagent that is deposited on reflecting point in advance is suspended in the reflecting point again, thereby allows being reflected in each reflecting point between deposited reagent and sample.Reaction in the reflecting point is monitored by detector.Moreover reaction can preferably can be controlled heating, according to the method for being set forth in the temperature control part below.

The more complicated duplicate of the common design that shows in Fig. 3 A is displayed among Fig. 3 B.Equipment shown in Fig. 3 B is that the cellular organization of level shown in a kind of Fig. 3 A and diverted flow passage 302 is repeated equipment repeatedly.Equipment shown in Fig. 3 B has shown that further protection passage 320 will be used in comprising of these equipment in the application that relates to heating (for example, thermal cycle).Protection passage 320 with respect to the example position fixes of flow channel 304 and branched bottom 306 to be shown at the amplification view described in the right plane of Fig. 3 B.Protection passage 320 overlapping diverted flow passage 306 and reflecting points 308.As mentioned above, current overprotection passage 320 between to the period of heating of equipment 300 with the local concentration of water in the increase equipment, thereby reduces evaporation of water in the solution from flow channel 306 and reflecting point 308.

The feature that begins the blind vias equipment discussed in this section minimizes the vestige of equipment, and makes a large amount of reflecting points will be formed on the equipment and be used for obtained high density.The such equipment that for example has 2500 reflecting points can easily be made, and (the aforementioned feature of 25mm * 75mm) can also use the equipment that utilizes the sidewalk for visually impaired people design to obtain very high density in the reflecting point to be assemblied on the standard microscope slide.For example, can obtain at least 50,60,70,80,90 or 100 reflecting point/cm easily ²Density or arbitrary integer density value betwixt.Then, concrete equipment has more high density scope, for example at 100 to 4000 reflecting point/cm ², or arbitrary integer density value betwixt.For example, some equipment have the density of 100,150,200,250,300,400,500,600,700,800,900 or 1000 reflecting point/cm2.Also can obtain to have at least 2000,3000 or 4000 reflecting point/cm ²Very highdensity equipment.This high density directly changes the very many reflecting points on the equipment into.Utilize the equipment of sidewalk for visually impaired people structure typically to have 10-100 reflecting point at least, perhaps arbitrary integer point betwixt.More specifically, equipment has 100-1000 reflecting point at least, perhaps arbitrary integer point betwixt.Higher density equipment can have more reflecting points, for example 1000-10000 reflecting point, perhaps arbitrary integer point betwixt at least.Therefore, depend on the whole dimension of equipment, concrete equipment has at least 100; 500; 1000; 2000; 3000; 4000; 5000; 6000; 7000; 8000; 9000; 10000; 20000; 30000; 40000; 50000 or 100000 reflecting points.

A large amount of reflecting points and available density are still made the result of the ability in very little well (well) or chamber.For example, chamber or well typically have the volume less than 50nL; In other example, less than 40nL, 30nL, 20nL or 10nL; And in another example, less than 5nL or 1nL.As object lesson, concrete equipment has 300 microns long, 300 microns wide and 10 microns dark wells.

The sidewalk for visually impaired people equipment that here provides can utilize the specific design feature and apply for the methodology described in PCT/US01/44549 (bulletin is WO02/43615) and the PCT/US02/10875 (bulletin is WO02/082047) at PCT, comprise the means that for example are used to fill dead end passage, liquid starting (priming), compression degasification starting, and the various means that are used for substitution gas during the filling of microfluidic channel.These two PCT be disclosed in this by standard with its combined all purposes that are used in full.

VI. Mixed Design

Another equipment makes the mixture of matrix and blind filling design.The design class of this type equipment is similar to the sidewalk for visually impaired people equipment shown in Fig. 3 A, except each bottom horizontal flow sheet passage is connected to himself sample inlet and bottom horizontal flow sheet passage do not interconnect via the perpendicular flow passage.Therefore, the sample that imports any given level flow channel fill level flow channel and be attached to reflecting point there only.Yet in the moving passage of the blind influx shown in Fig. 3 A, sample can flow between bottom horizontal flow sheet passage 304 via perpendicular flow passage 302.

The equipment example of this common equipment is displayed among Fig. 4.Equipment 400 comprises a plurality of bottom horizontal flow sheet passages 404, and each bottom horizontal flow sheet passage has a plurality of diverted flow passages 406 that stretch from it or self sample inlet 414.Control channel 410 overlapping each diverted flow passages 406, and diaphragm (valve) 412 separates control channel 410 with following diverted flow passage 406.When using the moving channels designs of blind influx, cause in the starting of 416 pairs of control channels of inlet to make diaphragm 412 deflections, and isolate reflecting point 408 to diverted flow passage 406.In the variation of this design, each bottom horizontal flow sheet passage 404 can be included in the inlet 414 of each end, thereby allows from two ends importing sample.

In some instances, reagent is deposited over reflecting point during device fabrication.Under considerable reaction condition, this makes great amount of samples tested with the short time, need not as the extra time loss of the desired reagent of matrix device.Replacedly, can the preparation feedback mixture before being injected at chip.In case the injection mixture, they can analyzed or further processing (for example, being heated).

By different samples being injected each bottom horizontal flow sheet passage, great amount of samples can be by rapid analysis.Suppose that reagent is deposited on reflecting point in advance.Provide easy method with the identical reagent existence that any given level flow channel is associated, implemented a large amount of reaction repeated to use each passage at each reflecting point place.If replace, then the reagent at the reflecting point place is different from given flow channel arbitrarily, and each sample is accepted various differential responses conditions in fact simultaneously then.

Therefore, the equipment that provides here is adjusted and is used for various dissimilar researchs.If research is included in the screening (for example, 100 samples are to 100 kinds of user's selective reagents) of considerable different samples under user's controlled condition, then matrix device provides the solution of usefulness.If yet research comprises that the sidewalk for visually impaired people design is useful to a kind of or analysis (for example, a sample is to 10000 reaction conditions) of limited quantity sample under a large amount of reaction conditions.At last, if people want to check quite a lot of number of samples at the defined reaction condition, needn't inject reagent (for example, 100 samples are to 100 kinds of reagent that limit in advance), then mixing apparatus is useful.

VII. Temperature control

A. Equipment and parts

The variation complexity (sophistication) of a large amount of different choice is utilizable, is used for controlling microfluidic device or entire equipment and selects the interior temperature in zone.Therefore, as used herein, the term temperature controller is meaned to refer to the equipment that can regulate the temperature in whole microfluidic device or the microfluidic device part or parts (for example, in the actual temp zone or the one or more tie points place in the type microfluidic device matrix of sidewalk for visually impaired people) widely.

Normally, this equipment is placed on the thermal cycle plate with this equipment of thermal cycle.A large amount of this plates are available by the commercial channel easily, comprise for example ThermoHybaid Px2 (Franklin, MA), MJ Research PTC-200 (South San Francisco, CA), and Eppendorf Part#E5331 (Westbury, NY), Techne Part#205330 (Princeton, NJ).

Array apparatus contacts with thermal control equipment, so that thermal control equipment is connected with the thermal control source, so that if the reaction temperature Sui at least one reaction chamber is the change of control source temperature and changing.

In different embodiment, heat-transfer devices can comprise for example semiconductor of silicon, can comprise reflecting material and/or can comprise metal.

Thermal control equipment can be suitable for active force is imposed on heat-transfer devices heat-transfer devices is pushed to the thermal control source.Active force can comprise magnetic, static or vacuum force in different embodiment.For example, in one embodiment, active force comprises by passage and applies vacuum force to heat-transfer devices that described passage is formed on the surface of thermal control equipment or heat-transfer devices.The vacuum that obtains between the surface of the surface of thermal control equipment and heat-transfer devices can be detected.This detection can be performed by the useful vacuum degree detector, and described vacuum detector is positioned in along the position of passage or away from the passage place of vacuum source position.When vacuum does not surpass preset value, can show that alarm maybe can meet to rearrange agreement.

Array apparatus can contact with thermal control equipment, by the use of one or more machineries or electromechanical positioning equipment.The execution of this method can be automatically control with monitoring.For example, this automatic control and monitoring can use automatic control system to carry out, and described automatic control system is exercisable to communicate with robot control system, are used to introduce thermal control equipment or remove array apparatus from thermal control equipment.Can also monitor the process of reaction.

The unit that comprises thermal control equipment can be provided.The system that comprises array apparatus and thermal control equipment can be provided.

In order to ensure the accuracy of thermal cycle step, in concrete equipment, usefully the sensor of detected temperatures is combined in each location of equipment.A kind of structure that is used for detected temperatures is a thermocouple.This thermocouple may be as the film wire on being patterned in following base material or manufactured as the lead that directly is incorporated into little manufacturing elastomeric material self.

Can also come detected temperatures by resistance variations.For example, the resistance variations in the thermal resistor can be calibrated to given variations in temperature, utilizes routine techniques described thermal resistor can be manufactured at following the semiconductor-based end.Replacedly, thermal resistor may directly be inserted little manufacturing elastomeric material.Another program by the resistance detection temperature is described in " MEMS Flow Sensors forNano-fluidic Applications " such as Wu, Sensors and Actuators A89152-158 (2001), this article are therefore combined in full with it by reference.This piece paper has been described the purposes of doped polycrystalline silicon structure to control and detected temperatures.Be used for polysilicon and other semi-conducting material, the temperature coefficient of resistance can accurately be controlled by characteristic and amount of impurities, thereby optimizes the performance of the sensor that is used for given application.

Thermocolour (thermochromatic) material is to can be used for detecting regional another type structure that goes up temperature in the augmentation apparatus.Especially, concrete material when it passes through different temperatures dynamically with change color renewablely.This material may be added into solution when its process different temperatures.Thermochromic materials can be formed in the following substrate or be bonded in the elastomeric material.Replacedly, thermochromic materials can particle form be added into sample solution.

The other method of detected temperatures is the use by infrared camera.The infrared camera that is connected with microscope can be used to determine the temperature curve of whole amplification structure.Elastomeric material will help such analysis to the infiltration of ray.

The other method of detected temperatures is the use by pyroelectric.Especially, some crystal material, these materials are also showed the piezoelectricity behavior particularly, show piezo-electric effect.This effect has been described the polarization of material crystals dot matrix and the phenomenon that material both end voltage height depends on temperature.This material can be bonded on substrate or the elastomer, and is used to detected temperatures.

Other electrical phenomena, for example electric capacity and inductance can be demonstrated with detected temperatures according to embodiments of the invention.

B. The verification of accurate thermal circulation

As fabrication portion is described in detail hereinafter, sidewalk for visually impaired people equipment has basalis, and reagent is placed on the described basalis.Comprise that the two-layer structure that comprises flow channel and control channel is superimposed on basalis, so that flow channel and deposited reagent align.The outside with basalis is placed in the substrate (for example glass) then.Usually, the reflecting point that takes place thereon of reaction is the about 100-150 micron on substrate/glass interface.Use the approximation of employed elastomer and glass in known thermal diffusivity equation and the equipment, people can calculate that temperature arrives the temperature required time that controller is attempted to keep in reflecting point.Temperature be can reach fast in the numbers illustrated that calculates shown in the table 1, employed thick many elastomers and glass (typical range that just, is used for equipment described here) in the equipment of approximate 100-150 micron are even use than reflecting point.

Table 1: calculate at instruction time place by the thermal diffusion length of PDMS and glassy layer.

	1 second	10 seconds	100 seconds
	1 second	10 seconds	100 seconds	PDMS	400μm	1.26mm	4.0mm
Glass	640μm	2.0mm	6.4mm	PDMS	400μm	1.26mm	4.0mm

Fig. 5 shows the speed of using sidewalk for visually impaired people equipment to arrive ideal temperature.

In another embodiment, the bispin oligonucleotide polymer that has known solution temperature (tm) by use can be measured temperature, can whether be that internal contrast coloring agent (for example SYBR Green (TM) or ethidium bromide) hybridization or sex change is used for determining that each cavity is constant scope on array wherein with its internal contrast indication oligonucleotide, wherein import the cavity of microfluidic device by the solution that will comprise the oligonucleotide that has coloring agent, the cavity of described microfluidic device has the reaction chamber array.In this embodiment, in the time of on temperature is elevated to solution temperature, the internal contrast coloring agent is changed into the single line oligonucleotide with its sex change (denataturation should be denaturation) that concerns to oligonucleotide.Replacedly, if temperature is higher than tm or is lowered, then dyestuff enter the internal contrast of the existing light oligonucleotide of quenching can be monitored.The use of dyestuff is provided for " oligonucleotide thermometer " in fact, and described " oligonucleotide thermometer " corresponding to the temperature change of the solution temperature of relative oligonucleotide and change characteristic, for example fluorescence.By designing or use the oligonucleotide of the solution temperature of selecting, can determine that in a similar manner the reaction chamber array changes the scope of temperature.

VIII. Detect

A. General introduction

The microfluidic device that here provides can utilize a large amount of different monitoring means.On the selection part to appropriate system information about the type of selecteed incident and/or reagent.Can design detector to detect a large amount of unlike signal types, include but not limited to indicate, molecule, electro-chemical activity molecule, enzyme, the cofactor of emission chemiluminescence, be connected to the signal of the enzyme of nucleic acid probe and enzyme substrate from radioisotope, fluorophor, chromophore, the close particle of electronics, magnetic particle, spin.

The illustrated detection technique that is suitable for microfluidic device use of the present invention includes but not limited to light scattering, multichannel fluoroscopic examination, UV and visible wavelength absorption, cold light, different reflectivity and confocal laser scanning.Operable other detection method comprises that flicker is near determination techniques, radiation chemistry detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time resolution energy transfer (TRET), fluorescence resonant energy transfer (FRET) and for example modification of bioluminescence resonant energy transfer (BRET) in concrete the application.Detection in addition selects to comprise resistance, resistivity, impedance and voltage detecting.

Detection occurs in " test section " or " surveyed area ".These terms and other relational language refer to the microfluidic device part that detection takes place.As above indicated, use the equipment that utilizes the sidewalk for visually impaired people design, the test section is the reflecting point as being isolated by the valve that is associated with each reflecting point normally.Be used for based on the test section of the equipment of matrix usually in zone, the crosspoint self of the flow channel in contiguous crosspoint or surround the zone in crosspoint and in the zone.

The test section can be connected with one or more microscopes, light stimulus equipment (for example laser instrument), photo-multiplier, processor and aforesaid combination, the signal that their cooperative detection are associated with concrete incident and/or reagent.Detected signal often is an optical signal, and it is detected by photodetector in the test section.Photodetector can comprise one or more optical diodes (for example avalanche diode), fibre-optic light guide conduit, for example photo-multiplier, microscope and/or video camera (for example CCD camera).

