CN102778432A - Thermal reaction device and using method thereof - Google Patents

Abstract

The invention relates to a M*N matrix microfluid device used for executing a matrix reaction, comprising a plurality of reaction units communicated with one of a sampler inlet or a reagent inlet via a through hole. The through hole is formed on an elastic material block of the device. A method of parallelly forming the through hole in an elastic material layer of the elastic material block of microfluid device is provided and comprises the steps of using a patterned photoresist mask and an etching agent to etch the area or a part of area in the elastic material layer of the elastic material block.

Description

Thermal reaction device and use its method

The application is that the application number of submitting on May 2nd, 2005 is 200580022583.7 (PCT/US2005/015352; 201010154616.6), denomination of invention divides an application for female case application of " thermal reaction device and use its method ".

Prioity claim

The application requires the right of priority of applying for below each: 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 on grand scale, implementing 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 advantages that further cost reduces and reduce operator's error because of artificial interference 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 the microfluidic device of having been made up to now.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 made.In addition, the fluid transport in many existing microfluidic devices requires with the adjusting to complicated electric field of the controllable way of using said 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 it is the importance in the biological chemistry in modern times, 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 possibly be used as analysis tool, to confirm in sample, whether to exist or lack interested objectives nucleic acid.Therefore, this equipment possibly be utilized the existence of the concrete pathogen of test (for example, virus, bacterium or fungi), 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 possibly be used to implement gene type analysis and gene expression analysis (for example, different genes expression study).Replacedly, can preparation method use this equipment be used for further analysis with the enough nucleic acid that increases, such as amplified production, cell type, dna fingerprint etc.In the range gene practical applications, 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 resilient material.

Concrete equipment is designed to implement thermal cycle reaction, and (for example, PCR), said equipment comprises one or more elastic valve, flows through the solution of equipment with adjustment.Therefore, the method for using this device designed to implement amplified reaction also is provided.

Some equipment comprise that blind influx moves passage, and the moving passage of said blind influx comprises the zone of playing the reflecting point effect.Concrete this equipment comprises the flow channel that is formed in the resilient 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.Said 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 point of crossing 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.Said equipment can further comprise a plurality of protection passages ideally, its be formed in the resilient material with overlapping flow channel and/or reflecting point in one or more.The protection passage is designed has perforation fluid stream therebetween, to reduce flow channel and the evaporation of reflecting point from said equipment.In addition, said 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 this device designed, 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 resilient 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 nucleotide.In miscellaneous equipment, reagent is nucleic acid-templated.

Various matrixes also are provided or based on the equipment of array.In these equipment some comprises: (i) more than first flow channel, and it is formed in the elastic substrates, (ii) more than second flow channel; It is formed in the elastic substrates, intersects 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 the solution in each reflecting point with isolated at the solution at another reflecting point place, and (iv) a plurality of protection passage; 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 temperature adjustment those (for example, thermal cycles of foranalysis of nucleic acids).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 resilient 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 to be 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 said 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 reaction mixture.Reaction in that one or more reflecting points are between first and second reagent is to be detected subsequently.

Another other method relates to provides microfluidic device, and said 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 said 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, in reflecting point, is heated 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 to be detected subsequently.

The other equipment that reduces the evaporation of fluid slave unit that is designed also is provided.Usually, this equipment comprises cavity, and said 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 through 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 through elastic body 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 complex of in the reflecting point of some microfluidic device, implementing foranalysis of nucleic acids also is provided.In below some these complex comprises one or more: reagent and the washing agent of retardance protein bound on resilient 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 the matrix design of cross-perpendicular and 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 planimetric map of example sidewalk for visually impaired people equipment.

Fig. 3 A is the planimetric map 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 planimetric map 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 point sample reagent in the 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 synoptic diagram 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.Said 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 (microtiter plate)), draw reagent, then through 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 intensity that is used for microfluidic device (chip) described in example 1 (the 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 to benchmark or wild-type allele CYP2D6*1.Allele 2 (Al-2) is the position control that is used for the FAM probe to 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 3 of Figure 13 and example.

Figure 15 is the synoptic 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 in chip, produces.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.

Par that Figure 19 describes to duplicate in every hole and positive polarity hole number chart relatively.

Figure 20 describes isothermal duplication configuration-SCORPION.

Figure 21 depicted example matrix microfluidic device planimetric map.

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

The stretch-out 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 planimetric map 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 in microfluidic device of the present invention, uses among some embodiment.

Figure 24 is the skeleton 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 cut-open 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 skeleton 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 matched on the carrier;

Figure 28 A is the skeleton view according to the integral carrier of the embodiment of the invention;

Figure 28 B be according to the present invention in the skeleton view of integral carrier 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.

Specify

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.Following reference offers the technician with the general definition of numerous terms used in the present invention: Singleton etc., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed.1994); THECAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walkered., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger etc. (eds.), Springer Verlag (1991); And Hale&Marham, THE HARPER COLLINS DICTIONARY 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 through its flowable circulation path.

If do not have specific indication, term " valve " to insert circulation passage and control channel in referring to therein and by the separated structure of elastomeric diaphragm, said 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 " be not commonly referred to as not with equipment on the reflecting point that is communicated with of other reflecting point fluids of existing.When being relevant to the sidewalk for visually impaired people and being used, isolating reflecting point is terminal zone, sidewalk for visually impaired people, and said 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 resilient material equipment, passes in and out to provide between outside port and one or more flow channel of fluid at equipment.Therefore, through hole can be used as sample and inputs or outputs, for example.

Term " elastic materials " and " resilient 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 polymkeric substance at temperature place between its glass transition temperature and the condensing temperature.Resilient material shows and elastic performance to stretch because polymer chain receives twisting motion easily to make main chain in response to acting force, presents previous shape there not being under the acting force main chain twine again.Usually, elastic body distortion when applying acting force, but when removing acting force, return back to its original shape then.Elasticity by resilient material is showed can be specialized by Young modulus.The disclosed here resilient material that is utilized in the microfluidic device typically has the Young 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 resilient material with the Young 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 conditions and possible adjuvant, have a large amount of maybe elastic body system, it can be used makes little valve of monolithic elasticity and pump.Material chosen typically depended on practiced 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 in the manufacturing of microfluidic device parts, can be used 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 at this by reference.