Can be manufactured in the microfluidic device detector is little, perhaps can be separating component.If detector exists as separating component and microfluidic device comprises a plurality of test sections, then can in single test section, detect at any given time.Replacedly, can use scanning system.For example, concrete automatic system is with respect to the microfluidic device scanning light source; The light of other system scan emission on detecting perhaps comprises multichannel detector.As the example that specifies, microfluidic device can be attached on the transferable platform or under microscope ocular and be scanned.Desired signal is delivered to processor then, is used for signal interpretation and processing.Can also utilize the photo-multiplier array.In addition, can utilize and have the optical system of collecting signal from all different test sections simultaneously and determining signal capabilities simultaneously according to various piece.

External detector is useful, because the equipment that provides is gone up fully or on a large scale by being the material manufacturing of optical transparency at detected wavelength place.These characteristics make equipment described here use a large amount of optical detection systems, and it is impossible that conventional microfluidic device based on silicon uses described optical detection system.

Concrete preferred detection device uses the CCD camera and is provided for the light path of big visual field and high-NA, so that the light quantity of collecting from each reaction chamber maximizes.About this point, CCD is used as photodetector array, and wherein each pixel or group of pixels are corresponding to reaction chamber rather than be used to produce array image.Therefore, optics can be changed so that image quality is lowered, or is focused on again to increase the optical system depth of field to collect more light from each reaction chamber.In a preferred embodiment, usefully adopt high aspect ratio or column cavity, concentrating the sample that will be addressed inquires to along the optical axis of optical system by detector, and preferably by image being focused on again to increase the depth of field.The use of low NA lens combination preferably uses bilateral to symmetry (symetrical) lens combination.Equally usefully use detector device, for example one or more CCD equipment, it has by the size of the elastomer block area of imaging or greater than will be by the elastomer block area of imaging.In the use that in low NA optics, links to each other, can realize improved detection sensitivity.

Detector can comprise the light source that is used to excite indicator, and described indicator produces detectable signal.Employed light source type depends in part on the type of the indicator that is excited.Suitable light source includes, but are not limited to laser instrument, laser diode and high density lamp.If the use laser instrument can use laser scans one cover test section or single test section.Laser diode can be fabricated in microfluidic device from one's body.Replacedly, laser diode can be fabricated in the contiguous microfluidic device that is used and in another equipment of placing, to implement thermal cycle reaction, so that from the directed test section that enters of the laser of diode.

The test section can comprise a large amount of non-optical methods in addition.For example, detector also can comprise, for example temperature sensor, conductive sensor, potential determination sensor (for example pH electrode) and/or amperometric sensor (for example, monitoring oxidation and reduce reaction).

The a large amount of external sensors that can buy on market can be used.Wherein many is fluorescence detector, because prepare fluorescence labeling reagent easily.The object lesson of available detector include but not limited to Applied Precision ArrayWoRx (Applied Precision Issaquah, WA).

B. The detection of nucleic acid to amplification

1. Intercalative dye (intercalation dye)

Only fluorescigenic concrete intercalative dye can be used to detect the bispin DNA amplification on the bispin DNA being bonded to.The example that is fit to dyestuff includes but not limited to that SYBRTM and Picro Green are (from OR, the Molecure Probe Co., Ltd of Eugene), ethidium bromide, propylidene, chromomycin, acridine orange, Hoechst 33258, Toto-1, Yoy-1 and DAPI (4 ', 6-diamidino-2-phenylindone hydrochloride) (4 ', 6-diamidino-2-phenylindole hydrochloride).Relating to other argumentation that intercalative dye uses equals Anal.Chem.66:1941-1948 (1994) by Zhu to provide it to rise by reference combined in full.

2. Detection method based on FRET

Such detection method is included in the main fluorogen centering of alms giver/be subjected to and detects from alms giver's (indicator) and/or be subjected to the change in fluorescence of main (quenching agent) fluorogen.Select the main fluorogen of alms giver/be subjected to so that the overlapping PLE of being led of alms giver's emission spectra.Therefore, when fluorogen is adjacent to each other to fully entering, can take place to shift from the alms giver to the energy of being led.This energy shifts and can be detected.

FRET and template expansion reactionThese methods are utilized usually and are used the primer (primer) that alms giver/be subjected to element of main centering indicates and use alms giver/the be subjected to nucleotides of another element sign of main centering.Relied between the expansion stage of reaction before the nucleotides that will indicate advances the alloy primer alms giver and be subjected to main being spaced apart enough far so that energy can not take place shift in template.Yet if the nucleotides that indicates is incorporated into primer and the space is enough low close, energy shifts generation and can be detected.These methods are reacted especially useful (seeing below) to implementing single substrate in the detection of SNP to expansion, and are described in the open WO97/22719 of US patent No.5945283 nuclear PCT.

Quantitative PT-PCRVarious what is called " in real time amplification " method or " real-time quantitative PCR " method also can be used to detect the target nucleic acid quantity that is present in the sample, by during amplification procedure self or measure the quantity of amplified production afterwards.It is an object lesson of real-time quantitative method that fluorogen (fluorogenic) nuclease is measured, and described method can be used equipment described here continuously.This method that the monitoring amplified production forms comprises uses two test constantlies that indicate the fluorogen oligonucleotide probes to the accumulation of PCR product---often be called as the approach of " TaqMan " method in the literature.

The probe that uses in this mensuration is lacked (ca.20-25 radix) polynucleotide (polynucleotide) typically, and it uses two different fluorogen dyestuffs to be labeled.5 ' terminal of probe typically is attached to the indicator dyestuff, and 3 ' terminal is attached to the photoinitiator dye of quenching (quenching dye), although dyestuff also can be attached to other position of probe.Designing probe is to have at least and the real sequence complementation of probe binding site on target nucleic acid.The PCR primer that also comprises nuclear downstream, upstream in reactant mixture, described PCR primer are equipped with receives the zone that becomes the side with the probe binding site in year.

When not touching probe, energy takes place between two fluorophor shift, and quenching agent (quencher) is to the emission quenching from indicator.During the extension phase of PCR, pass probe by for example 5 ' nuclease of the nucleic acid polymerase of Taq nuclease, thereby from the polynucleotide quenching agent, discharge indicator, thereby cause the increase of the indicator emission concentration that can measure by suitable detector.

The routine a kind of detector of fluorogen emission that is specially adapted for measuring as producing in fluoremetry is ABI 7700, and it is by Foster City, and the Applied Biosystems Co., Ltd of CA makes.The employed computer software of this instrument can write down the fluorophor concentration of indicator, and the light of quenching in amplification procedure.Then, these numerical value that are recorded can be used to calculate the increase of standard indicator emission concentration on continuous foundation, and the final mRNA quantity that is amplified of adding up.

Be described about the other details that is used for the theoretical agent operation of the concentration fluorogen of definite amplified production in real time, for example authorize the US patent No.5210015 of Gelfand, authorize Livak etc. US patent No.5538848, authorize US patent No.5863736 and the Heid of Haaland, C.A. the Genome Research that waits, 6:986-994 (1996); Gibson, the GenomeResearch of U.E.M etc., 6:995-1001 (1996); Holland, the Proc.Natl.Acad.Sci.USA88:7276-7280 of P.M etc., (1991); And Livak, the PCR Methods and Applications357-362 (1995) of K.J. etc., its each its full text is combined by reference.

Therefore, the gene-amplification course of reaction, the dye quantity of increase is restricted, and is accompanied by the increase of following in the signal.

Can also use for example above-mentioned mutual contrast dye with different approaches, with the quantification PCR method.As mentioned above, these dyestuffs select and excellently are bonded to bispin DNA (for example, SYBR GREEN), and then only produce signal after restriction.Therefore, when amplified reaction carried out, accelerating of dyestuff was limited, and accompanied by the increase of following in the signal that can be detected.

Molecular labeling: use molecular labeling, the change of probe structure causes the formation of detectable signal when it hybridizes complementary region to amplified production.Self comprises two parts probe: in the part at 5 ' end place with at the another part at 3 ' end place.These parts are in the side of the probe portion that the probe binding site is quenched, and are complementary each other.One terminal part typically is attached to indicator dye, and all the other end parts are attached to the photo etching dyestuff of quenching usually.

In solution, both ends can be hybridized mutually to form hairpin loop (hairpin loop), in this structure, the indicator and the photo etching dyestuff of quenching be in enough approaching in so that from the fluorogen of indicator dye by the photo etching dyestuff of quenching effectively by the light of quenching.The ground that compares, hybridization probe produces lineament, and in described structure, the light path degree of quenching is lowered.Therefore, change by the emission of monitoring two kinds of dyestuffs, possible is the formation of indirect control and supervision amplified production.The method of this type probes and use thereof is further described, for example, by Piatek, the Nat.Biotechno1.16:359-63 of A.S. etc. (1998); Tyagi, S. and Kramer, F.R, Nature Biotechnology 14:303-308 (1996); And Tyagi, the Nat.Biotechnol.16:49-53 of S etc. (1998), its each combined here by being used for quoting of all purposes in full with it.

Invader(invader): invader is measured (the 3rd wave technology (Third WaveTechnologies/Madison, WI)) be used to SNP genetic typing and use oligonucleotide, the specification signal probe, described signal probe is complementary to target nucleic acid (DNA or RNA) or polymorphy point.Second oligonucleotide is specified oligomeric invader, comprises 5 identical ' nucleotide sequence, but 3 ' nucleotide sequence comprises nucleotides polymorphy (polymorphism).Oligomeric invader interference signal probe is to the combination of target nucleic acid, so that 5 ' end of signal probe is comprising pantomorphic nucleotides place formation " tablet (flap) ".This species complex specifies endonuclease (endonuclease) to be confirmed by structure, but is called fissility enzyme preparation (cleavase enzyme).But 5 ' tablet of fissility enzyme segmentation nucleus thuja acid.The tablet that discharges combines with the 3rd probe that receives the FRET sign, thereby but forms another double structure of being confirmed by the fissility enzyme preparation.Specifically, but the fissility enzyme preparation divides the fluorogen away from the photo etching of quenching, and then produces fluorescence signal.Be used for the SNP genetic typing, signal probe will be designed to hybridize mutually with benchmark (wild type) allele or distortion (saltant) allele.Different with PCR, the linear amplification of signal is arranged, described amplification does not have the amplification of nucleic acid.Further details is enough to guide those skilled in the art to pass through for example Neri, Advances in Nucleic Acidand Protein Analysis 3826:117-125 such as B.P., 2000 and provide.

Nasba: the amplification (NASBA) based on nucleotide sequence is to use RNA as the detection method that detects template.The primer complementation of RNA is comprised this primer permission of sequence that is used for T7 promoter point (promoter site) (RT) combines with the reverse transcriptase (reverse transcriptase) of template ribonucleic acid and interpolation, with produce from 3 ' to 5 ' the complementation rotation.RNase H is added subsequently, falls RNA with digestion, the remaining cDNA that singly revolves.Then, the second edition of primer can be in conjunction with singly selecting cDNA and making bispin cDNA.Add t7 rna polymerase, with by the RNA of first primer from the T7 promoter point generation that is incorporated into the cDNA sequence more editions.All endonuclease capables of mentioning locate to work at 41 ℃ (referring to, Compton for example, J.Nucleic Acid Sequence-basedAmplification, Nature 350:91-91,1991).

Scorpion。This method is for example by the Nucleice Acids Research of Thelwell N etc., 28:3752-3761,2000 descriptions, so its by reference its to be used for all purposes in full combined, and Figure 20 described its scheme, and wherein Scorpion probe mechanism is as follows.Step 1: the initial sex change of target and Scorpion backbone sequences.Step 2: with the Scorpion primer annealing to target.Step 3: prolong the Scorpion primer and produce double-stranded DNA.Step 4: the double-stranded DNA that produces in the denaturing step 3.The strand target molecule of Scorpion primer is adhered in this generation.Step 5: by cooling, the Scorpion probe sequence is bonded to its target in intramolecular mode.Because the intermolecular of complementary object chain has advantage in conjunction with this.Scorpion (as shown in figure 24) comprise be contained in by 3 of probe ' and 5 ' side on special probe sequence in the hairpin loop that forms of complementary backbone sequences.Be attached to 5 ' terminal fluorophor and be connected to the part of 3 of ring ' end (methyl red usually) quencher.Hairpin loop is linked to 5 ' end of primer by PCR terminator sequence (stop device).After primer extended in the pcr amplification process, specific probe sequence can be bonded to its complementary series in the same chain of DNA.This hybridisation events is opened hairpin loop so fluorophor no longer by quencher and the increase that can be observed signal.The PCR terminator sequence prevents to read over, and this is readed over and may cause hairpin loop to be opened under the situation that lacks the specificity target sequence.This reading over will cause detecting non-specific PCR product, for example, and primer dimer or mispriming incident.

3. Capacitive DNA detects

DNA concentration and electric capacity have linear relationship between changing, and this electric capacity changes by the nucleic acid passage of crossing over the 1khz electric field and causes.Found that this relation is not rely on species.(seeing that for example Sohn waits (2000) Proc.Natl.Acad.Sci.U.S.A.97:10687-10690).Like this, in some equipment, the nucleic acid in the flow channel (for example, the almost flow channel of annular of Fig. 1 or the reaction chamber of Fig. 2) stands such field to determine the concentration of amplified production.Can be instead, the solution that comprises amplified production is removed and stands such electric field then.

IX. Implement the composition of the synthetic of reaction

The reaction of being undertaken by microfluidic device that herein discloses is typically implemented with intensified response by some additive.Therefore, for example, in the example of reflection deposition equipment within it, these additives can be at reactive site for example by one or more reagent contaminations.One cover additive is the closed reagent of protein binding site on the sealed elastic matrix of materials.A variety of such synthetics can be used and comprise multiple different albumen (for example, gel and various albumin, for example bovine serum albumin) and glycerine.

Detergent additives also can be useful.Can use any washing agent in the multiple different washing agent.Example comprises, but is not limited to SDS and various Triton washing agent.

In the special example of nucleic acid amplification reaction, can comprise the number of different types additive.A kind of is the reinforcing agent that promotes amplified reaction.Such additive is including but not limited to the reagent (for example, tetramethyl-ammonium chloride) of reagent (for example, betaine) that reduces secondary structure in the nucleic acid and minimizing misguidance incident.

Find that also some polymer enzyme strengthens effect in the carrying out of some amplified reaction.For example, though use from Thermus aquaticus (Applied Biosystems, Foster city, CA) AmpliTaq Gold polymerase obtains good result, use Finnzyme in some cases, Espoo, the reaction that the DyNAzyme polymerase of Finland is improved.This polymerase is from Thermophilic Bacteria, Thermus brockianus.The polymerase of the example that other can be used is including but not limited to rTH polymerase XL, and it is Thermus thermophilus (Tth) and Thermococcus litoralis (Tli), have a liking for the combination of superthermal primitive bacteria pyrosoccus woesei (Pwo) and Tgo DNA polymerase.Merging two or more polymer enzymes in reactant mixture is useful to the degree of accuracy or the susceptibility that increases the PCR reaction.Other reagent for example the adding of DMSO especially those comprise internal contrast dyestuff person and are added to can further assist in the PCR mixture and implement reaction.Use glycerine or PEG solution to be used to contrast the enforcement that row liquid also can improve the PCR reaction.

React that to comprise in the nucleic acid amplification reaction be other useful details about additive using some equipment that discloses herein, provide in the example 1 hereinafter.

X. Exemplary applications

Because microfluidic device provided herein can be manufactured to the reactive site that comprises enormous quantity, this equipment is useful in very multiple screening and analytical method.Usually, this equipment can be used for detecting the reaction that reacts with between the kind that produces detectable signal, or in case and the product of another species interaction generation detectable signal.Consider their uses under all kinds temperature control system, equipment also can be used in temperature controlled analysis of a plurality of dissimilar needs or the reaction.

A. Nucleic acid amplification reaction

The equipment of Pi Luing mainly can be used to carry out any kind nucleic acid amplification reaction herein.Therefore, for example, amplified reaction can be a linear amplification, (using single primer amplification) and index amplification (that is the amplification of being implemented by forward direction and reverse primer collection).

When using the blind vias type equipment to implement nucleic acid amplification reaction, the reagent that typically is deposited in the reactive site is that those implement the necessary reagent of required type amplified reaction.Usually some or all that this means following material deposits: for example primer, polymerase, nucleotides, metal ion, buffer solution and cofactor.It is nucleic acid-templated guiding the sample that enters reactive site in such example.Yet interchangeable, template can be deposited and amplifing reagent flows into reactive site.As discussed above, when matrix device is used to carry out amplified reaction, comprise nucleic acid-templated sample by perpendicular flow channel flow and amplifing reagent by the bottom horizontal flow sheet passage or vice versa.

Though PCR may be best known amplification technique, this equipment is not limited to and carries out pcr amplification.Other type can effective amplified reaction including but not limited to (i) ligase chain reaction (LCR) (seeing Wu and Wallace, Genomics 4:560 (1989) and Landegren etc., Science241:1077 (1988)); (ii) transcription amplification (seeing Kwoh etc., Proc.Natl.Acad.Sci.USA86:1173 (1989)); The (iii) sequence replicating of self keeping (being Guatelli etc., Proc.Natl.Acad.Sci.USA, 87:1874 (1990)); (iv) based on the sequence amplification (NASBA) (seeing Sooknanan, R and Malek, L, BioTechnology 13:563-65 (1995)) of nucleic acid.Each above-mentioned reference is combined by reference at this for all purposes with their integral body.

Use any detection method that is used to detect the DNA of amplification described above can finish detection to the amplified production that produces.

B. Snp analysis and Genotyping

1. General introduction

A lot of with or the relevant disease of the genome change of host's body or infectious body be the result of minority nucleotides change, usually comprise the change of single nucleotides.The change of single nucleotides like this refers to the polymorphic or simple SNPs of single nucleic acid, and the site that SNP produces is typically referred to as polymorphic site.Equipment described herein can be used for detecting the evaluation of the nucleotides of now so polymorphic site.As the expansion of this ability, this equipment can be used for the Genotyping analysis.Genotyping comprises determines whether dliploid organism (being the organism that each gene has two copies) comprises the allele (being heterozygote) of a copy of two copies (the pure and mild body of reftype) of reference allele, each reference and a variation or comprise allelic two copies (being the pure and mild body of anomaly) that make a variation.When carrying out the genetic analysis analysis, method of the present invention can be used to detect single variant sites.Yet as further describing in the following diversification part, this method also can be used for detecting the genotype of individuality or a plurality of different DNA gene locus in same gene or different genes or their combination.

The reactive site that the equipment that is used for carrying out the Genotyping analysis is designed to use suitable size appears at reactive site with each copy of two allele guaranteeing the dliploid experimenter from the statistics viewpoint with the DNA concentration that can work.Otherwise it is the result of pure and mild body that analysis may produce the assorted and son of prompting, does not only appear at reactive site owing to the second allelic copy.Following table 2 has indicated the DNA concentration with various examples of using equipment described herein to be used to appear at the number of the genome copy in the 1nl reaction volume.

Table 2: the number that appears at the genome copy in the 1nl volume with the DNA concentration of indication

Volume (nl)	[DNA](μg/μL)	N
Volume (nl)	[DNA](μg/μL)	N	1	0.33	100
1	0.10	32	1	0.33	100
1	0.10	32	1	0.05	16
1	0.01	3	1	0.05	16
1	0.01	3	1	0.003	1

Usually, since the ratio at random of sample, the possible errors during the copy number that occurred before amplified reaction begins decision detects.Use the Genotyping analysis of some equipment typically to carry out, although at present inventors' each reactive site of having carried out success with each concentration has the Taqman reaction of term single gene group with sample with about 0.1 μ g/ μ L DNA concentration.

2. Method

Genotyping can be used various distinct methods and be carried out.In these methods, the result who obtains "Yes" or "No" usually is just enough, that is, detecting only needs to answer the problem whether a certain allele exists.Therefore, the necessary primer of allele or the nucleotides that only use to detect that polymorphic site may occur can be analyzed.Yet, more typically, comprise the primer and the nucleotides that detect each the possible allelic appearance of polymorphic site.It below is the example of suitable method.

Single base-pair extends (SBPE) reactionThe SBPE reaction is to analyze the technology that specificity grows up for carrying out Genotyping.Although developed several SBPE reaction, total method be quite similar.Typically, these analyses comprise and will hybridize with the primer of target nucleic acid complementation, like this 3 of primer ' and terminal closely near 5 of variant sites ' or adjoin with it.Extend under the situation of the non-extensible nucleotides that has one or more marks and polymerase and carry out described non-extensible nucleotides and the nucleotides complementation that occupies variant sites.Non-extensible nucleotides is nucleotide analog, and it is in case in conjunction with the further extension that enters under the effect of primer prevention polymerase.If the non-extension nucleotides that adds is in variant sites and nucleotides complementation, the non-extension nucleotides of mark is bonded to 3 of primer ' end to produce the extension products of mark so.Therefore, the primer of extension provides nucleotides to appear at the indication of target nucleic acid variant sites.Such method and correlation technique are for example in United States Patent (USP) the 5th, 846,710; 6,004,744; 5,888,819; 5,856,092 and 5,710,028 and WO92/16657 number in describe.

The detection of extension products can use the FRET detection method of describing in above-mentioned test section that is used for extension to detect.Like this, for example, use equipment described herein, comprise the reagent mixture of a member in the donor/acceptor fluorophor of mark, the non-extension nucleotides of one to four kind of mark (if comprise, by not isolabeling) and polymerase and be introduced into (or before by point sample) to reactive site more than a kind of non-extension nucleotides.The sample that comprises template DNA then is introduced into reactive site and extends to allow producing template.The extension products of any formation detected by the formation of FRET signal (see, for example, United States Patent (USP) the 5th, 945,283 and PCT publication WO97/22719).Optionally make the reaction heat circulation increase signal to use temperature-controlled process and device described above.

Quantitative PCRThe Genotyping analysis also can use the quantifying PCR method of more early describing to carry out.In this case, be included as reagent, also have primer, nucleotides and polymerase with the different label probes of each complementation of allelic form.Yet reaction can only use single probe to carry out, and is owing to lack specific alleles or only ambiguous owing to producing in the reaction failure although whether this lacks signal.For typical diallelic example, be possible wherein for two allele in polymorphic site, usually comprise both not probes of isolabeling in reagent mixture, each is all perfect complementary with one of allele, also has amplimer, nucleotides and polymerase.The sample that comprises target dna is introduced into reactive site.If the probe allele complementary with it is present on the target dna, produces amplification so, thereby cause as at the detectable signal described in the above detection.Based on the kind of the unlike signal that obtains, can determine the evaluation of polymorphic site nucleotides.If two kinds all are detected, two kinds of genes all exist so.Thermal cycle in the course of reaction is implemented as described in above temperature control part divides.

B. Gene expression analysis

1. General introduction

Gene expression analysis relates to the level of measuring one or more gene expressions in the specific cells.This mensuration can be qualitatively, but normally quantitative.In the otherness gene expression analysis, gene level is compared with homologous genes expression levels in the another kind of cell (control cells) in a kind of cell (for example detecting cell).Can carry out very multiple such comparison.Example including but not limited to, between health and the diseased cells, do not treat between the individual cell, be exposed to the cell of specific poisonous substance and not comparison between the exposed cell or the like with a kind of cell of individuality of drug therapy and another.The different gene of expression can be used as the target of sign and/or treatment between detection and the control cells.For example, if find certain group gene rise in diseased cells more than healthy cell, such gene can be used as the sign of this disease and may be used as the basis of diagnostic test.These genes also can become target.The strategy of treatment disease can comprise the program of the expression decreased that causes up-regulated gene.

The design of the equipment of Pi Luing herein is useful for facilitation several genes expression analysis.Because this equipment comprises the reactive site of enormous quantity, the gene of enormous quantity and/or sample can be simultaneously detected.Use the moving channel unit of blind influx, for example, can detect hundreds of or thousands of expression of gene levels simultaneously.This equipment has also made things convenient for the differential gene expression analysis.By matrix design, for example, the sample that obtains from healthy cell can detect at a flow channel, and carries out at close close passage from the sample of diseased cells.This feature has strengthened the simplicity of detection and result's accuracy, because this two sample carried out under identical device and the same terms in the identical time.

2. Sample preparation and concentration

In order to detect a gene or a plurality of gene transcription level (thereby with expression), obtain to comprise this gene or genetic fragment the mRNA transcription product sample of nucleic acid or from the nucleic acid of mRNA transcription product.Be meant that from the nucleic acid of mRNA transcription product mRNA transcription product or its sequence are finally as template for this nucleic acid synthetic.The cDNA that obtains from the mRNA reverse transcription, the RNA that transcribes from this cDNA, the RNA that transcribes from the DNA of this cDNA amplification, from the DNA of amplification are from the mRNA transcription product and detect such transcription product and indicated the existence and/or the abundance of original transcription product the sample like this.Like this, suitable sample including but not limited to, gene or a plurality of gene transcription product, the cDNA that obtains from the mRNA reverse transcription, the cRNA that transcribes from cDNA, the RNA that transcribes from the DNA of gene magnification, from the DNA of amplification.

In some method, total mRNA that sample of nucleic acid separates from biological specimen; In other example, sample of nucleic acid is the total RNA from biological specimen.Term " biological specimen " as use herein, refers to from organism or the sample that for example obtains cell, biological tissue and the liquid from organic component.In some method, sample is from human patients.Such sample comprises liquid, serosal fluid or their cell of saliva, blood, haemocyte (for example leucocyte), tissue or fine needle biopsy sample, urine, peritonaeum.Biological specimen also can comprise the part of tissue and for example fetch the frozen portions that is used for the histology purpose.Usually provide two samples to be used for the purpose of comparison.Sample can be, for example, and from different cell or tissue types, from Different Individual or from the identical source samples (for example drug therapy and contrast) of accepting different treatments.

Any non preference opposes that the RNA isolation technics of mRNA separation can be used to the purification of such RNA sample.For example, the method of separate nucleic acid and purification is at WO97/10365, WO97/27317, Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, first's the 3rd chapter, Theory and Nucleic AcidPreparation, (P.Tijssen, ed.) Elsevier, N.Y. (1993), Laboratory Techniques inBiochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, the 3rd chapter in the first, Theory and Nucleic Acid Preparation, (P.Tijssen, ed.) Elsevier, N.Y. (1993), with Sambrook etc., Molecular Cloning:A LaboratoryManual, Cold Spring harbor Press, N.Y. (1989), Current Protocols in MolecularBiology, (Ausubel, F.M. etc., eds.) John Wiley ﹠amp; Sons describes in detail among the Inc., New York (1987-1993).The tissue samples of enormous quantity can use technology well known in the art easily to handle, and for example comprises, and Chomczynski, P. be at United States Patent (USP) the 4th, 843, the one step RNA separable programming of describing in No. 155.

In the gene expression analysis that uses described equipment, the key factor that influences the result is the concentration of sample amplifying nucleic acid.When the low copy number order, disturb relevant with copy number purpose square root.Therefore, think that acceptable error level has determined required copy number.Required copy number has been determined the concentration of required DNA in the specific sample volume.Although not necessarily best, quantitative reaction can be implemented with the error level that reaches 50%, and is preferably lower.Suppose that 1 receives and rises volume, it is as shown in table 3 to obtain the required DNA concentration of particular error level.As directed, as can to work with microfluidic device concentration for example 1 is received and is risen volume and have enough gene expression product copy numbers by what some equipment used.

Table 3: gene expression---DNA is quantitative

Mistake (%)	N (copy number)	Volume (nL)	[DNA](10-
Mistake (%)	N (copy number)	Volume (nL)	[DNA](10-	2	2500	1	4.2
10	100	1	0.17	2	2500	1	4.2
10	100	1	0.17	25	16	1	0.027
50	4	1	0.0066	25	16	1	0.027

Further some that calculate in the equipment that confirms use 1nL reactive site provided herein comprises enough DNA to obtain expression of results accurately.Especially, typical mRNA preparation procedure produces about 10 μ g mRNA.Proved 1 to 10,000 copy that typically in each cell, has each mRNA.Among the mRNA that expresses in any specific cells, about four information the most common comprise about 13% of total mRNA level.Like this, this high expressed information comprises 1.3 μ g mRNA (each is 4 * 10 ^-12Gram molecule or about 2.4 * 10 ¹²Copy).According to aforementioned expression scope, expect that rare information is with 2 * 10 ^-8The level of copy exists.If the mRNA sample is dissolved among the 10 μ l in standard analysis, the concentration of rare information is about 2 * 10 ⁷Copy/ul; This concentration is corresponding to every 1nl hole 20,000 copies (or 4 * 10 ¹¹M).

3. Method

Because expression analysis typically changes quantitative analysis, one of typically use in the quantitative real-time PCR method described above and to finish detection.Like this, the reagent of if use the Taqman method, introducing (or point sample) before reactive site can comprise one of following material or all: primer, label probe, nucleotides and polymerase.If use intercalative dye, reagent mixture typically comprise one of following material or whole: primer, nucleotides, polymerase and intercalative dye.

D. Name unitization

Equipment based on array described herein (see, for example, Figure 1A, 1F, 2,3A and 3B and the text of following) be designed in essence in order to implement the enormous quantity amplified reaction simultaneously.Yet these characteristics can easily further expand by carry out multiple analysis (for example, Genotyping and expression analysis) in each reactive site.

Even can implement polynary amplification at the single reaction position, by for example use multiple primer in the thermal cycle process, each is specific interesting target nucleic acid specificity.The probe in detecting that the existence of different amplified productions can be used mark differentially is to carry out the quantitative RT-PCR reaction or by using the molecular marker of mark (seeing above-mentioned) differentially.In such method, the probe design of each variant ground mark is only to hybridize with specific amplification target.Select employed different probe by wisdom, analyze and to be carried out, wherein difference is marked in the single reaction at different wave length and is activated and/or detects.Further guidance about the suitable fluorescently-labeled selection that is applicable to such method comprises: Fluorescence Spectroscopy (Pesce etc., Eda.) Marcel Dekker, New York, (1971); White etc., Fluorescence Analysis:Apractical Approach, Marcel Dekker, New York, (1970); Berlman, handbook of Fluorescence Spectra ofAromaticMolecules, second edition, Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.) .Pergamon Press, Oxford, 19723; And Haugland, handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Eugene (1992).