Term " nucleic acid ", " polynucleotide " and " oligonucleotide " are used the condensate form of the nucleotide 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 through methylating and/or gland (capping), and the unchanged form of polynucleotide.Especially; Term " nucleic acid ", " polynucleotide " and " oligonucleotide " comprise polydeoxyribonucleotide (comprising the 2-deoxy-D-ribose), polyribonucleotide (comprising D-ribose), are purine or the N-of pyrimidine base or any type polynucleotide of C-glucosides; And other polymkeric substance that comprise non-nucleotide main chain (nonnucleotidic backbone); For example polyamide (such as peptide nucleic acid (PNA)) with gather morpholino (polymorpholino) (can from Corvallis Oregon (Kang Walisi city Oregon) Anti-Virals company limited buy the same) polymkeric substance and other composition sequence specific nucleic acid polymkeric substance with Neugene; Suppose that polymkeric substance 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 can combine the target nucleic acid of complementary series, uses complementary base right usually, uses hydrogen bond to constitute usually, thereby forms duplex.Probe combines or hybridization " probe binding site ".Probe can indicate by detecting, to allow the detection easily of probe, particularly once probe hybridization to its complementary target.For example, the sign that is attached to probe can comprise various different sign the known in the art arbitrarily, and they can detect through chemistry or physical means.The suitable sign that can be attached to probe includes but not limited to that radioisotope, fluorophore, chromophore, quality indication, electron density particle, magnetic particle, spin indicate, molecule, electrochemical activity molecule, enzyme, accessory factor 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 nucleotide.Other probe is long at least 20,30 or 40 nucleotide.Another probe is longer slightly, at least 50,60,70,80,90 nucleotide long.Another probe is longer, at least 100,150,200 or more a plurality of nucleotide 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 nucleotide, and more typically between 10 to 30 nucleotide are long, changes.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 of the dna sequence dna that will be increased ' end, 3 ' " downstream primer " with the fill-in hybridization of 3 of the dna sequence dna that will be increased ' end.

" perfect complementation " primer has the sequence of the complete complementation on the whole length of primer, and does not have off resonance.Primer typically is perfect complementary to the part (subsequence) of target sequence." off resonance " refers to the nucleotide in the primer and is not complementary point with nucleotide 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 nucleotide 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 to all the other nucleic acid specially.Referring to M.Kanehisa, Nucleic Acids Res.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, fluorophore, chromophore, quality indication, electron density particle, magnetic particle, spin indicate, molecule, electrochemical activity molecule, enzyme, accessory factor, 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 the reagent of some inherent feature (for example, size, shape or color), said characteristic allow it not have conjugated to indicate and to be 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 and restriction fragment length polymorphic, variable number is connected repetition (VNTR ' s), hypervariable zone, microsatellite (minisatellite), dinucleotide repeat, tetranucleotide repeats, the insertion element of simple sequence repetition and 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 that in the group of selecting, the most frequently occurs is called as the wild type form sometimes.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 said polymorphic point is the position that changes between the allele sequence.Allelic highly conserved sequence (for example, in the member less than crowd 1/100 or 1/1000, changing) is arranged before or after this position usually.Mononucleotide is polymorphic usually owing to replacing another to produce at nucleotide 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 nucleotide about the insertion of reference allele and produce.

" reagent " broadly refer to any in reaction applied agents.Reagent can comprise self can be monitored single agents (for example, when it is heated monitored material) or both or the potpourri of more reagent.Reagent can be lived (for example, cell) or abiotic.The reagent exemplary that is used for nucleic acid amplification reaction comprises but is not limited to: damping fluid, metallic ion, polymerase, primer, template nucleic acid, nucleotide, mark, dyestuff, nuclease and the like.The reagent that is used for enzyme reaction comprises, for example, and substrate, accessory factor, connection enzyme, damping fluid, metallic ion, suppressant and catalyzer.Being used for reagent based on the reaction of cell comprises but is not limited to cell, cell-specific dyestuff and is bonded to the aglucon (for example, activator and antagonist) of cell receptor.

" aglucon " is meant any molecule, and for the molecule (that is, anti-aglucon) of this molecule another specificity of existence or non-specific binding to this aglucon, said combination is owing to the identification of anti-aglucon to the 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 combines concrete 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; Said 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 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, with the defined reaction lattice array at the place, point of crossing.Therefore, this device designed 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 horizontal flow passage, thereby allows the convertible sealing of the various flow channels in the matrix.Therefore, although sidewalk for visually impaired people equipment is designed under different situations, use the limited quantity sample to implement macromethod, yet matrix device is configured under the limited quantity situation, great amount of samples 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, flow channel, control channel, valve and/or the pump for example made by elastomeric material.In some instances, in fact, entire equipment is by the elastomeric material manufacturing.Therefore, this equipment obviously is different from form and function by the principal feature based on the conventional microfluidic device of the made of silicon.

Said Equipment Design makes them can be combined and be utilized with a large amount of different heating system.Therefore, said equipment is useful implementing to require in the temperature controlled various analysis.In addition, these microfluidic devices that in heat application, use can combine other design feature, being to minimize from the evaporation of the sample of reflecting point.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 resilient material that increases the said equipment of formation through said 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 possibly be suitable for realizing thermo-contact with microfluidic device.Wherein microfluidic device is by the base material of for example microslide 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 part that preferably reacts therein with the vacuum source of supplying with microfluidic device suction.Replacedly, can firm thermal transfer plate be bonded to microfluidic device, said 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 said equipment effectiveness here, especially their purposes in the temperature controlled any reaction of needs of being reflected at.Yet, should be understood that said equipment is not limited to these concrete application.Said 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 at least part by elastomeric material structure and use single or the soft lithography 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, both are combined with the full text that it is used for all purposes at this by reference for it) structure.Make in this way, microfluidic device can be designed, and the solution of said 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 the entering flow channel or from flow channel, withdraw, and through exciting force being affacted control channel control channel are associated with flow channel.Be deflected the degree that gets into flow channel or from flow channel, withdraw through the control barrier film, 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.

Here the equipment that is provided has combined 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 in temperature control reaction (for example, thermal cycle reaction), use this equipment, described more in detail as hereinafter then, elastomeric device typically is fixed to supporting and goes up (for example, microslide).Then, can the structure that obtain 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, can this equipment be placed on the thermal cycle plate of any amount.

Because this equipment uses various different detection systems can easily monitor the reaction of any position on microfluidic device in fact by being the transparent resilient material manufacturing of relative light.Yet great majority detect and typically to occur in reflecting point from being in (for example, in point of crossing that comprises flow channel or the zone at the cecum of flow channel).This equipment is also meaned by the fact of substantial transparent made: 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 said equipment or with said device separates but with detecting device with the region alignment of equipment to be detected, can implement to detect.

B. protect passage

In order to reduce the evaporation of sample and reagent here, protect passage can be formed in the said equipment in a large number from the elasticity microfluidic device that provides.Protection passage and the similar part of control channel are that typically they are formed in the elastomer layer, and said 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 resilient 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 (typically being water) flows into.Come from the spreadable elastic body aperture of going into adjacent flow channel or reflecting point of the water vapor 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 in this context, being used: with under the same conditions into and out of or compare through the flow of solution of flow channel or reflecting point; Into and out of or flow of solution through 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 through 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 betwixt any integral body 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 with condition under reduce from the sample of said equipment and evaporation of reagents extremely less than 50%, in other example less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%.Therefore, for example, comprising 40 round-robin typical case PCR reaction 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 betwixt arbitrary integer density value.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 appears as from the measured interval between 1mm to 1 μ m of the nearest outer edge of outer edge to adjacent lines of a line, perhaps betwixt arbitrary integer level of density.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 separate channels network, perhaps the smaller channels of branch's control channel.The said equipment of the protection extensible mistake of passage perhaps only has concrete zone or a plurality of zone of said equipment.Typically, the protection passage is close to and above flow channel and reflecting point, is provided with, and 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 specified hereinafter.