Several genes somatotype and expression analysis can selectively carry out at each reactive site.If use for example TaqMan of quantifying PCR method, the primer of the interesting target DNA zones of different that is used to so to increase is comprised in the single reaction position.Use is used for the probe of the mark differentially in each zone and distinguishes the product that forms.

E. The non-nucleoside acid analysis

Though to carrying out very multiple foranalysis of nucleic acids is useful, equipment also can be used to multiple other purposes.As prompting more early, equipment can mainly be used to analyze reacting to each other between two or more species, described reaction produce detectable signal or can with the product that can detect reagent reacting, this can detect reagent in case can produce signal with reaction.

Therefore, for example, this equipment can be used to a plurality of screenings and use the detection reagent that has specific required activity with identification.As special example, this equipment can be used to screening compounds, screens its activity as the substrate or the inhibitor of one or more enzymes.In such analysis, the compound of detection and other necessary enzyme analytical reagent (for example, buffer solution, metal ion, cofactor and substrate) are introduced in (if not depositing before) reactive site.Introduce enzyme sample and reaction (if detection compound is a substrate) then or inhibitory reaction (if detection compound is an inhibitor) is detected.Such reaction or inhibition can be finished by standard technique, for example directly or indirectly detect the forfeiture of substrate and/or the appearance of product.

Equipment with enough a large amount of flow channels and reactive site also can be used to carry out cell analysis to detect the interaction between cell and one or more reagent.For example, some analysis relates to and determines whether particular cell types is present in the sample.Be used to finish the cell-specific dyestuff that an example is to use preferentially and some cell type reacts of this analysis.Therefore, such dyestuff can be introduced into reactive site and add cell then.The dyeing of cell can use the standard microtechnic to detect.As another example, the compound of detection can screened triggering or is suppressed the ability of cell effect, for example signal transduction bypass.In such analysis, the compound of detection is introduced into reactive site and adds cell then.Check that then reactive site is to detect the formation of cell effect.

Set forth in No. the 60/335th, 292, the unexamined that further being set forth in of the application of relevant device and such equipment submitted to November 30 calendar year 2001 and the common all U.S. Provisional Applications, it is whole by with reference to combined with it for all purposes herein.

XI. Make

A. General aspect

As previously mentioned, utilize single and multilayer soft lithography (MSL) technology and/or sacrifice layer method for packing, the microfluidic device that ordinary construction provides.Basic MS L nibbling method is included in a series of elastomeric layers of casting on little processing mold, removes this layer from mould, then this layer is melted in together.In the sacrifice layer method for packing, it is desirable place that the photoresist layer pattern is deposited over passage.These technology and the use in producing microfluidic device thereof are discussed in detail by following document, for example (2000) " Integrated Elastomer FluidicLab-on-a-chip-Surface Patterning and DNA Diagonstics " such as (2000) Science 288:113-116, Chou such as Unger, at the Hilton Head of Solid StateActuator and Sensor Workshop, S.C. meeting paper is concentrated and in the open WO01/01025 of PCT, wherein each here by reference device be used for all purposes in full and combined.

Briefly, the front manufacture method relates at first by using photoresist (Shipley SJR5740) photoetch to make on silicon chip and (for example is used for top layer, elastic layer with control channel) and the mould mould of bottom (elastic layer that for example, has flow channel).By spin coating speed control channel height accurately.By after developing, photoresist being exposed to UV light, form the photoresist passage.Hot reflux is handled and protection is disposed and typically is implemented, and with M.A.Unger, H.-P.Chou, T.Throsen, A.Scherer and S.R.Quake, Science (2000) 288:113, here it is in full and combined by reference for it.Then, mixing two silicone elastomers (GE RTV 615) is come down in torrents on top mold by precession bottom die and quilt respectively.Here, also have, can utilize the thickness of spin coating control bottom condensate fluid layer.The local solidification top layer cures with 80 ℃ in baking oven and is peeled off from mould after 25 minutes, aligns with bottom and assembles.Use and finally cured irreversibly two-layer in 80 ℃, 1.5 hours in conjunction with this.In case peeled off from the bottom silicon master tooling, this RTV equipment typically uses HCL to be disposed (0.1N, 30 minutes, 80 ℃).The effect of some Si-O-Si combination of splitting is played in this disposal, thereby exposes the hydroxyl groups (hydroxy group) that makes passage more hydrophilic.

Then, this equipment can preferably be sealed to bearing airtightly.This bearing can be in fact by any materials manufacturing, although the surface should be smooth to guarantee excellent sealing, because the sealing that forms is mainly because of cohesive force.The example of suitable bearing comprise peel off, plastics etc.

The equipment that forms according to previous methods produces the substrate (for example, slide) that forms a wall in the flow channel.Replacedly, in case the equipment that removes from master tooling just is sealed to thin elastic diaphragm so that flow channel by integral sealing in elastomeric material.Therefore, the elastic devices that obtains can preferably be connected to base support.

B. Utilize the equipment of sidewalk for visually impaired people design

1. Layer structure

Microfluidic device based on the sidewalk for visually impaired people design typically forms by three layers, and reagent is deposited over reflecting point during manufacture in described microfluidic device.Bottom is a reagent deposition layer thereon.Bottom can be formed by various elastomeric materials, as in the MLS method of quoting as proof in the above described in quote.Typically, material is dimethyl silicone polymer (PMDS) elastomer.Based on the location of this device and the desirable reflecting point that is used for concrete equipment, people can determine the location on suitable reagent should point sample bottom thereon.Because PMDS is hydrophilic, then the moisture point of deposition shrinks and moves the very little point of formation.The reagent of deposition is deposited so that do not form covalent bond between reagent and surface of elastomer, in case be used to melt in sample solution because reagent as discussed previously is imported in the reflecting point.

All the other of equipment are two-layer to be to form the layer of flow channel and form control and the layer of preferred protection passage.This two-layer basis is produced at the previous described conventional method of this part, then, the double-layer structure that obtains is placed on the top of ground floor, and reagent has been deposited on the described ground floor.The object lesson of the composition in three layers following (forming A): ground floor (sample layer) 30: 1 (on the weight) to forming the ratio of B; The second layer (flow channel layer) 30: 1; And the 3rd layer (key-course) 4: 1.Yet what can imagine is also to utilize other composition and the ratio of elastomeric element.Use in conjunction with two or multilayer and or other method of substrate can comprise and use binding agent or plasma combination that preferred air plasma combination is to form other parts of elastic material block or microfluidic device.In certain embodiments, with the extra play that other layer is connected by through hole inside, the size that the preferred elastomeric layer can be used to increase the reaction density of every unit area in the elastic material block or reduce elastic material block.

In this processing procedure, the reagent of reflecting point and deposition aligns, so that reagent is positioned in the suitable reflecting point.Fig. 6 is the photo of a cover from four jiaos of interceptings of equipment; These photos show: utilize previous methods, the reagent of deposition can accurately be aligned with reflecting point.These photos show that protection passage and reflecting point are positioned in the end of diverted flow passage.White circle indication deposited reagent is with respect to the position of reflecting point.As indicated, each reagent point is in the restriction of reflecting point well.

2. Point sample (spotting)

Utilize the point sample technology of the reagent sample applicator that on market, can buy and the various structures of use of any amount, can deposited reagent.The example of the suitable sample applicator that can be utilized in the equipment preparation comprises pin sample applicator, ultrasonic sample applicator, automatic little pipettor, electrophoresis pump, inkjet printing machine equipment, drips black printer and some osmotic pump.The sample applicator that can buy on market comprises CartesianTechnologies MicroSys 5100 (Irvine, CA), Hitach SPBIO (Alameda, CA), Genetix Q-Array (United Kingdom (Great Britain)), Affymetrix 417 (Santa Clara, CA) and Packard Bioscience SpotArray (Aeriden, CT).Usually, the very little reagent point of deposition; Usually the point less than 10nl is deposited, in another example less than 5nl, 2nl or 1nl, in another example, less than 0.5nl, 0.25nl or 0.1nl.

The US patent No.5445935 of use Foder etc.: name is called the method described in " Array of oligonucleotideon a solid substrate " and can also forms the material array, and wherein for example the oligonucleotide probes usage space light of SNP probe guiding photoetch is synthesized in situ.This array also will be used as the substrate or the substrate of microfluidic device among the present invention, comprise one or preferred unnecessary one oligonucleotide probes on the substrate so that will be arranged in corresponding to for example blind filling of the substrate region chamber of reflecting point in a known location.In the situation of separating microfluidic structures, for example depicted in figure 15 here a kind of, be depicted as along the reflecting point of the square box of serpentine (serpentine), flow channel will comprise a plurality of different SNP probes, preferably be suitable for the set of the SNP probe of identification monomer from the summation of monomer, if so that comprise fluid sample from the nucleotide sequence in a plurality of monomers, be imported into the serpentine flow channel herein, with a plurality of valves that are connected with the serpentine flow channel, so that the time caused the serpentine flow channel separated by starting, isolate each reflecting point mutually thereby isolate, in each reflecting point, to comprise the segment of fluid sample.Can carry out amplification that sample is formed to increase molecular number, for example nucleic acid molecules is used to be bonded to the SNP probe array that is positioned in each reflecting point.In certain embodiments, each reflecting point along the serpentine flow channel will be identical array, just have identical arrangement SNP probe, and in another embodiment, will have different series SNP probe along two or more reflecting points of serpentine flow channel.Other separated flow channels configuration also may be used, for example branch and/or branch branch system or the like.Other permutation technology, point sample for example described here, same being used to form is positioned at along the array in the separable reflecting point of serpentine or common flow channel (for example branch).

Following example is explained, to further specify the concrete aspect of equipment disclosed herein and method.Described example is not considered to limitation of the present invention.

Example 1

Signal strength signal intensity is estimated

I. Introduce

The purpose of this cover experiment is that proof uses the microfluidic device of the design of setting forth can carry out the PCR reaction of success herein, and signal intensity ratio Macro TaqMan reacts big by 50%.

II. Microfluidic device

Three layers of microfluidic device of use MSL program manufacturing are designed and make, and are used for carrying out the experiment that following example is described.Fig. 7 A illustrates the cross-sectional view of this equipment.As shown, equipment 700 comprises layer 722, forms flow channel within it.This liquid level 722 is sandwiched between cover layer 720 and the bottom sealant 724, and cover layer 720 comprises control and protective layer.724 layers of side that forms flow channel of confining bed.Consequent three-decker is fixed to basic unit 726 (in this example, slide plate or cover plate), and basic unit 726 provides structural stability, increases the heat conduction, and helps prevent from the evaporation of microfluidic device 700 bottoms.

Fig. 7 B illustrates sketch map and control/protective layer 720 inner control passages of the design of flow channel in the fluidized bed 722 and protects the sketch map of the design of passage.Equipment 700 comprises ten independently flow channels 702, and each has the inlet 708 of oneself, and branch's blind vias 704, and each blind vias 704 has the reactive site 706 of 1nl.Equipment 700 comprises the network of control line 712, and it separates reactive site 706 when applying enough pressure.Also comprise a series of protection passages 716 and evaporate reactive site 706 to prevent liquid; Liquid is introduced by inlet 718.

II. Test configurations

Implement the PCR reaction in equipment 700, described PCR reaction uses β actin primer and TaqMan probe with the exon 3 from human male's genome DNA (Promega, Madison WI) amplification β actin gene.The TaqMan reaction comprises following composition: and 1 * TaqMan buffer A (50mM KCI, 10mM Tris-KCI, 0.01MEDTA, 60nM Passive Reference1 (PR1), pH8.3); 3.5-4.0mM Macl; 200nM dATP, dCTP, dGTP, 400nMdUTP, exciting albumen forward direction primer of 300nM and reverse primer; The β actin probe of 200nMFAM mark; 0.01U/ul AmpEraseUNG (Applied Biosystems Foster city, CA); 0.1-0.2U/ul DyNAzyme (Finnzyme, Fspoo, Finland (Finland)); 0.5%Triton-x-100 (Sigma, St.Louis, MO); 0.8ug/ul gel (Calbiochem, San Diego, CA); 5.0% glycerine (Sigma, St.Louis, MO); Deionized water and male sex's genome DNA.Add reacted constituent to generate 25ul overall reaction volume.Comprise negative control (negative control) in every suit PCR reaction, negative control comprises all TaqMan reacted constituents of removing outside the target dna.

In case TaqMan reaction sample and contrast are produced, then use the gel that is connected to 1 milliliter of syringe to load pipette tip, they are injected in the microfluidic device 700.Pipette tip is filled the reaction sample, is injected into fluid by 708 then.By manually buffer brake being imposed on syringe, flow channel 702 is filled, and is filled until all whole sidewalk for visually impaired people 704 and reactive site 706.Control line 712 is filled deionized water, and is compressed into 15-20psi after all samples are loaded influent stream line 702,704.The control line 712 of compression is energized with valve-off with sample and is isolated in the 1nl hole 706.Then, guiding channel 716 is filled deionized water, and then be compressed to 5-7psi.Mineral oil (15ul) (Sigma) is placed on the flat board of thermo cycler, then microfluidic device/cover plate 700 is placed on the thermo cycler.Microfluidic device 700 thereby by thermal cycle uses initial ramp and three steps or two step thermal cycling curves.

1. initial ramp to 95 ℃, and keep 1 minute (1.0 ℃/s to 75 ℃, 0.1 ℃/sec (second) is to 95 ℃).

Three step thermal cycles continued for 40 weeks (92 ℃ continue 30 seconds, and 54 ℃ continue to continue 1 minute in 30 seconds and 72 ℃) or;

3. two step thermal cycles continued for 40 weeks (92 ℃ continue 30 seconds and 60 ℃ continue 60 seconds)

MicroAmp (microampere) pipe (the Applied Biosystems Foster city that has residual reactant mixture, CA),, they and the reacting phase that carries out indicate the MacroTaqMan reaction for being distinguished in microfluidic device, be placed on 9700 (the Applied Biosystems of GeneAmp PCR system, the Foster city, CA) in, and in 9600 models by thermal cycle.Macro TaqMan reaction is used as the macroscopic view contrast of the reaction of carrying out in the microfluidic device.The thermal cycle agreement be configured to microfluidic device in be complementary, except the initial ramp ratio is not for Macro T aqMan reaction controls.

In case finish thermal cycle; the contrast and protective wire is depressurized and this sheet be transferred to the glass slide plate (VWR, West Chester, PA) on then this sheet be placed ArrayWoRx scanner (the Applied Precision that enters carriage with improvement; Issaquah, WA).Fluorescence intensity is measured under three kinds of different excitation/emission wavelength: 475/510nm (FAM), 510/560nm (VIC), and 580/640nm (Passive Reference 1 (PR1)).Use fluorescence and signal and the background intensity measured in each 1nl hole in the Array Works software video picture microfluidic device.Use Microsoft Excel file analysis result then, calculate the FAM/PR1 ratio of β actin TaqMan reaction.For traditional MacroTaqMan, the computational methods of describing in the agreement (TaqMan PCR kit agreement) that the positive sample of target dna uses manufacturer to provide are measured.Signal strength signal intensity is calculated by the FAM/PR1 ratio that the FAM/PR1 ratio with contrast removes sample.Successful reaction is defined as sample ratio and is higher than 99% confidence threshold value level.

III. The result

Beginning, (Applied Biosystems, Foster city CA) are used in the TaqMan reaction and with the reaction ratio of Macro TaqMan reaction 5.0-14.0 and compare AmpliTaq Gold, produce the FAM/PR1 contrast ratio of 1.5-2.0.Although the result is positive, need to strengthen signal strength signal intensity.Therefore, because the heat endurance of DyNAzyme polymerase, correction and the opposing of impurity strengthened, AmpliTaq Gold polymerase is replaced by the DyNAzyme polymerase.0.025U/ the standard MacroTaqMan DyNAzyme concentration of μ l is used in the micro-fluid experiment.Polymerase is changed into the FAM/PR1 contrast ratio that DyNAzyme has produced 3.5-5.8.Signal strength signal intensity is enhanced, but is difficult to obtain stable result.Because known some albumen sticks to PDMS, has increased the concentration of polymerase and has comprised the surfaction additive.Detected two kinds of DyNAzyme that concentration increases with every nl100pg in the microfluidic device or 10pg genomic DNA, 8 * (0.2U/ μ l) and 4 * (0.1U/ μ l) are used for the normal concentration of MacroTaqMan.Add gel, glycerine and 0.5%Triton-x-100 and adhere to PDMS to prevent polymerase.Reaction result with Macro TaqMan contrast in the microfluidic device (sheet) illustrates at Fig. 8.

Microfluid TaqMan reaction ratio range from 4.9-8.3 Macro TaqMan reaction range from 7.7-9.7.Therefore, the signal strength signal intensity of TaqMan reaction reaches 87% of Macro TaqMan reaction in the sheet.4 * and 8 * DyNAzyme between do not have significant difference.This result confirms that PCR reaction can carry out in microfluidic device, signal strength signal intensity greater than with 50% of Macro TaqMan reacting phase ratio.This result is consistent at least four times trial.

Example 2

The reagent point sample

I. Introduce

The purpose of this experiment is to confirm point sample PCR reaction successful in the microfluidic device.Term in the text " spotted " is meant the placement of droplet reagent (spot) on matrix, and this matrix is combined into the part of microfluidic device then.Normally implemented the subclass of the required reagent mixture of PCR by the reagent of point sample.

II. Process

A. The point sample of reagent

The conventional point sample of reagent is printed by contact and is implemented.Reagent is got on the metal needle from a cover hole, source, and by pin contact target matrix is deposited.This print procedure is further summarized in Fig. 9.As shown, reagent is by (for example titer plate) obtains from the source, and contacts with matrix by the pin that makes loading then and be printed.Washing step is included in and shakes vacuum drying then in the deionized water.The system that is used to print the reagent spot is Caresian Techologies MicroSys 5100 (IrvineCA), uses TeleChem " ChipMaker " print needle, although can use other system as mentioned above.

The pin that uses is TeleChem ChipMaker 4 pins, and it is combined with TURP and cuts the slit (see figure 9) with the volume that increases picked-up (and therefore the quantity of printable spot).Under the operating condition of using (typically, 75% relative humidity and about 25 ℃), the every loader cycle of every pin is printed and is surpassed 100 spots.In the above conditions, the volume of the reagent of point sample on PDMS matrix is the 0.1nl level.

The size of needle tip is 125 * 125 μ m.The final spot of dry reagent comes down to (little of diameter 7 μ m) less than this, and the size of pin still determines the lower limit of the spot spacing that can obtain easily.Obtainable spacing has determined in the final equipment minimum hole-to-pitch of holes.Use such equipment and aforesaid method, can obtain the array of 180 μ m spacings.The array that is structured in the active gage trends towards having from 600 to 1300 microns spacing.

Only use a pin to carry out point sample at a time.Yet the system of use has can hold the needle section that reaches 32 pins.Print standard-sized (20 * 25nm level array size) cost and be lower than 5 minutes.

B. The combination of the sheet of point sample

Mobile and the key-course of PCR equipment is according to normal MSL suite described above.Microfluidic device design is identical with described in the example 1 that.Simultaneously, comprise A: the hypothallus of the 150 μ ms thick PDMS of B composition than 30: 1, by rotation lining bare silicon wafer, and solidified 90 minutes at 80 ℃ then.

The blank hypothallus of the PDMS that solidifies (sealing/basic unit 724 of Fig. 7 A) is used as the target of reagent point sample.The pattern of point sample is printed on still on the matrix on the blank wafer, and the reagent that is used for the point sample of PCR reaction is the Auele Specific Primer and the probe of the specific gene that is about to be amplified.The reagent of point sample comprises volume ratio 1: 1: 1 300nM β actin forward direction primer (FP), the reverse primer of 300nM nM β actin (RP) and 200nM β actin probe (Prb).In some cases, it is useful further adjusting chemical property by the mixture that concentrates point sample.Found to adjust concentration so that primer and concentration and probe concentration equal or be higher than a little the prescription value generation uniformity good result of conventional macroscopic view.Therefore, the reagent of point sample is concentrated into 3 times and 4 times of macroreaction concentration.Reagent be concentrated in the relative ratio that carries out and do not change FP: RP: Prb in the Centrivap heating and the centrifugal separator of finding time.The increase of spot concentration produces when the appropriate final concentration of reagent when the 1nl reaction volume is suspended again.The reagent of point sample need not be restricted to primer and probe; Also needn't all threes (FP, RP and Prb) by point sample.Can have only probe or even one of primer by the application of point sample.Sample primer/the probe set of point sample is that the experiment of TaqMan β actin and TaqManRNAse-P is carried out.

On hypothallus after the point sample process, in conjunction with flow and key-course (be Fig. 7 A layer 720 and 722) aligns with speckle patterns and contacts.Use further and toast 60-90 minute matrix is bonded to the remainder of sheet at 80 ℃.After the sheet assembling, flow channel and sheet that all the other compositions of PCR reaction (describing in example 1) are injected into sheet are carried out thermal cycle described in example 1.

III. The result

Use wherein primer (forward and reverse primer) and probe molecule by the equipment of point sample, carry out the PCR reaction continuously and repeatedly.The data example of the sub-chip of class is displayed among Figure 10, carries out reaction in this chip continuously.The continuous P CR reaction that the reagent of point sample produces as limits in example 1.Use 2 rank and 3 rank thermal cycle agreements to carry out reaction continuously.

Example 3

Genetic typing

I. Introduce

The purpose of testing below is to set forth to utilize for example microfluidic device described here or chip can implement the genetic typing test.Particularly, these tests are designed to determine whether the reaction of implementing has enough sensitivity and guarantee that other primer except that the β actin/probe set can be executed in the microfluidic device in this equipment.

II. Method/result

A. Rnase P test

(AppliedBiosystems, Foster city CA), produce detectable result so that other primer/probe set to be described to carry out Rnase P TaqMan reaction described in example 1 in microfluidic device.Rnase P reaction also requires more high sensitivity, detects single copy gene (2 copy/genome) because contrast Rnase P primer/probe set with β actin primer/probe set.The set of β actin detects single copy β actin gene and several pseudogene, and their each genomes are concentrated approximate 17 parts altogether.Use as described in Example 1 same composition operation Rnase P reaction, except β actin primer/probe set by Rnase P primer/probe sets conjunction generation.In addition, Rnase P primer/probe sets is combined in 4 * manufacturer recommendation numerical value place and is used, to strengthen fluorescence signal.VIC dyestuff conjugated to the probe that is used for Rnase P, and is paid close attention at the VIC/PRI ratio analysis.The result of one of four kinds of tests is displayed among Figure 11.

The VIC/PRI/ contrast ratio that is used for Macro TaqMan reaction is 1.23.The corresponding ratio that is used for microfluidic device TaqMan reaction is 1.11 and 1.21.The ratio of genome dna sample is be sure of on the threshold value being higher than 99% in the microfluidic device.In addition, the signal strength signal intensity of TaqMan reaction is 50% and 93.7% of Macro TaqMan reaction in the microfluidic device.Contrast TaqMan reaction has standard deviation .006 and .012 in the microfluidic device, and the reaction continuity on microfluidic device has been described.Therefore, determine that the TaqMan reaction is sensitive in the chip, enough detect each genomic 2 copy.

B. The DNA dilution test

In order further to determine the sensitivity of TaqMan reaction in the microfluidic device, the dilution of genome DNA is tested, use β actin primer/probe set.Reaction is formed usually and the same composition the described in the example 1, the dilution of use 4 * DyNAzyme and genome DNA.Genome DNA is diluted under the 0.25pg/nl, and it is corresponding to 1 part of approximate every nl.A kind of result of dilution series is displayed among Figure 12.

According to Poisson distribution, if the average criterion number is one, then 37% of the hole sum should be negative.

Hole count

5,6 and 7 is lower than the threshold value of calculating, therefore bears.This suggestion: the β actin TaqMan reaction in the micro-fluid chip can detect the mean value of every nl portion.Therefore, the reaction sensitivity in the microfluidic device is enough to carry out the genetic typing test.

C. The genetic typing test

Because the TaqMan in the microfluidic device can detect the low target number, test SNP (mononucleotide polymorphic) in advance, so use predetermined AllelicDiscrimination bag (Applied Biosystems at CYP2D6P450 cytochromes gene, Foster city CA) carries out genetic typing.Described bag comprises a primer collection and two probes; Sign is used for the FAM of wild type or reference allele, and CYP2D6*1 and sign are used for the CYP2D6*3 sudden change or the allelic VIC that makes a variation.Be used for along the allelic positive polarity contrast of each of genome DNA, PCR product testedly, use and the same terms described in the example 1 at equipment.Result from a test is displayed in Figure 13 and 14, and this test is repeated three times at least, with checking result and argumentation reliability.

As shown in Figure 13, (Allele 1 by the Al-1 of the average VIC/PRI/ contrast ratio of generation 3.5 and 2.2 respectively, the CYP2D6*1 wild-type allele) and genome DNA (100pg/nl), indication genome DNA is the positive polarity that is used for the CYP2D6*1 wild-type allele.These numerical value are on the threshold value limit that is used to react.The signal strength signal intensity of TaqMan reaction is respectively 59% and 40% of Macro TaqMan contrast in the microfluidic device.Should the position in the VIC passage negative Al-2 (Allele 2, CYP2D6*3 sudden change or variation allele) is displayed on some signals on the contrast (1.5), may leak the into VIC passage of detector because of FAM fluorescence.Using improved detection to handle can minimize leakage.

Al-2 positive polarity contrast provides 3.0 average VIC/PRI/ contrast ratio, its for the MacroTaqMan signal 37% and on the threshold value limit of calculating (seeing Figure 14).The genome sample that is used for the CYP2D6*3 mutation allele is born, because the allelic frequency of CYP2D6*3 is low expected result.Moreover the leakage that shows some Al-1, VIC probe enters the FAM passage of detector.Generally speaking, the SNP detection reaction is successful in microfluidic device.

Example 4

Use the PCR verification of gel electrophoresis

I. Introduce

When but the system of selection that confirms DNA cloning occurs in the microfluidic device, use the test of detected through gel electrophoresis PCR product to be performed.The same with described in the example 1 formed in PCR reaction, except the TaqMan probe be omitted with the β actin forward primer by conjugated to FAM.

II. Process

A. Microfluidic device

Three layers of microfluidic device that use MSL to handle manufacturing are designed and make, and are used to be implemented in the test described in this example; Figure 15 shows the schematic diagram of this design.Equipment 1500 generally comprises sample area 1502 and control zone 1504.Sample area 1502 comprises 341 1nl reflecting points 1508, and the rectangle that this reflecting point is arranged by the moving passage 1506 of longshore current is represented, and flow channel 1506 comprises and enters the through hole 1510 and the through hole 1512 of going out.Control zone 1504 comprises three control flow channels 1514, and each flow channel comprises 1nl reflecting point 1518, equally by rectangle with to enter through hole 1516 represented.Be applied in when entering in the through hole 1524 at enough pressure, control gauze 1522 is isolated each reflecting points 1508,1518.Comprise a series of protection passages 1520, to stop the outside of fluid evaporator to reflecting point 1508,1518.This equipment is three-layer equipment (seeing Fig. 7 A) with the same described in the example 1.Entire chip is placed on the cover glass.

B. Test configurations

Make 3 temperature curves described in the use-case 1, load and thermal cycle microfluidic device 1500.Residual reaction sample by thermal cycle, uses the thermal cycling curve the same with being used for microfluidic device 1500 in GeneAmp 9700.After thermal cycle was done, product was reproduced.In order to reproduce the DNA of amplification, with 3 μ l water injecting sample through holes 1506, and from going out through hole 1512 taking-up 3-4 μ l products.Product from equipment 1500 and Marco reaction is used 2 μ l ExoSAP-IT (USB, Cleveland, OH) processed, ExoSAP-IT is made up of DNA Exonuclease I and ShrimpAlkaline Phosphatase, to remove excessive nucleotides and primer.The Marco product was diluted to 1: 106 from 1: 10.Product in the equipment 1500 is dehydrated, and is suspended in again in the 4 μ l formamides.

III. The result

At polyacrylamide gel, two products are analyzed together with negative contrast.Figure 15 shows the gel electrophoresis result.In Figure 16, observe the right suitable big or small DNA band of 294 tape bases on length.

Be illustrated in the left-hand side of gel from the product of Marco reaction, and corresponding to about 294 tape bases to (the desired size of β actin PCR product).Negative contrast lacks the PCR product.Similarly, the product that obtains from this equipment provides the β actin PCR product of expectation.Therefore, target dna is amplified in microfluidic device.

Example 5

Bulk is cut apart

Polymerase chain reaction (PCR) becomes the main tool in the molecular biology.The combination of itself and sensitivity (amplification of a dna single molecule), specificity (distinguishing single tape base mismatch) and dynamic range (real-time testing equipment 105) make its become existing in one of the most powerful analysis tool.Described: the PCR performance improves when reaction volume reduces: we carry out 21000 simultaneous PCR reactions on single micro-fluid chip, react in the volume of 90pL and have the sensitivity of single temperature molecule at each.