The solution that flows through the protection passage comprises the arbitrary substance that can reduce the water evaporation.Said 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 WS in fact, and suitable in this case solution includes but not limited to water and buffering solution (for example, TaqMan buffer solution and PBS).Suitable blocking agent comprises for example mineral oil.

The protection passage typically is formed in the elastic body MSL technology of quoting as proof above the utilization 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 to 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 horizontal flow passages; Said a plurality of vertical and horizontal flow passage intersects to form the tie point array each other; Because different samples and reagent (or reagent set) can be imported into each flow channel, so great amount of samples can be to be tested with the high-throughput form under considerable reaction conditions.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 horizontal flow passage.Said different ground makes the valve location only pass the perpendicular flow passage or only pass flowing of horizontal flow passage to allow selection.Because such equipment allows to be relevant to the elasticity of selection of type and quantity and the amount of reagent and the type of sample, so these equipment very well are suitable for implementing to analyze, people attempt under considerable reaction conditions, to screen great amount of samples therein.Matrix device preferably the join protection passage to help to stop sample and evaporation of reagents.

The present invention is provided for the high-density matrix design, and said 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 through 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 the reagent fluid passage in the second layer 2120 through 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 through interface channel 2150, and said 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 through 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; Said 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 through the interface channel, and said interface channel has related betwixt interface valve, are 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 through 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 said 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 through sample channel; And the reagent inlet is connected with reagent chamber through reagent passage; Sample channel part and reagent passage part be by location approximately in parallel with each other, and each has the container value of being mutually related, and is 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, through 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 characteristic in the elastic material block, and said method comprises step: first elastomeric layer is provided; On the first resilient 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 resilient material laminar surface; First elastomeric layer is applied etching reagent do not have the first resilient material laminar surface by the pattern covers of reacting photo anti-corrosion agent material with etching; Thereby remove the first resilient material layer region that does not have by the pattern covers of reacting photo anti-corrosion agent material, and remaining corresponding to the resilient material layer pattern that reacts the photo anti-corrosion agent material pattern.In the concrete preferred embodiment of this method, said method comprises that removal reacted the step of photo anti-corrosion agent material pattern; Through splicing tape being applied to elastomeric layer and having reacted the surface of photo anti-corrosion agent material pattern; Cause and implement to remove; Then from elastomeric layer separating adhesive band, simultaneously some or all reacted the photo anti-corrosion agent material pattern by surface removal from resilient material; 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 through through hole.

Microfluidic device of the present invention can further be integrated into vehicle equipment; Said 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 feeding (charge) like valve of the present invention described here and encouraging.Another preferred embodiment; Be used for having the equipment of platen to the accumulator charging with to the automatic system employing of valve excitation; Said platen coupling is on one or more surfaces of microfluidic device, and wherein platen has two or more at least mouthfuls that are communicated with to fluid in control vacuum or pressure source, and can comprise the mechanical part that is used to handle the microfluidic device part; For example, but be not limited to non-return valve.

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 characteristics: well (well) or accumulating tank, its with elastic material block through being formed in the carrier or at least one passage phase fluid of use carrier is communicated with; Accumulator and elastic material block are through 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, and wherein fluid flow port is preferably addressable for automatic vacuum or pressure source, for example above-mentioned automatic system; Wherein automatic source further comprises the platen of port; Said port and fluid flow port are complementary, and sealing fluid connects between the automatic system to form, and is used for hydrodynamic 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 made that is different from carrier another part in the zone, is selected for other parts that the region material of improving heat conduction and scattering parameter is different from carrier.Be used to improve heat conduction and include, but are not limited to silicon with the preferred material that scatters parameter, 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, the heat conduction of carrier and scattering portion are used for to the elastomer block carrier heat control through heat conduction and scattering portion being provided.In a preferred embodiment; Through 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 portion, attracts and the heat conduction of maintenance carrier and the position of scattering portion to apply.In a preferred embodiment; The heat conduction of carrier and scattering portion do not have reality to contact with carrier; But be associated with carrier and elastic material block; Through only heat conduction and scattering portion being bonded to elastic material block, and remaining in heat conduction and the scattering portion gap at edge, 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 possibly be substituted; Use the microfluidic device of any known 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; Said 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 said control channel 118 can be at single inlet 114 places by all excitations.

Similarly, going valve 108 regulates the liquid inventory in the horizontal directions; These are by 116 controls of control channel, and said control channel 116 is by 112 excitations of single control inlet.Such as Figure 1A demonstration, the control channel 116 of regulating row valve 108 depends on that the position changes on width.When control channel 116 cross-perpendicular flow channels 102, control channel 116 is enough narrow, so that it can not be deflected into perpendicular flow path 10 2 when it is energized, runs through liquid inventory wherein thereby reduce basically.Yet the width of control channel 116 is increased when its overlapping horizontal flow path 10 4; The flow of solution that this makes the diaphragm of control channel enough pass horizontal flow path 10 4 with obstruction greatly.

In operation, reagent R1-R7 is introduced into their horizontal flow path 10s 4 separately and is spurted into their perpendicular flow path 10s 2 separately with sample S1-S7.Therefore with at each perpendicular flow path 10 2 samples reagent in each horizontal flow path 10 4 mix intersecting 106 places, and said 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 horizontal flow path 10 4.The different IPs acid template is imported into perpendicular flow path 10 2.In some was analyzed, being imported into maybe be different between flow channel as the primer (primer) of reagent mixture part, and said reagent mixture is imported in each horizontal flow 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 in analysis, to operate with devices illustrated more specifically.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).Shown in Figure 1B, through closed row valve 110 with open capable valve 108 and begin to analyze, cross horizontal flow path 10 4 to allow flow of solution, block simultaneously and flow through perpendicular flow path 10 2.Reagent R1 is imported into horizontal flow path 10 4 then and then flows through the length of horizontal flow path 10 4 fully, so that all reflecting points 106 are filled.Flow of solution through external pump can obtain passing horizontal flow path 10 4 still more typically obtains through peristaltic pump being incorporated into the resilient 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, shown in Fig. 1 D, open capable valve 108 and allow S1 to mix with the S2 distribution and with R1.Therefore, in each point of crossing or reflecting point 106 zones, obtain the potpourri 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 potpourri to react then, and through 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 well 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 common ground among two figure.Equipment 150 differences shown in Fig. 1 F are that 4 pairs of horizontal flow path 10s are connected to public inlet 124.This uses the unique single injection port that gets into 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 gets into 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 horizontal flow path 10 2,104 that can be ideally be connected to public inlet 120,124 through change is changed.

Equipment shown in Fig. 1 F also comprises the separating controlling feeder connection 128 of regulating control channel 130 and another control channel inlet 132 of regulating control channel 134; Said control channel 130 can be used to arrange the flow of solution that flows to outlet 132, and said control channel 134 is adjusted to the flow of solution of outlet 136.Additionally, equipment 150 join protection passages 138.In concrete 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 walked around and is got in the following horizontal flow path 10 4, so that flow of solution is interrupted.

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

V. sidewalk for visually impaired people design

A. general introduction

Utilize the sidewalk for visually impaired people device designed to have concrete characteristics.At first, equipment comprises one or more flow channels, comes out from said one or more flow channel branch in one or more sidewalk for visually impaired people.As stated, 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 said equipment is by the resilient material manufacturing.Resilient material is enough porous, so that the air in flow channel and the sidewalk for visually impaired people can be escaped through these holes when solution is imported into passage.The hole of the material that in other microfluidic devices, utilizes lacks the use of having got rid of the sidewalk for visually impaired people, if because in somewhere, longshore current body path through hole is not provided then air in the sidewalk for visually impaired people no longer overflows holding when also being injected into.

The 3rd characteristics are that during in reflecting point, making, one or more reagent are deposited on (the other details about manufacturing process that sees below) on the elastomeric stratum non-covalently.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 made, be deposited over each reflecting point place.Below this reagent for example comprises some or whole: the dyestuff of primer, polymerase, nucleosides, accessory factor, metallic ion, buffering agent, interpolation etc.In order to make high-throughput maximization, be selected that the different primers of zones of different are deposited over each reflecting point in the 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.Through equipment being placed on the thermal cycle plate and, can accomplishing 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 (linker).Various link thing models can be utilized, for example photochemistry/to the non-persistent link thing of light, to hot destabilization connector 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 in some is measured, can be used 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 Agent Discovery Today.NVOC (6 nitro black false hellebore oxygen base carbonic acyl radical) link thing is the example (seeing, for example WO90/15070 and WO92/10092) that suitable photochemistry links thing with other NOVC peer link thing.Peptide with proteinase 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 resilient 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.

This operation of 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 mixes mutually through the reagent of diffusion preventing in each reflecting point 208 dissolvings.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 concrete design 300, each horizontal flow passage 304 is enclosed within its terminal quilt and connects straight two perpendicular flow passages 302.A plurality of diverted flow passages 306 stretch away from each horizontal flow passage 304.Diverted flow passage 304 in this concrete 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 intersect with horizontal flow passage 302,304 so that sample to enter the mouth 314 such as allowing flow of solution to cross level and perpendicular flow channel network, and finally via diverted flow passage 306 each reflecting points 308 of entering.

Therefore, in operation, sample is injected into inlet solution is imported each reflecting point.In case reflecting point is filled, then through port Extrusion Control channel start 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 detecting device.Moreover preferably may command heating of reaction is according to the method for being set forth in the temperature control part below.

The more complicated duplicate of the common design that in Fig. 3 A, shows 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 passages 306 and reflecting point 308.As stated, 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 characteristic of the blind vias equipment that begins in this section to discuss minimizes the vestige of equipment, and makes a large amount of reflecting points will be formed on the equipment and be used for quilt is obtained high density.The such equipment that for example has 2500 reflecting points can easily be made, and (25mm * 75mm) aforementioned characteristic can also be used and utilize the sidewalk for visually impaired people device designed to obtain the 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 betwixt arbitrary integer point.More specifically, equipment has 100-1000 reflecting point at least, perhaps betwixt arbitrary integer point.Higher density equipment can have more reflecting points, 1000-10000 reflecting point at least for example, perhaps betwixt arbitrary integer point.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 concrete 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 microfluid passage.These two PCT are disclosed in this and are combined to be used for all purposes through standard in full with it.

VI. Mixed Design

Another equipment makes the amalgam 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 horizontal flow passage is connected to himself sample inlet and horizontal flow passage do not interconnect via the perpendicular flow passage.Therefore, the sample that imports any given level flow channel is fill level flow channel and the reflecting point that is attached to the there only.Yet in the moving passage of the blind influx shown in Fig. 3 A, sample can flow between horizontal flow 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 horizontal flow passages 404, and each horizontal flow 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 Channel Design 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 horizontal flow passage 404 can be included in the inlet 414 of each end, thereby allows the importing sample from two ends.

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

Through different samples are injected each horizontal flow passage, great amount of samples can be by express-analysis.Suppose that reagent is deposited on reflecting point in advance.With the identical reagent existence that any given level flow channel is associated easy method is provided, has implemented a large amount of reaction repeated to use each passage at each reflecting point place.If replacement, 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 used for various dissimilar researchs by adjustment.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 conditionss) of limited quantity sample under a large amount of reaction conditionss.At last, if people want to check quite a lot of number of samples to 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 through the commercial channel easily, for example comprise 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 heat control source, so that if the temperature of reaction Sui at least one reaction chamber is the change of Controlling Source temperature and changing.

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

Thermal control equipment can be suitable for acting force is imposed on heat-transfer devices heat-transfer devices is pushed to the heat control source.Acting force can comprise magnetic, static or vacuum force in different embodiment.For example, in one embodiment, acting force comprises through passage and applies the vacuum force to heat-transfer devices that said passage is formed on the surface of thermal control equipment or heat-transfer devices.The vacuum tightness that between the surface of the surface of thermal control equipment and heat-transfer devices, obtains can be detected.This detection can be performed by the useful vacuum degree detecting device, and said vacuum tightness detecting device 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 arrange agreement again.

Array apparatus can contact with thermal control equipment, through 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 said 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 degree of 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 thermopair.This thermocouple possibly or made as the lead that directly is incorporated into little manufacturing resilient material self like the film wire on being patterned in following base material.

Can also come detected temperatures through resistance variations.For example, the resistance variations in the thermal resistor can be calibrated to given temperature variation, utilizes routine techniques can said thermal resistor be manufactured at following the semiconductor-based end.Replacedly, thermal resistor possibly directly inserted little manufacturing resilient material.Another program through the resistance detection temperature is described in " MEMS Flow Sensors for Nano-fluidic Applications " such as Wu; Sensors and Actuators A89152-158 (2001), therefore this article is combined with it by reference in full.This piece paper has been described the purposes of doped polycrystalline silicon structure to control and detected temperatures.Be used for polysilicon and other semiconductor material, the temperature coefficient of resistance can through characteristic accurately be controlled with amount of impurities, thereby optimization is used for the performance of the sensor of 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 possibly be added into solution when its process different temperatures.Thermochromic materials can be formed in the following substrate or be bonded in the resilient material.Replacedly, thermochromic materials can particle form be added into sample solution.