Figure 17 a-17d has described the single and double microfluidic device of cutting apart, wherein multilayer soft light etching (MSL) (1) is used to produce the elasticity micro-fluid chip, this elasticity micro-fluid chip plays the effect of Movable valve, with each bulk in several fluid samples be isolated into the reaction volume of a large amount of isolation.At the sample remarks as entering the mouth after 1703, described inlet 1703 is connected (Figure 17 b) with channel isolation system of branch 1705 in the microfluidic device 1701, and the 90pL volume 1709 of 2400 each samples is by keeping apart along the isolated shut off valve 1707 of single microfluidic channel (Figure 17 d).Then, chipset on dull and stereotyped thermo cycler by thermal cycle, and imaging in the fluorescence number of degrees device that on market, can buy.

By concentration that changes template DNA and the quantity that measurement provides the hole of positive polarity Taqmantm signal, the PCR performance in the assessment chip.We find, observe digital amplification (Figure 18 a and 18b) when the par of every hole duplicate is low.Even par duplicate is lower than at 1 o'clock in every hole, also observes and strengthen positive polarity and negative polarity signal clearly; This means even single duplicate of target may have good amplification.The quantity in positive polarity hole is consistent with the control quantity of calculating, has to distribute by Poisson (Poisson) 〉=1 target version (Figure 19).This results verification: even this system also has constant amplification according to the single goal version.Even the fluorescence signal intensity from microfluid TaqmantmPCR is that comparable each reaction of a macroreaction comprises more than＞104 template version at macroscopical PCR reaction with same DNA concentration.

The main source of perhaps observed fidelity (fidelity) is the valid density of target: the unimolecule in the 90pL volume is richer than 55000 times than the unimolecule in the 5 μ L volumes.Because counting nt, target molecule do not change (just, nt=1) and the molecular number ns that can produce side reaction (just, primer-dimer in the sample and incomplementarity dna sequence dna) with the linear ratio of volume (just, ns ∝ V), target is inversely proportional to the ratio and the volume of side reaction: nt/ns ∝ 1/V.Because side reaction is the main cause of PCR accident (4), the advantage that then reduces reaction volume is conspicuous.

Reported (5) in front from single pcr amplification that copies of template.Yet, in macroscopical volume, realize often (for example need changing the thermal cycle agreement from the current method of the reliable amplification of single copy, the long extension time, many circulations), to wrong primer (mispriming) and nonspecific amplification (for example, " thermal starting " PCR (thermal excitation of polymerase), " booster " PCR, additive, to reduce nonspecific hybridization etc.) precautionary measures, and almost use always two the circle PCR make, wherein the part among the PCR be used as second the reaction in template.Mutually on the contrary, this system realizes amplification reliably from single copy, uses standard conditions to leave dividing plate primer and probe and individual pen standard thermal cycle agreement.As intactly sealing, also be subjected to the influence of environmental pollution hardly.Than macroscopical volume (, the ability of doing a large amount of PCR reaction simultaneously provides 1 chip clear and definite logic, that cost and time benefit have 21000 reactions to 219 96 orifice plates that separate, and the time that is associated, equipment and tracking foundation structure).

This principle of a large amount of separation that the use digital pcr is read can be used to the absolute quantity of aimed concn in the sample.For example, what can be used is to provide the hole number of aforesaid specific allele or a plurality of allele positive polaritys simply by statistics, to storage (pool) the sample genetic typing of genome DNA.Since to the enhancing resistance of side reaction, in the mutant (being relevant to the problem of cancer detection) in quantizing wild type DNA, should be still spendable.Rule by the concentration of isolating may be still useful in other reaction, and the detection to unimolecule, bacterium, virus or cell is interested (for example, being used for the ELISA reaction of protein detection) therein.US patent No.6446706, name that the US patent No.6143496 of Brown etc., name are called " Method of sampling; amplifying and quantifying segment of nucleic acid; polymerase chain reactionassembly having nanoliter-sized chambers and methods of filling chambers " and Vogelstein etc. are called " Digital PCR " and have described digital pcr, thereby its both combined by quoting in full with it.Use the obtainable little volume of microfluidic device to allow highly-parallelization and very high target one background concn ratio.High target-background concn ratio allows unimolecule amplification fidelity.These factors are proposed to be used in PCR, and are the smaller the better.

The invention provides the method and apparatus that is used for implementing digital pcr at microfluidic environment, described method comprises step: be provided at the wherein microfluidic device of fluid passage, described fluid passage has two or more relevant betwixt valves, the fluid passage can be isolated into two or more reflecting points or cavity when valve is activated; Introducing comprises the polymeric sample of at least one target nucleic acid, starter gate valve goalkeeper fluid sample is isolated into two or more parts, wherein at least a portion comprises the target nucleic acid condensate, and another part does not comprise the target nucleic acid condensate, amplification target nucleic acid condensate, and the flow channel quantity partly of determining to comprise target molecule.In a preferred embodiment, microfluidic device comprises elastomeric material, more preferably comprises the one deck at least that comprises elastomeric material.In concrete preferred embodiment, microfluidic device further comprises deflectable barrier film, wherein deflectable barrier film can be deflected into the fluid passage or deflection goes out the fluid passage, with a part and another part in fluid stream in the control fluid passage and/or the buffer fluid passage, preferably wherein deflectable barrier film is complete for the microfluidic device layer, have the passage or the import and export that are formed on wherein therein, and preferably wherein forming deflectable barrier film, the first passage in the ground floor of this place's microfluidic device is overlapping by the institute of the second channel in the second layer.In certain embodiments, sample fluid comprises implements all required parts of amplified reaction, and in other embodiments, before introducing sample fluid, microfluidic device comprises at least one parts of amplified reaction.In certain embodiments, microfluidic device further comprises detection reagent, preferably to useful (complimentary) the one or more nucleic acid polymers of small part target nucleic acid polymer, preferably multiple different IPs acid polymer spatially is arranged in the reflecting point or cavity of microfluidic device.

Can obtain amplification by the thermal cycle reaction of for example PCR or by adiabatic reaction, described adiabatic reaction for example is described among the U.S. Patent application No.10/196740 by Van Ness etc., it is announced is US2003/0138800A1, and its purpose that is used to instruct adiabatic amplification to put case in full is combined by reference once more for it.

The present invention further uses fluorescent dye to be used for the protein microcalorimetric method and measures, SYBER green (TM) for example, to measure the conformation change of protein, for example sex change is if the protein deformation temperature changes when especially interacting in second half family (moiety) of protein and for example ligand or compound or other protein.Use the other advantage of SYBR green (TM) to be: it is used at the wavelength place lower than other UV scope dyestuffs, thereby reduces typically relevant with many plastic materials background problems.

Figure 22 B has described the expanded view of complete microfluidic device 2899, described microfluidic device 2899 comprises the parts shown in Figure 22 A, and further comprise elastic material block 2808, described elastic material block 2808 is attached or more preferably uses binding agent bonded and more preferably directly bonded, does not preferably use binding agent to the elastic material block position 2807 of substrate 2800 to form complete microfluidic device 2899 (Figure 22 C).Be to be in the one or more passages that are communicated with one or more through hole 2814 phase fluids in elastic material block 2808, next described through hole 2814 provides the fluid between elastic material block internal channel and the substrate internal channel to be communicated with, and described fluid connection produces well (well) 2805 then and is communicated with well (well) 2805 and the fluid between elastic material block 2808 internal channels that substrate 2800 is provided in well array 2806a-d.Accumulator well (well) top 2809 and 2810 is attached to accumulator well (well) 2801 and 2802 to form accumulator chamber 2815 and 2816.Accumulator well (well) top 2809 and 2810 comprises valve 2812 and 2811 respectively, and it is preferably and is used to guide and keep gas to enter the check valve of accumulator chamber 2815 and 2816 under pressure.In the time of in being present in accumulator chamber 2815 and 2816, valve 2811 is suitable for being positioned in gully 2802 and 2804 inboards apart from contacting valve 2811 and 2812 in order to keep liquid with 2812.Valve 2811 and 2812 can preferably be opened by machinery by sheet extrusion, pin etc. in preferred check valve, overcoming the power of closing automatically of check valve, thereby to allow reducing the fluid pressure that is comprised in the accumulator chamber from accumulator chamber release pressure.

Figure 22 D has described the plane of microfluidic device 2899 and well (well) 2805, wherein fracture is positioned by contiguous well (well) substrate, preferred bottom, or the side of well (well) 2805 selectively, be used for entering passing through of the fluid that is formed on substrate 2800 passages from well, preferably in 2805 pairs of mistakes of well (well) on the side of substrate 2800.In concrete preferred embodiment, substrate 2800 is molded, and has groove therein, and described groove is fabricated in the passage by the sealant of preferred binder film or sealant.

Substrate 2800 machine associated components can be by the polymer manufacturing, for example polypropylene, polyethylene, Merlon, high density polyethylene (HDPE), polytrifluorochloroethylene PTFE or Teflon (R), glass, quartz or metal (for example aluminium), transparent material, polysilicon or the like.Accumulator well (well) top 2809 and 2810 further can comprise import spiral 2812, import spiral 2812 can be removed to import or to remove gas or liquid from accumulator chamber 2815 and 2816, preferably, valve 2812 and 2811 can be activated to discharge otherwise remain on fluid pressures in accumulator chamber 2815 and 2816.Use recess 2817 to assist to proofread and correct the position of microfluidic device in Other Instruments, for example, the instrument Figure 22 D that is used to operate or analyze microfluidic device or carry out reaction therein further describes the chamber for hydrating 2850 around elastic material block zone 2807, described chamber for hydrating can use hydration lid 2851 to cover to form moistening chamber, to help the control to the elastic material block ambient humidity, light and temperature.Being added into chamber for hydrating 2851 by for example volatile liquid of water can increase humidity, preferably passes through sorbing material or sponge humidification.Can preferably use polyvinyl alcohol (polyvinyl alcohol).The ratio that is preferred for the polyethylene alcohol and water of humidification sorbing material or sponge by change can obtain humidity control.Can also control hydration by the moisture control unit that uses for example moistening encapsulation of HUMIDIPAKTM, described moisture control unit for example uses permeable and sealing bag liquid non-permeate of steam to hold to have the salting liquid of the salinity that is suitable for keeping the ideal humidity value.Referring to the US patent No.6244432 of Saari etc., be used to by reference here to comprise that all purposes of specific purpose of moisture control unit and method are combined.Hydration lid 2850 is preferably transparent, with in order not cover visual situation during use in elastic material block.Similarly, the part of the substrate 2800 below elastic material block zone 2807 is preferably transparent, but also fuzzy or reflect.

Figure 22 E has described the cross-sectional view of substrate 2800, has the elastic material block 808 that is positioned in elastic material block zone 2807 along sealant 2881, and described sealant 2800 is attached to the side of substrate 2800 on the opposite of elastic material block 2808.Well (well) 2805 is in during elastic material block 2808 fluids are communicated with, by first port 2890, passage 2870 and second port 2892 and then enter the groove of elastomeric layer 2808, described groove by last end face 2897 sealings of substrate 2800 to form passage 2885.Sealant 2881 forms passage 2870 by groove molded or that be manufactured into substrate 2800 basal surfaces 2898.Sealant 2881 is transparent material preferably, for example polystyrene, Merlon or polypropylene.In one embodiment, sealant 2881 is flexible, splicing tape for example, and can be attached to substrate 2800 by bonding, for example use bonding agent or heated sealant or mechanical attachment, for example by compression.The material that is used for sealant 2881 preferably is easy to form the fluid sealing that has groove, the fluid passage that has minimum leakage with formation.Sealant 2881 can be further by the additional support layer (not shown) supporting that is rigidity.In another embodiment, sealant 2881 is rigidity.

The closed details of fluid has been showed interface between the elastic material block 2808 of substrate 2800 and the elastic material block zone 2807.Described at Grossman wait unexamined patent application US11/043395, wherein be used for here all purposes of stating with there scanning and combined, and be used for disclosing about the purpose of integrated carrier with the further details of the use of automatic starting device, can form elastic material block by the multilayer elastic material that is combined together to form elastic material block.Preferably, first elastomeric layer with the groove that is formed on the there is bonded to second material layer with the groove that is formed on the there, is formed on the elastic material block of groove there with formation.The groove of first elastomeric layer is blocked in whole or in part, to be formed on the passage in first elastomeric layer.When elastic layer was bonded to substrate, the groove that is formed in second material layer was similarly blocked in whole or in part, to form the passage in second elastomeric layer, had the microfluidic device that is formed on the multilayer that wherein has passage thereby form.

In Figure 23, first elastomeric layer 2920 and second elastomeric layer 2923 are bonded in the elastic material block that has passage 2907 (being formed by first groove 2901 and second elastomeric layer, 2923 end faces) together with formation, described first elastomeric layer 2920 has and is formed on the basal surface that wherein has first groove 2901, and described second elastomeric layer 2923 has top surface that has second groove 2905 and the basal surface that is formed on wherein.Substrate 2800 is attached to the basal surface of second elastomeric layer 2923, to form passage 2909 by the top surface 2897 of substrate 2800 and the basal surface of second elastomeric layer 2923.Port 2892 can use the passage 2909 of second elastomeric layer to connect the passage 2872 of substrate 2800, and passage 2909 parts form by the top surface of substrate 2800.Replacedly as shown in Figure 23, via through hole 2950, port 2892 uses the passage 2907 of first elastomeric layer 2920 of elastic material block 2808 to connect the passage 2872 of substrate 2800.Through hole 2950 approximately is formed perpendicular to substrate surface 2897, is preferably formed in second elastomeric layer 2923, before itself and elastomeric layer 2920 are bonding, and more specifically after first and second elastic layers are bonded in together.Referring to unexamined and the commonly assigned US temporary patent application No.60/577715 that is proposed on March 29th, 2004 by Unger, it is combined via constituting the special-purpose purpose of using automatic laser ablation system and method by being used for all purposes and instruction.The exemplary method that is used to make through hole comprises that little manufacturing forms second elastomeric layer 2923 simultaneously, CO is specially controlled, used to laser ₂The laser drill of laser instrument, use laser drill, the machine drilling of excimer laser and get core, preferably wherein use the robot hole-drilling system to carry out boring, preferably have x, the hole-drilling system in automatic stage of y.

Figure 23 describes another cross-sectional view that preferably uses of through hole, and described through hole is arranged in microfluidic device described here.Microfluid piece 2921 comprises and has the ground floor groove that is formed on wherein the ground floor 2920 of when being bonded to the second layer (or passage) 2907 and has the ground floor 2923 of therein second layer groove when being bonded to substrate (or passage) 2950.Two second layer passages are in the fluid connection of passing the ground floor passage, use two or more through holes 2950.Preferably, at least one through hole 2950 is in during hole 2999 other fluids in the substrate 2800 are communicated with, and passes base groove 2892 (perhaps passage, if the sealant (not shown) is incorporated in to substrate 2800).At least one second layer passage 2909 is overlapping by ground floor passage 2907, is not in the fluid connection.In the embodiment shown in Figure 23 because the fluid passage in one deck can be on the fluid passage between two parties in one deck or under advance, so obtain the reaction of the higher density of every cellar area in the microfluidic device and/or detect band.The fragment of melting chamber 2989 is arranged, be used to collect the fragment that produces by laser ablation through hole 2950.Use two-layer casting method fragment chamber 2989 can be cast in the layer 2920, wherein after the first photoresist layer is patterned and develops, the second photoresist layer is superimposed on first pattern, and second pattern is formed on the pattern of the first photoresist layer, can be differing heights so that make the photoresist area of the pattern.On another, can construct multilayer for one, with the pattern of the height that changes.Different photo anti-corrosion agent materials can also be used, so that for example can flow when being heated again in the photoresist upper strata, yet lower floor is by go out the photo anti-corrosion agent material manufacturing that can not flow again basically in identical heating-up temperature.