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

Another method of detected temperatures is the use through pyroelectric.Especially, some crystal material, these materials are also showed the piezoelectricity behavior particularly, show piezoelectric effect.This effect has been described the polarization of material crystals dot matrix and the phenomenon that material voltage height depends on temperature.This material can be bonded on substrate or the elastic body, and is used to detected temperatures.

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

B. accurate thermal round-robin verification

As fabrication portion is described in detail hereinafter, and sidewalk for visually impaired people equipment has basalis, and reagent is placed on the said basalis.Comprise that the two-layer structure that comprises flow channel and control channel is overlapped on the basalis, so that flow channel and deposited reagent align.The outside with basalis is placed in the substrate (for example glass) then.Usually, reacting the reflecting point that takes place above that is the about 100-150 micron on substrate/glass interface.Use the approximate value of employed elastic body 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 elastic bodys 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 through the thermal diffusion length of PDMS and glassy layer.

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

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

In another embodiment; The bispin oligonucleotide polymkeric substance that has known solution temperature (tm) through 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 confirming that each cavity is constant scope on array wherein with its internal contrast indication oligonucleotide; Wherein import the cavity of microfluidic device through the solution that will comprise the oligonucleotide that has coloring agent, the cavity of said 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 the internal contrast of the existing light oligonucleotide of quenching of dyestuff entering can be monitored.The use of dyestuff is provided for " oligonucleotide thermometer " in fact, and said " oligonucleotide thermometer " corresponding to the temperature change of the solution temperature of relative oligonucleotide and change characteristic, for example fluorescence.Through designing or use the oligonucleotide of the solution temperature of selecting, can confirm 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 meanss.On the selection part to appropriate system is the information about the type of selecteed incident and/or reagent.Can design detecting device to detect a large amount of unlike signal types, include but not limited to indicate, molecule, electrochemical 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, hyperchannel fluoroscopic examination, UV and visible wavelength absorption, cold light, different reflectivity and confocal laser scanning.Operable other detection method comprises the modification of flicker near determination techniques, radiation chemistry detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time resolution energy transfer (TRET), fluorescence resonant energy transfer (FRET) and biological example fluorescence 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 refer to the microfluidic device part that detection takes place with other relational language.As above indicated, to use and utilize the sidewalk for visually impaired people device designed, 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 equipment testing of matrix part usually in zone, the point of crossing self of the flow channel of contiguous point of crossing or surround the zone of point of crossing 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.Signal to be detected often is a light 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 detecting device is little, perhaps can be separating component.If detecting device 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, 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 confirming signal capabilities simultaneously according to various piece.

External detector is useful, because the equipment that provides is gone up by being the made of optical transparency in wavelength to be detected fully or on a large scale.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 said optical detection system.

Concrete preferred detection device uses CCD camera and the light path that is provided for 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, optical device 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, with the concentrated sample that device to be detected is addressed inquires to along the optical axis of optical system, and preferably through 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 with the size of the elastomer block area that is formed images or greater than the elastomer block area that will be formed images.In the use that in low NA optical device, links to each other, can realize improved detection sensitivity.

Detecting device can comprise the light source that is used to excite indicator, and said indicator produces detectable signal.Employed light source type depends in part on the type of the indicator that is excited.Suitable light source comprises, but is 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, implementing thermal cycle reaction, is got into the test section so that point to from the laser of diode.

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

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

B. to the detection of nucleic acid of 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 company limited 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 and provides it to rise by reference in full to be combined.

2. based on the detection method of FRET

Such detection method is included in the main fluorophore centering of alms giver/receive and detects from alms giver's (indicator) and/or receive the change in fluorescence of main (quenching agent) fluorophore.Select the main fluorophore of alms giver/receive to so that the overlapping PLE of being led of alms giver's emission spectrum.Therefore, when fluorophore is adjacent to each other to abundant 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 reaction.These methods are utilized usually and are used primer (primer) that alms giver/receive element of main centering indicates and use alms giver/the receive nucleotide of another element sign of main centering.Relied between the expansion reaction period before the nucleotide that indicates is advanced the alloy primer in template, the alms giver with receive main being spaced apart out enough far so that energy can not take place shift.Yet, if the nucleotide that indicates be incorporated into primer and space enough low near, 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-PCR.Various 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, through during amplification procedure self or measure the quantity of amplified production afterwards.It is an object lesson of real-time quantitative method that fluorophore (fluorogenic) nuclease is measured, and said method can be used equipment described here continuously.This method that the monitoring amplified production forms comprises uses two test constantlies that indicate the fluorophore oligonucleotide probes to the accumulation of PCR product---in the document of being everlasting, be called as the approach of " TaqMan " method.

The probe that in this mensuration, uses typically is short (ca.20-25 radix) polynucleotide (polynucleotide), and it uses two different fluorophore dyestuffs to be indicated.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, complementary to have at least with the real sequence of probe binding site on target nucleic acid.The PCR primer that in reaction mixture, also comprises nuclear downstream, the upper reaches, said 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 through for example 5 ' nuclease of the nucleic acid polymerase of Taq nuclease, thereby from the polynucleotide quenching agent, discharge indicator, thus the increase of the indicator emission concentration that causes measuring by suitable detector.

The routine a kind of detecting device of fluorophore emission that is specially adapted for measuring as in fluorometric assay, producing is ABI 7700, and it is by Foster City, and the Applied Biosystems company limited of CA makes.The employed computer software of this instrument can write down the fluorophor concentration of indicator, and the light of in amplification procedure, quenching.Then, the increase that these can be used to calculate standard indicator emission concentration on continuous foundation by numerical values recorded, and the final mRNA quantity that is increased of adding up.

Other details about being used for the theoretical agent operation of the concentration fluorophore of definite amplified production in real time is described; 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., it is combined in full its each 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 stated, these dyestuffs select and excellently are bonded to bispin DNA (for example, SYBR GREEN), and then only after restriction, produce signal.Therefore, when amplified reaction carried out, accelerating of dyestuff was limited, and accompanied by the increase of following in the signal that can be to be detected.

Molecular labeling: use molecular labeling, the change of probe structure causes the formation of detectable signal when it hybridizes the 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, indicator and the photo etching dyestuff of quenching be in enough approaching in so that from the fluorophore of indicator dye by the photo etching dyestuff of quenching effectively by the light of quenching.The ground that compares, hybridization probe produces aligned structure, and in said structure, the light path degree of quenching is lowered.Therefore, change through 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, through Piatek, the Nat.Biotechnol.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), here quilt is combined its each through being used for quoting of all purposes in full with it.

Invader (invader): invader is measured (the 3rd wave technology (Third Wave Technologies/Madison; WI)) be used to the SNP genetic typing and use oligonucleotide; The specification signal probe, said signal probe is complementary to target nucleic acid (DNA or RNA) or pleomorphism point.Second oligonucleotide is specified oligomeric invader, comprises 5 identical ' nucleotide sequence, but 3 ' nucleotide sequence comprises nucleotide pleomorphism (polymorphism).The combination of oligomeric invader interference signal probe to target nucleic acid is so that 5 ' end of signal probe is comprising pantomorphic nucleotide place formation " tablet (flap) ".This species complex specifies endonuclease (endonuclease) to confirm 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 fluorophore 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, said 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 Acid and 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 combine singly to select cDNA and make bispin cDNA.Add t7 rna polymerase, with through 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-based Amplification, 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 it is used for all purposes in full and is combined by reference; And Figure 20 described its scheme, and wherein Scorpion probe mechanism 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: through cooling, the Scorpion probe sequence is bonded to its target with intramolecular mode.Because this has advantage the intermolecular combination of complementary object chain.Scorpion (shown in figure 24) comprise be contained in by 3 of probe ' with 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 through PCR terminator sequence (stop motion mechanism).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 possibly cause hairpin loop under the situation of lack of specific target sequence, to be opened.This reading over causing detecting non-specific PCR product, for example, primer dipolymer or mispriming incident.

3. capacitive DNA detection

DNA concentration and electric capacity have linear relationship between changing, and this electric capacity changes by the nucleic acid passage of crossing over the lkhz 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 confirm 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 complex of reaction

The reflect typical ground that is undertaken by microfluidic device that here discloses is implemented with intensified response by some adjuvant.Therefore, for example, in the example of reflection deposition equipment within it, these adjuvants can be at reactive site for example by a kind of or more reagent contamination.One cover adjuvant is the closed reagent of protein binding site on the sealed elastic matrix of materials.A variety of such complexs 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 adjuvant.A kind of is the reinforcing agent that promotes amplified reaction.Such adjuvant comprises but is not limited to reagent (for example, betaine) that reduces secondary structure in the nucleic acid and the reagent (for example, tetramethyl-ammonium chloride) that reduces the misguidance incident.

Also find some polymer enzyme reinforced effects 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 comprises but is 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.In reaction mixture, merge two kinds or more the more the polymers enzyme be useful to 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 potpourri 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 adjuvant using some equipment that discloses here, 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 approach.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 that here discloses mainly can be used to carry out any kind nucleic acid amplification reaction.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, nucleotide, metallic ion, damping fluid and cofactor.The sample of guiding entering reactive site is nucleic acid-templated 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 through perpendicular flow channel flow and amplifing reagent through the horizontal flow passage or vice versa.

Though PCR possibly be best known amplification technique, this equipment is not limited to and carries out pcr amplification.Other type can effective amplified reaction comprises but is 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 sequence replicating of (iii) 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, BioTechnology13:563-65 (1995)) of nucleic acid.Each above-mentioned reference is combined through reference at this for all purposes with their integral body.

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

B.SNP analyzes and Genotyping

1. general introduction

A lot of with or the relevant disease of genome change of host's body or infectious body be the result of minority nucleotide change, usually comprise the change of single nucleotide.The change of single nucleotide 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 nucleotide of now so polymorphic site.As the expansion of this ability, this equipment can be used for the Genotyping analysis.Genotyping comprise confirm dliploid organism (being the organism that each gene has two copies) whether comprise two copies (the pure and mild body of reftype) of reference allele, each reference a copy and a variation allele (being heterozygote) 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 like what further describe 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 device design that is used for carrying out the Genotyping analysis 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 for the reactive site that uses suitable size.Otherwise, analysis possibly produce prompting assorted with son be the result of pure and mild body, only do not 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
			1	0.33	100
1	0.10	32
			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 before amplified reaction begins, occurs 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 carried out.In these methods, the result who obtains " being " or " denying " 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 nucleotide that only use to detect that polymorphic site possibly occur can be analyzed.Yet, more typically, comprise the primer and the nucleotide that detect each the possible allelic appearance of polymorphic site.It below is the example of suitable method.

Single base-pair extends (SBPE) reaction.The 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 hybridizing with the complementary primer of target nucleic acid, like this 3 of primer ' and terminal tight near 5 of variant sites ' or adjoin with it.Extend in and have one or more carry out said non-extensible nucleotide and the nucleotide complementation that occupies variant sites under the situation of non-extensible nucleotide and the polymerase of multiple labeling.Non-extensible nucleotide is nucleotide analog, and it stops the further extension under the polymerase effect in case combine to get into primer.If the non-extension nucleotide that adds is complementary in variant sites and nucleotide, the non-extension nucleotide of mark is bonded to 3 of primer ' end to produce the extension products of mark so.Therefore, the primer of extension provides nucleotide 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 with 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 a member's in the donor/acceptor fluorophor of mark reagent mixture, the non-extension nucleotide 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 nucleotide.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 with PCT publication WO97/22719).Optionally make the reaction heat circulation increase signal with the device that uses temperature-controlled process and above description.

Quantitative PCR.The Genotyping analysis also can use the quantifying PCR method of more early describing to carry out.In this case, involved with each complementary different label probe of allelic form as reagent, also have primer, nucleotide and polymerase.Yet reaction can only use single probe to carry out, and is because lack of specific allele 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 in reagent mixture, comprise both not probes of isolabeling, each is all perfect complementary with one of allele, also has amplimer, nucleotide 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 confirm the evaluation of polymorphic site nucleotide.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 a kind of or level of multi-gene expression more in the specific cells of measuring.This mensuration can be qualitatively, but normally quantitative.In the otherness gene expression analysis, gene level is compared with the middle homologous genes expression levels of another kind of cell (control cells) in a kind of cell (for example detecting cell).Can carry out very multiple such comparison.Example comprises but is 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 expression different gene 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 possibly 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 Equipment Design that here discloses 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 to be 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.Through matrix design, for example, the sample that obtains from healthy cell can detect at a flow channel, and from the sample of diseased cells closely near passage carry out.This characteristic 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 prepares 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 like this, 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, suitable sample comprises but is 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 " like what use here, 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 for 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 in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes; The 3rd chapter, Theory and Nucleic Acid Preparation in the first; (P.Tijssen, ed.) Elsevier, N.Y. (1993) and Sambrook etc.; Molecular Cloning:A Laboratory Manual; Cold Spring harbor Press, N.Y. (1989), Current Protocols in Molecular Biology, (Ausubel; F.M. etc.; Eds.) John Wiley & 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 said 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 confirmed the concentration of required DNA in the sample-specific 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 through what some equipment used.

Table 3: gene expression---DNA is quantitative

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

Some that further 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 in any specific cells, expresses, 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 ^-12Grammol 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/μ l; This concentration is corresponding to every 1nl hole 20,000 copies (or 4 * 10 ¹¹M).