Flow channel of the present invention depends on that the application that they are wanted selectively is designed to have the varying cross-section size and dimension, presents different advantages.For example the shape of cross section of current downflow passage can have curved upper surface, perhaps along its whole length or be in the zone that is arranged on below the transversal passage.This curved upper surface helps valve seal, as following.That membrane thicknesses section of realizing by the present invention and flow channel cross comprise is rectangle, trapezoidal, circular, oval-shaped, paraboloidal, hyperbola with polygonal, also have the upper shape part.More complicated shape of cross section for example has the embodiment of bump (protrusion) or the embodiment that has depression in flow channel, also uses the present invention to be implemented.

In addition, although the present invention main in conjunction with flow channel wherein wall and ceiling is made of elastomer and the ground of flow channel is described by the embodiment that following substrate constitutes, the invention is not restricted to this concrete location.The wall of passage and ground also may be formed in the following substrate, have only ceiling in the flow channel by elastomeric construction.The exciting force that applies of response, this elastomer flow channel ceiling will give prominence to admission passage downwards, thus material mobile of flow channel passed in control.Usually, the monolithic elastomer structure is preferred for microfluidic applications.Although what come in handy is, adopt the passage that is formed in the substrate, wherein this structure has a plurality of advantages.For example, comprise that the substrate of fiber waveguide can be configured, so that fiber waveguide guides to light the side of microfluidic device specially.

Microfluidic device of the present invention can be used as independent equipment, or preferably can be used as by the parts in the system provided by the present invention.Figure 24 has described to be used to start the robotic station's of microfluidic device perspective view.Automatically pneumatic control and accumulator loading terminal 3200 comprise and accept gulf (bay) 3203, are used to keep microfluidic device 3205 of the present invention, for example the type of being described in Figure 22 A-22E.Platen 3207 is suitable for contacting top 3209 of microfluidic device 3205.Platen 3207 has therein in the port of microfluidic device 3205 alignment, provides hole and accumulator to the microfluidic device 3205 with the fluid pressure with preferred gas pressure.In one embodiment, the motion of pressure case 3207 by arm 3211 is pushed above microfluidic device 3,205 3221, described arm 3211 is suspended on the pivot 3213 and by piston 3215 and is energized, and described piston 3215 at one end is connected to 3211 and be connected to platform 3217 at the other end.Detect piston along the sensor of piston 3215 and move and will go to controller in the information about piston position, preferably the controller under the computer (not shown) control of following software scripts (software script).Platen detector 3219 detects microfluidic devices 3205 in the existence that receives 3203 inboards, gulf, preferably can detect into the fluid clavicle 3205 correct location.For example make by the side of light being departed from microfluidic device 3205 and use up existence and the position of detecting microfluidic device 3205, this may realize.Platen 3207 can robotize ground, be lowered by pneumatically, electrically or the like.In certain embodiments, platen 3207 is manually descended with bonding apparatus 3205.

In one embodiment, the streamline of sensing platen 3207 is positioned in the arm 3211 and is connected to fluid pressure source, preferably the automatic pneumatic pressure source under controller control.Pressure source will be controlled the interior port of platen surface (not shown) that fluid pressure offers platen 3207, and the controlled compression fluid is supplied to microfluidic device 3205.By adopting platen 3207 to be connected to the universal joint (gimbal joint) 3223 of arm 3211, realize microposition at least in part, so that platen 3207 can be around the axle gimbal of vertical microfluidic device 3205 upper surfaces 3221 to platen 3207.

Figure 25 has described the side view of cutting open of platen 3207, and described platen 3207 is pushed to the upper surface 3221 of microfluidic device 3205.Platen 3207 is pushed to microfluidic device 3205 upper surfaces 3221, to be formed between microfluidic device 3205 and the platen face 3227 or the fluid sealing between 3227 of equipment 32056 parts and surface.In one embodiment, platen face 3227 comprises or by the compliant materials manufacturing, elasticity elastomer for example, preferred durable rubber etc.In the platen 3207 the separation of the fluid pressure line, the preferred gas pressure line, each position is complementary on the upper surface 3221 of itself and microfluidic device 3205.What also illustrate is that check-valves (check valve) purifies actuator 3233, described check-valves purification actuator 3233 preferably pneumatically is energized and when being energized pin 3231 is pushed check-valves 2812 downwards, to open and to alleviate fluid pressure, perhaps open of the introducing of resistance permission fluid by check-valves 2718 by overcoming it.In one embodiment, platen 3207 has first and second and purifies actuator 3233, and itself and check-valves 2811 and 2812 mesh (seeing Figure 22 B).

In another embodiment, chip or equipment 3205 are manufactured with normally closed container and/or boundary valve.In this embodiment, accumulator needn't keep valve to turn-off in the training period.At interface and/or container value is ideal when being opened, with force applications to carrier or equipment 3205 bore regions.Be used for whole or most of other times, valve will remain closed so that each chip test is disconnected from each other, and/or reagent on the chip and protein hole is disconnected from each other.

Figure 25 has described the cutaway view of platen 3207, and described platen 3207 is pushed to the upper surface 3221 of microfluidic device 3205, wherein by platen surface 3227 contact in the spine 3250 of upper surface 3221 is being formed on pressure chamber 3255 above the well array 2806.Then, fluid pressure preferred gas pressure is applied to pressure chamber 3255, by fluid is imported cavity 3255 from the pressure line of the arm 3211 of tracing back to loading terminal (chargingstation) 3200.Adjust pressure by the pressure regulator relevant, preferably can change the electric controlled variable pressure regulator of output pressure, preferably under computer control by the signal that sends according to the loading terminal controller with loading terminal 3200.Fluid pressure in the pressure cavity 3255 next fluids in the drive hole 2805 passes passage in the substrate 2800, and then enter in the passage and/or cavity of elastic material block 2808, with the deflectionable part of filling channel or cavity or excitation elastic material block 2808, but preferred foregoing deflection barrier film valve.

Figure 27 A and 27B have described the specific embodiment of system 3500, more specifically, have described the specific embodiment of interface plate 3520.In Figure 27 A, interface plate 3520 is coupled to integrated chip and carrier 3400, seals the mode in its a certain zone with fluid.Particularly, fluid sealing is set in demarcation strip 3520 and carrier 3400 and the chip between one or more zones, for example the first protein zone 3430, the second protein zone 3432, first well area 3420, second well area, 3422 boundary accumulators 3460, check-valves 3465, container accumulator 3450 and/or check-valves 3455.In one embodiment, demarcation strip 3520 offers zone 3420,3422,3430,3432 with the fluid sealing, and offers accumulator 3450 and 3460.In one embodiment, demarcation strip 3520 provides one or more check valve actuator 3570, sees as best in Figure 27 B.

In certain embodiments, the demarcation strip 3520 fluid sealing that all are desirable offers carrier 3400 and microfluidic device.When doing like this, demarcation strip 3520 can comprise seal washer 3580.Seal washer 3580 can comprise material widely, includes but not limited to silica gel, elastomer etc.In certain embodiments, packing ring 3580 comprises compliant materials, seals to help going out to form fluid in the position of wanting.By this way, system 3500 can offer the pressure of wanting the appropriate area in chip and the carrier 3400.In other embodiments, demarcation strip 3520 is two or more plate member.For example, each can be coupled to separating plate 3520 by fluid zone on carrier 3400 and microfluidic device or port, and described separating plate 3520 is suitable for assembling described port or zone.System 3500 will comprise the demarcation strip 3520 of necessary amount then, be used for each port or zone.In addition, in certain embodiments, be coupled to concrete demarcation strip 3520, and all the other zones or port are coupled to separation demarcation strip 3520 more than a zone or port.Other combination of demarcation strip and carrier/chip area and port also falls within the scope of the present invention.

In one embodiment, the operation of system 3500 comprises a carrier 3400 is loaded in the receiving station 3510.In certain embodiments, carrier 3400 comprises the microfluidic device that is coupled to the there, and reagent and the protein that will want before carrier is placed into receiving station 3510 are loaded into carrier hole.In other embodiments, carrier 3400 is placed into receiving station 3510, uses reagent and protein to load subsequently.Carrier 3400 further can be mounted with the hydration fluid.The hydration fluid can be placed in the chamber for hydrating 3440.After carrier 3400 was loaded into system 3500, demarcation strip 3520 was lowered or is transferred on the contrary in conjunction with carrier 3400.Plate 3520 can be manually, robot ground or be lowered on the contrary to use down the part or all of fluid sealing of sheet/carrier 3400.The hydration fluid is provided for boundary accumulator 3460 and/or container accumulator 3450, and the pressure source that is coupled to demarcation strip 3520 by use is applied to accumulator 3450,3460 with convenient pressure and drives in chip.In specific embodiment, system 3500 automatically performs this technology, and described technology took place in about 20 (20) hours after the hydration fluid is added into carrier 3400 in specific embodiment.The result is to use the hydration fluid that chip is fully loaded, with operation chip container and/or dividing valve, as reaching the described more fully and previous application of combination by reference here of this patent here.

By desirable pressure being imposed on appropriate seal chip area, will comprise that the solution of reagent and sample is distributed into chip around suitable inlet.For example, will impose on first and second well area 3420 and 3422, and make driving reagent enter chip at the pressure between about 1psi (pound/square inch) and the about 35psi.Similarly, will impose on first and second well area 3420 and 3422, and make kinesin matter enter chip at the pressure between about 1psi (pound/square inch) and the about 35psi.In specific embodiment, this took place within about 60 (60) minutes after chip loads the hydration fluid.In case solution is driven the hole of wanting, chamber to the chip, accumulated storehouse or the like, open boundary valve in the chip by discharging check-valves 3465 in the boundary accumulator 3460.In specific embodiment, check-valves 3465 is released, and to open the dividing valve in the chip, system's 3500 excitations this moment are in conjunction with the check valve actuator 3570 of check-valves 3465.In certain embodiments, check valve actuator 3570 comprises the pin that is suitable in conjunction with check-valves 3465, binding post etc., to discharge the pressure in the boundary accumulator 3460.In alternative embodiment, check-valves 3465 is by manual release or open.

After making the solution mixed ideal time, use for example conventional mixing or repeat to open and close dividing valve to increase dispersion or other technology that fluid moves, closed dividing valve in the chamber.Pressure is imposed on actuator 3450 and/or 3460, with boundary valve and the container valve that remains closed.Carrier 3400 can be removed from system 3500, is used for described technology here and cultivates or store or use.Actuator 3450 and 3460 makes pressure continue the time of wanting, from a few hours to a couple of days, to stop or to help to stop container or dividing valve to open.In specific embodiment, actuator 3450 and 3460 keeps the chip internal pressure on desirable threshold pressure, is enough to keep container and/or dividing valve closure.In one embodiment, actuator 3450 and 3460 keep-ups pressure and continued on threshold pressure at least two (2) days, at least seven (7) days etc.Actuator 3450 and 3460 keeps the long cultivation temperature that depends in part on of the time of desired pressure.Depend in part on the long and/or condition of culture of desirable cultivation period, carrier 3400 can come and go to system 3500, in order to repeat charging or repeated compression actuator 3450,3460.By this way, cultivation period can

In specific embodiment, integrated carrier 3400 and microfluidic device are suitable for carrying out the experiment of wanting according to the embodiment of the invention, the system of the application of the invention.More specifically, as shown in Figure 26 A, system 3500 comprises one or more receiving stations 3510, and each is suitable for received vector 3400.In specific embodiment, system 3500 comprises four (4) receiving stations 3510, although provide still less in the embodiment of selection of the present invention or the station 3510 of greater number.Figure 26 B is depicted in carrier 3400 and the equipment that is combined and is provided with in the station 3510, is in below the demarcation strip 3520.In Figure 26 B, demarcation strip 3520 is suitable for downward transmission, so that demarcation strip 3520 is in conjunction with the upper surface and the microfluidic device thereof of carrier 3400.In certain embodiments, stand 3510 and platen 3520 be similar to station 3200 and platen 3207.Demarcation strip 3520 comprises one or more ports 3525, is used for being coupled with the zone that is suitable for holding at carrier 3400 fluid, pressure etc.In certain embodiments, demarcation strip 3520 comprises two-port, three ports, four ports, five-port, six ports, seven ports, eight ports, nine ports, ten ports etc.In a preferred embodiment, demarcation strip 3520 is coupled to and is used for providing two lines wanting six lines in zone and be used to provide the machinery of excitation check-valves 3455 and 3465 to carrier 3400 with pressure.

Shown in Figure 26 A, system 3500 further comprises processor, and described in one embodiment processor is the processor that is associated with notebook or other computer equipment 3530.Computer equipment 3530 comprises the memory that is suitable for preserving the software that is used to carry out desirable technology of the present invention, scheme etc.In addition, computer equipment 3530 comprises the research and analysis result's who is used to present microfluidic device screen 3540, and in one embodiment, system 3500 uses the GUI displays.System 3500 is coupled to one or more pressure sources, and for example pressure fluid, gas etc. are used for the same microfluidic device that passes to, and described microfluidic device fluidly is coupled to demarcation strip 3520.

Figure 27 A and Figure 27 B have described the specific embodiment of system 3500, more specifically, have described demarcation strip 3520.In Figure 27 A, demarcation strip 3520 is coupled to integrated chip and carrier 3400, seals the wherein mode in a certain zone with fluid.Particularly, fluid seals between the one or more zones that are set in demarcation strip 3520 and carrier 3400 and the chip, for example the first protein zone 3430, the second protein zone 3432, first well area 3420, second well area 3422, boundary accumulator 3460, check-valves 3465, container accumulator 3450 and/or check-valves 3455.In one embodiment, demarcation strip 3520 offers zone 3420,3422,3430,3432 with the fluid sealing, and offers accumulator 3450 and 3460.In one embodiment, demarcation strip 3520 provides one or more check-valves accumulations 3570, sees as best in Figure 27 B.