3. method

Because expression analysis typically changes quantitative test, one of typically use in the above quantitative real-time PCR method of describing to accomplish and detect.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, nucleotide and polymerase.If use intercalative dye, reagent mixture typically comprise one of following material or whole: primer, nucleotide, polymerase and intercalative dye.

D. diversification

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 through in each reactive site, carrying out multiple analysis (for example, Genotyping and expression analysis).

Even can implement polynary amplification at the single reaction position, through for example in the thermal cycle process, using multiple primer, each is specific interesting target nucleic acid specificity property.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 through 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 through wisdom, analyze and to be able to carry 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 of Aromatic Molecules, second edition, Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.) .Pergamon Press, Oxford, 19723; And Haugland, handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 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. 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, but said reaction produce detectable signal or can with the reaction product of detectable reaction, but should detectable in case can produce signal with reaction.

Therefore, for example, this equipment can be used to a plurality of screenings and use the detectable that has specific required activity with identification.As special example, this equipment can be used to screening compounds, screens its activity as substrate a kind of or more multienzyme or suppressant.In such analysis, the compound of detection is introduced into other necessary enzyme analytical reagent (for example, damping fluid, metallic ion, cofactor and substrate) 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 a suppressant) is to be detected.Such reaction or inhibition can be accomplished through 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 a kind of or more reagent.For example, some analysis relates to and confirms whether particular cell types is present in the sample.Be used to accomplish 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.Like another example, the compound of detection can be triggered or suppress the ability of cell effect, for example signal transduction bypass by screening.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, its herein for all purposes with its integral body through with reference to being combined.

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 by following document in detail; For example (2000) " Integrated Elastomer Fluidic Lab-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 State Actuator 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 manufacturing approach relates at first through using photoresist (Shipley SJR5740) photoetch on silicon chip, to make 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).Through spin coating speed control channel height accurately.Through after developing, photoresist being exposed to UV light, form the photoresist passage.Hot reflux is handled and the protection disposal 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 combined in full 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 after 25 minutes with 80 ℃ in baking oven and from mould, is peeled off, and aligns with bottom and assembles.Using finally to cure in 80 ℃, 1.5 hours irreversibly combines this two-layer.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 that some Si-O-Si that splits combines 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 material 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, microslide) 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 resilient material.Therefore, the elastic devices that obtains can preferably be connected to base support.

B. utilize the sidewalk for visually impaired people device designed

The 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 said microfluidic device.Bottom is a reagent deposition layer above that.Bottom can be formed by various resilient materials, as in the MLS method of quoting as proof in the above described in quote.Typically, material is dimethyl silicone polymer (PMDS) elastic body.Based on the location of this device and the desirable reflecting point that is used for concrete equipment, people can confirm the location on suitable reagent should point sample bottom above that.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 between reagent and surface of elastomer, do not form covalent bond, 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 the layer and the layer of formation control with preferred protection passage that forms flow channel.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 said ground floor.The object lesson of the composition in three layers (is formed A to forming the ratio of B) as follows: ground floor (sample layer) 30: 1 (on the weight); 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 to combine two or multilayer with or other method of substrate can comprise and use cementing agent or plasma to combine that preferred air plasma combines, to form other parts of elastic material block or microfluidic device.In certain embodiments, with the extra play that other layer is connected through 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 four jiaos of interceptings of a cover slave unit; 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 reagent sample applicator that on market, can buy and the point sample technology of using various structures of any amount, can deposited reagent.The example of the suitable sample applicator that in the equipment preparation, can be utilized 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 on market, can buy comprises CartesianTechnologies Micro Sys 5100 (Irvine; CA), and Hitach SPBIO (Alameda, CA); Genetix Q-Array (United Kingdom (Great Britain)); Affymetrix 417 (Santa Clara, CA) with 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.

Use the US patent No.5445935 of 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 direct light of SNP probe is etched in original position and is synthesized.This array also will be used as the substrate or the substrate of microfluidic device among the present invention, in a known location, comprise one or preferred unnecessary one oligonucleotide probes on the substrate so that will be arranged in corresponding to the for example blind filling of the substrate region chamber of reflecting point.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 from the summation of monomer the set of the SNP probe of identification monomer,, be imported into the serpentine flow channel herein if so that comprise fluid sample from the nucleotide sequence in a plurality of monomers; With a plurality of valves that are connected with the serpentine flow channel; So that the time caused the serpentine flow channel separated, isolate each reflecting point mutually thereby isolate, in each reflecting point, to comprise the segment of fluid sample by starting.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 possibly 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.Said example is not considered to limitation of the present invention.

Example 1

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 here, and signal intensity ratio Macro TaqMan reacts big by 50%.

II. microfluidic device

Use three layers of microfluidic device of MSL program manufacturing to be designed and to make, be used for carrying out the experiment that following example is described.Fig. 7 A illustrates the cross-sectional view of this equipment.As shown in, equipment 700 comprise the layer 722, form flow channel within it.This liquid level 722 is sandwiched between overlayer 720 and the bottom sealant 724, and overlayer 720 comprises control and protective seam.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 heat conduction, and helps prevent from the evaporation of microfluidic device 700 bottoms.

Fig. 7 B illustrates the sketch map of the design of flow channel in the fluidized bed 722 and the sketch map of the control/protective seam 720 inner control passages and the design of protection 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 in case the solution stopping evacuator body goes out reactive site 706; Liquid is introduced through inlet 718.

II. test configurations

In equipment 700, implement the PCR reaction, said 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 buffering agent 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 through 708 then.Through manually buffer brake being imposed on syringe, flow channel 702 is filled, and is filled with reactive site 706 until all whole sidewalk for visually impaired people 704.Control line 712 is filled deionized water, and is loaded influent stream line 702 at all samples, is compressed into 15-20psi after 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 ℃) perhaps;

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 reaction mixture; CA); In order they and the reacting phase that in microfluidic device, carries out to be distinguished and to be indicated that MacroTaqMan reacts, and is placed on GeneAmp PCR system 9700 (Applied Biosystems, 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 completion thermal cycle; Contrast with 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 of carriage that entering has 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 computing method of describing in the agreement (TaqMan PCR kit agreement) that the positive sample of target dna uses manufacturer to provide are measured.Signal intensity is calculated through 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. 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 enhancing signal intensity.Therefore, because the thermal stability of DyNAzyme polymerase, correction and the opposing of impurity strengthened, the AmpliTaqGold polymerase is replaced by the DyNAzyme polymerase.0.025U/ the standard Macro TaqManDyNAzyme 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 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 adjuvant.Detected two kinds of DyNAzyme that concentration increases with every nl 100pg or 10pg genomic DNA in the microfluidic device, 8 * (0.2U/ μ l) and 4 * (0.1U/ μ l) are used for the normal concentration of Macro TaqMan.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 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 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 through contact and is implemented.Reagent is got on the metal needle from hole, a cover source, and through pin contact target matrix is deposited.This print procedure is further summarized in Fig. 9.As shown in, reagent is by (for example titer plate) obtains from the source, and contacts with matrix through the pin that makes loading then and quilt is 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 (Irvine CA), uses TeleChem " ChipMaker " print needle, although can use other system as stated.