In certain embodiments, demarcation strip 3520 is sealed to carrier 3400 and microfluidic device with whole perfect fluids.Do like this, demarcation strip 3520 can comprise seal washer 3580.Seal washer 3580 can comprise material widely, includes but not limited to silica gel, elastomer etc.In certain embodiments, packing ring 3580 comprises compliant materials, seals to help going out to form fluid in the position of wanting.By this way, system 3500 can offer the pressure of wanting the appropriate area in chip and the carrier 3400.In other embodiments, demarcation strip 3520 is two or more plate member.For example, each can be coupled to separating plate 3520 by fluid zone on carrier 3400 and microfluidic device or port, and described separating plate 3520 is suitable for assembling described port or zone.System 3500 will comprise the demarcation strip 3520 of necessary amount then, be used for each port or zone.In addition, in certain embodiments, be coupled to concrete demarcation strip 3520, and all the other zones or port are coupled to separation demarcation strip 3520 more than a zone or port.Other combination of demarcation strip and carrier/chip area and port also falls within the scope of the present invention.

In one embodiment, the operation of system 3500 comprises one or more carriers 3400 is loaded in the receiving station 3510.In certain embodiments, carrier 3400 comprises the microfluidic device that is coupled to the there, and reagent and the protein that will want before carrier is placed into receiving station 3510 are loaded into carrier hole.In other embodiments, carrier 3400 is placed into receiving station 3510, is mounted with reagent and protein subsequently.Carrier 3400 further can be mounted with the hydration fluid.The hydration fluid can be placed in the chamber for hydrating 3440.After carrier 3400 was loaded into system 3500, demarcation strip 3520 was lowered or is transferred on the contrary with in conjunction with carrier 3400.Plate 3520 can be manually, robot ground or be lowered on the contrary with the part or all of fluid sealing of following sheet/carrier 3400.The hydration fluid is provided for boundary accumulator 3460 and/or container accumulator 3450, and the pressure source that is coupled to demarcation strip 3520 by use is applied to accumulator 3450,3460 with convenient pressure and drives in chip.In specific embodiment, system 3500 automatically performs this technology, takes place in about 20 (20) hours after the hydration fluid is added into carrier 3400 in technology described in the specific embodiment.The result is that chip is fully loaded the hydration fluid, with operation chip container and/or dividing valve, as reaching the described more fully and previous application of combination by reference here of this patent here.

By desirable pressure being imposed on, will comprise that the solution of reagent and sample scatters into chip around the chip area of the appropriate seal of suitable inlet.For example, will impose on first and second well area 3420 and 3422, and drive reagent and enter chip at the pressure between about 1psi (pound/square inch) and the about 35psi.Similarly, will impose on first and second well area 3420,3422 at the pressure between about 1psi and the about 35psi, kinesin matter enters chip.In specific embodiment, this takes place within about 60 (60) minutes after chip loads the hydration fluid.In case solution is driven to the well of wanting, chamber in the chip, accumulates storehouse or the like, open boundary valve in the chip by discharging check-valves 3465 in the boundary accumulator 3460.In specific embodiment, check-valves 3465 is released, and to open the dividing valve in the chip, system's 3500 excitations this moment are in conjunction with the check valve actuator 3570 of check-valves 3465.In certain embodiments, in order to discharge the pressure in the boundary accumulator 3460, check valve actuator 3570 comprises the pin that is suitable in conjunction with check-valves 3465, binding post etc.In alternative embodiment, check-valves 3465 is by manual release or open.

After making the solution mixed ideal time, use for example conventional mixing or repeat to open and close dividing valve to increase dispersion or other technology that fluid moves, closed dividing valve in the chamber.Pressure is imposed on actuator 3450 and/or 3460, with boundary valve and the container valve that remains closed.Carrier 3400 can be removed from system 3500, is used for described technology here and cultivates or store or use.Actuator 3450 and 3460 makes pressure continue the time of wanting, from a few hours to a couple of days, to stop or to help to stop container or dividing valve to open.In specific embodiment, actuator 3450 and 3460 keeps the chip internal pressure on desirable threshold pressure, is enough to keep container and/or dividing valve closure.In one embodiment, actuator 3450 and 3460 keep-ups pressure and continued on threshold pressure at least two (2) days, at least seven (7) days etc.Actuator 3450 and 3460 keeps the time span of desired pressure to depend in part on cultivation temperature.Depend in part on desirable cultivation period length and/or condition of culture, carrier 3400 can come and go to system 3500, in order to repeat charging or repeated compression actuator 3450,3460.

In certain embodiments, described integrated chip carrier (ICC) and the elastomer chip that is attached to the there are used as here, to promote polymerase chain reaction (PCR).Yet when using the PCR sheet to carry out PCR, the pyroconductivity of plastics ICC may be not enough to produce uniform heat-field in hidden reaction array in the elasticity chip.For example, although the ICC described in Figure 28 A is useful to the crystallization of protein in other technology, can not heat transferred be coupled to chip there full and uniformly.In addition, the operation of the ICC among Figure 28 A may require its reservation is constrained on the platen.As shown in Figure 29 A, the application of convergent force can have a negative impact to chip.Therefore, in some embodiments of the invention, use and provide improved uniform heat-field, and improved ICC reservation method is depicted among Figure 29 B in conjunction with the described ICC of Figure 28 B.

In certain embodiments, the elasticity chip is designed, so that be connected the outer edge that is positioned in the elasticity chip with the fluid of ICC.By this way, ICC part needn't since in the fluid transmission.A kind of such embodiment is described among Figure 28 A.In certain embodiments, not with the contacted elasticity chip in ICC surface in the zone be positioned at the place that the reaction chamber array is positioned.This conversion zone 3810 comprises elasticity chip and Heat Conduction Material, and elasticity chip and Heat Conduction Material are mated the downside (additionally, the elastomer block side will contact the parts of plastics of ICC) at chip.The size of conversion zone 3810 and shape can change within the scope of the invention, and an embodiment described in Figure 29 C has shown the relation between ICC and the chip.

By this way, have the thermal impedance of minimum or reduction, heat energy (for example, from PCR equipment) can be passed to elastomer block.In certain embodiments, Heat Conduction Material comprises silicon (Si).In specific embodiment, use silicon, with employed similar or identical in the semi-conductor industry from polishing and level and smooth silicon wafer.Can also use other material of low thermal impedance within the scope of the invention, depend on the attribute of the heating curve of looking for.In certain embodiments, Heat Conduction Material has (just, influencing the material that temperature changes fast, even good thermal conductor, for example copper) low in calories.In certain embodiments, use polished silicon, strengthening reflection effect and to increase the quantity of the light that can collect by the detector that uses in the system, or in real time or as the PCR reaction in the distal point analysis.These benefits also can be improved adiabatic reaction.

Use ICC3800, a kind of cleaning of carrying out PCR can realize, is complementary by conversion zone and the thermal control source that makes elastomer block.The thermal control source can comprise together with other PCR equipment, the platen that is heated, separation thermal source together.In certain embodiments, ICC is fitted into the Standard PC R equipment that receives flat reaction tray and/or has flat hot pressing dish, wherein is used for can being used based on the adapter of the various standards of the PCR of pipe.Yet each these structures depend on dish usually to lower compression, to obtain the thermo-contact of good (evenly), as shown in Figure 29 A.In some embodiments of the invention, elastic material sheet subtend lower compression can not be revised, because the deformable and cause undesired fluid behavior in flexure strip of flexible valve and flexible cavity.

In other embodiments, be bonded on other hot material that exposes the elastomer block downside with coupling, reducing or avoided adverse effect to lower compression to ICC by using vacuum chuck (vacuum chuck).By this way, when vacuum is applied to chuck, between vacuum chuck and ICC hot material, produce tight seal.In an embodiment as shown in Figure 29 B, vacuum chuck 3950 comprises or by being one or more material manufacturings of good conductor of heat, described good conductor of heat is incorporated in to the thermal control source, for example one or more Peltier (amber ear card) device.In one embodiment, use a plurality of thermal controls to be created in the hot tonsure of reacting in the array.

In the use, ICC is positioned in vacuum chuck 3950 tops, and reduces downwards or be transferred, so that the hot part 3920 among integrated chip 3910 and the ICC3930 contacts with vacuum chuck 3950.Moreover in one embodiment, hot part 3920 comprises silicon.Hot part 3920 is described to be coupled to elastomer block or sheet 3910, uses real-time this couplings such as binding agent in certain embodiments.As shown, piece 3910 is in conjunction with ICC3930, and in one embodiment, gap 3940 is maintained between hot part 3920 and the ICC3930.Gap 3940 is owing to isolate ICC3930 with the heat of the thermal source of for example chuck or platen 3950.In addition, in one embodiment, gap 3940 allows some warpages of piece 3910 and/or hot part 3920.By this way, gap 3940 in these embodiments helps to form sealing when chuck 3950 is applied vacuum, by one or more vacuum ports 3960 so that hot part 3920 is pulled to chuck 3950.Can monitor the vacuum quantity that obtains, between chuck 3950 and hot part 3920, need the good thermo-contact made from variation veritably.In certain embodiments, the one or more ports in the vacuum chuck are used and are used to monitor vacuum.In the specific embodiment shown in Figure 29 D and Figure 30, the one or more ports that are used to monitor vacuum will be positioned in the far-end of vacuum ports and by the path fluid communication with each other, the preferred tortuous and narrow path of described path.By this way, between the heat part among one side of vacuum chuck-ICC difference can with heat part among the opposite side-ICC of vacuum chuck between compare, can solve vacuum loss problem (if having) to determine the effort that on vacuum chuck, reapposes ICC.Folding can be the iterative processing of automatic or manual or some of them combination.

Figure 30 describes an embodiment according to chuck of the present invention.Can change the attribute of zigzag channel within the scope of the present invention, the attribute of described zigzag channel is used to obtain the accurate measurement to different vacuums on the hot subregion of vacuum chuck, and promotes vacuum evenly or relatively equably using ICC heat part.By using this scheme, the elastomer block that is used to carry out the PCR reaction can be in the good of PCR equipment and contact, if will use usually to lower compression, then not to elasticity (deflectable) function hazard partly of elastomer block.

Although the present invention here is described with reference to wherein specific embodiment, during but change on a large scale, various variation and alternative quilt are meaned and are disclosed in front, and will be understood that: in some instances, not breaking away from the described scope of the invention does not have the purposes of corresponding further feature, and features more of the present invention will be used.For example, except the above-mentioned brakes based on pressure, optionally the moving system of static and mangneto also is implemented.Also possible is, encourages described equipment, produce fluid in the control channel and flow by being applied in based on heat energy, or by thermal expansion or by the gas generation from liquid.In addition, in another embodiment, use centrifugal force that protein and reagent driving are entered chip.Therefore, do not break away from true scope of the present invention and aim and can make the many improvement that are suitable for concrete situation or material at the present invention's instruction.Its purpose is, the invention is not restricted to be used to carry out specific embodiments of the invention as the specific embodiment is disclosed, but the present invention will comprise all embodiment and the equivalent that falls in the claim scope.

Quote:

1.Unger etc., Science 288,113-116 (2000)

2. be fully ventilative by " blind filling " sample loading passage and control line-PDMS, drive gas out of passage, remaining complete by the liquid filling they with the liquid of several pound/square inches (psi) compression.Referring to Hansen etc., PNAS 99,16531-16536 (2002)

3. the 294bp fragment of people β actin gene is used 5 ' exonuclease analysis (Taqman) amplification.Forward direction and reverse primer sequence be 5 respectively '-TCACCACACTGTGCCCATCTACGA-3 ' and 5 '-CAGCGGAACCGCTCATTGCCAATGG-3 '.Fig. 1 b is to use the FRET probe based on TAMRA, sequence 5 '-(FAM) ATGCCC-X (TAMRA)-CCCCCATGCCATCCTGCGTp-3 ' takes.Data among Fig. 1 c are to use based on the probe of black-quencher and take, because a large amount of these primer probe sets begin and can commerce provide.Reaction comprises 1 * Taqman buffer A (50nMKCL, 10nM Tris-Hcl, 0.01MEDTA, 60nM Passive Reference 1, pH 8.3), 4mM MgCl2,200nMdATP, dCTP, dTTP, 400nMdUTP, 300nM forward direction primer, the reverse primer of 300nM, the 200nM probe, and 0.01U/ μ lAmperase UNG (all from Applied Biosystems, the Foster city, CA), 0.2U/ μ lDyNAzyme (Calbiochem, San Diego, CA), 5.0% glycerine, deionized water and people's male gene group DNA (Promega).

4. quantitative PCR technique.About " gene quantification " chapters and sections, LJ McBride, K Livak, M Lucero, etc., editor, Francois Ferre, Birkauser, Boston, MA, p97-110,1998.

See E.T.Lafally, I.Medntz, R.A.Mathies, Anal Chem73 (3), 565-570 (2001), and B.Vogelstein, K.W.Kinzler, PNAS96,9236-9241 (1999).

Be understood that, here shown in example and embodiment only be used for illustration purpose, wherein various changes of Chan Shuing or change and will give those skilled in the art suggestion, and being comprised in the scope of the application's aim and clause and additional claims.Here whole publication, patents and patent applications of enumerating are used for all purposes and combined by using in full with it, and on same degree, indication is the same specially and individually with each single publication, patents and patent applications quilt, and is combined by using.

Claims

1. microfluidic device, it comprises:

A) ground floor (2110) and the second layer (2120);

B) reaction member (2160) in the second layer (2120), described reaction member comprises sample chamber (2180) and reagent chamber (2180), described sample chamber and described reagent chamber are in the mutual fluid connection by boundary passage (2150), described boundary passage has the dividing valve that both are associated with it (2140), is communicated with the fluid of controlling between described sample chamber and the described reagent chamber;

C) the sample fluid passage in the second layer, described sample fluid passage and the described sample chamber of described reaction member are in during fluid is communicated with; With

D) the reagent fluid passage in ground floor, the reagent fluid passage in the described ground floor is connected to reagent fluid passage in the second layer via through hole (2130), and the reagent fluid passage in the described second layer ends at the reagent chamber of reaction member.

2. microfluidic device described in claim 1, comprise a plurality of reaction members (2160), each reaction member is associated with reagent fluid passage in the ground floor that is connected to the reagent fluid passage in the second layer via through hole (2130), and the reagent fluid passage in the described second layer ends at the reagent chamber of reaction member.

3. the described microfluidic device of arbitrary as described above claim, wherein the part of the part of the described sample fluid passage in the described second layer and described reagent fluid passage is positioned and each has its both container valve that are associated approximately parallel to each other, and the fluid that is used to control by wherein is communicated with.

4. the microfluidic device in the claim 3, wherein said sample fluid channel container valve (2170) and described reagent fluid channel container valve (2170) operationally are in the connection mutually by common container control channel.

5. the microfluidic device described in claim 4, wherein said common container control channel is along approximately being positioned perpendicular to one of the described sample fluid passage in the second layer and described reagent fluid passage or both lines.

6. one kind is utilized the microfluidic device contact sample solution of one of claim 3-5 and the method for reagent solution, comprising:

A) sample solution is introduced in the sample chamber, and reagent solution is introduced in the reagent chamber, wherein, described sample solution is introduced into by sample channel, described reagent solution is introduced into by reagent passage, and, wherein, in introducing step, dividing valve is closed, and container value is opened; Then

B) closing containers valve and open dividing valve, thus sample solution and reagent solution are in contact with one another.