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 conditions of using (typically, 75% relative humidity and about 25 ℃), the every loader cycle of every pin is printed and is surpassed 100 spots.Under above condition, 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, the lower limit of the spot spacing that the size of pin still determines to obtain easily.Obtainable spacing has determined hole minimum in the final device-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

Flowing and the normal MSL suite of key-course of PCR equipment according to above description.Described in the microfluidic device design and routine 1 that is identical.Simultaneously, comprise A: the hypothallus of the 150 μ ms thick PDMS of B composition than 30: 1, through 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 increased.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 through the potpourri 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 consistance 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 produce when reagent at the appropriate final concentration of 1nl reaction volume when suspending 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 align with speckle patterns with key-course (be Fig. 7 A layer 720 and 722) and contact.Use further and toast 60-90 minute matrix is bonded to the remainder of sheet at 80 ℃.After sheet was assembled, flow channel and sheet that all the other compositions of PCR reaction (in example 1, describing) are injected into sheet were carried out thermal cycle described in example 1.

III. 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, in this chip, carries out reaction continuously.The continuous P CR reaction that the reagent of point sample produces as in example 1, limits.Use 2 rank and 3 rank thermal cycle agreements to carry out reaction continuously.

Example 3

Genetic typing

I. introduce

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

II. method/result

A.Rnase P test

(Applied Biosystems, Foster city CA), produce detectable result so that other primer/probe set to be described described in example 1, in microfluidic device, to carry out Rnase P TaqMan reaction.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 kinds of pseudogenes, and their each genomes are concentrated approximate 17 parts altogether.Use as example 1 described in 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 to 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 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, confirm that the TaqMan reaction is sensitive in the chip, enough detect each genomic 2 copy.

The B.DNA dilution test

In order further to confirm 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 is formed with the same quilt described in the example 1, uses the dilution of 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 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 a mean value of every nl.Therefore, the reaction sensitivity in the microfluidic device is enough to carry out the genetic typing test.

C. 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 Allelic Discrimination bag (Applied Biosystems to CYP2D6P450 cytochrome gene; Foster city CA) carries out genetic typing.Said 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 to be 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 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), possibly leak the into VIC passage of detecting device 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 gets into the FAM passage of detecting device.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

Use MSL to handle three layers of microfluidic device making and be designed and make, be used to be implemented in the test described in this example; Figure 15 shows the synoptic 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 entering through hole 1510 and goes out through hole 1512.Control zone 1504 comprises three control flow channels 1514, and each flow channel comprises 1nl reflecting point 1518, and is represented with entering through hole 1516 by rectangle equally.In the time of in enough pressure is applied in to entering through hole 1524, 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.The same described in 1 of this equipment and example is three-layer equipment (seeing Fig. 7 A).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, reaction 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.Reaction product from equipment 1500 and Marco reaction is used 2 μ l ExoSAP-IT (USB; Cleveland; OH) be processed, ExoSAP-IT is made up of DNA Exonuclease I and ShrimpAlkaline Phosphatase, to remove excessive nucleotide 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. result

To 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 from this equipment, obtains provides the β actin PCR product of expectation.Therefore, target dna is increased 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 kinds of fluid samples be isolated into the reaction volume of large-amount isolation.At the sample remarks as entering the mouth after 1703; Said 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 microfluid passage (Figure 17 d).Then, chipset by thermal cycle, and forms images in the fluorescence number of degrees device that on market, can buy on dull and stereotyped thermo cycler.

Through 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 duplicate is low in every hole.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 possibly have good amplification.The quantity in positive polarity hole is consistent with the control quantity of calculating, has to distribute through 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 each reaction of comparable-macroreaction comprises more than＞104 template version to macroscopical PCR reaction with same DNA concentration.

The main source of the fidelity that perhaps is observed (fidelity) is the effective concentration 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-dipolymer 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 need changing thermal cycle agreement (for example, long extension time, many circulations), (for example to wrong primer (mispriming) and nonspecific amplification from the current method of the reliable amplification of single copy; " warm start " PCR (thermal excitation of polymerase), " supercharger " PCR, adjuvant; To reduce nonspecific hybridization etc.) preventive measure, 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 receive 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 through 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 through the concentration of isolating maybe 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 reaction assembly having nanoliter-sized chambers and methods of filling chambers " and Vogelstein etc. are called " Digital PCR " and have described digital pcr, thereby both are combined through quoting in full with it its.Use the obtainable little volume of microfluidic device to allow highly-parallelization and very high target-background concentration ratio.High target-background concentration ratio allows unimolecule amplification fidelity.These factors are proposed to be used in PCR, and are more little good more.

The present invention is provided in microfluidic environment, implementing the method and apparatus of digital pcr; Said method comprises step: be provided at the wherein microfluidic device of fluid passage; Said fluid passage has two or more relevant betwixt valves, can the fluid passage 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; Starting 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 confirming 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, has 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 detectable; Preferably to useful (complimentary) one or more nucleic acid polymers of part target nucleic acid polymkeric substance at least, preferably multiple different IPs acid polymer spatially is arranged in the reflecting point or cavity of microfluidic device.

Can obtain amplification through the thermal cycle reaction of for example PCR or through adiabatic reaction; Said 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 it is used to instruct the purpose that case is put in adiabatic amplification to be combined in full 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 being used than the low wavelength of other UV scope dyestuffs, thereby reduces typically relevant with many plastic materials background problems.

Figure 22 B has described the stretch-out view of complete microfluidic device 2899; Said microfluidic device 2899 comprises the parts shown in Figure 22 A; And further comprise elastic material block 2808; Said elastic material block 2808 is by attached or more preferably use cementing agent bonded and more preferably directly bonded, preferably do not use cementing 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 said through hole 2814 provides the fluid between elastic material block internal channel and the substrate internal channel to be communicated with, and said 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 the check valve that under pressure, is used to guide and keep gas entering accumulator chamber 2815 and 2816.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 through sheet extrusion, pin etc. in preferred check valve; Overcoming the power of closing automatically of check valve, thereby be comprised in the hydrodynamic pressure in the accumulator chamber to allow from the accumulator chamber relief pressure to reduce.

Figure 22 B has described the stretch-out view of complete microfluidic device 2899; Said microfluidic device 2899 comprises the parts shown in Figure 22 A; And further comprise elastic material block 2808; Said elastic material block 2808 is by attached or more preferably bonded and more preferably directly bonded, preferably do not use cementing 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 said through hole 2814 provides the fluid between elastic material block internal channel and the substrate internal channel to be communicated with, and said fluid connection produces hole (well) 2805 then and is communicated with hole (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 the check valve that under pressure, is used to guide and keep gas entering accumulator chamber 2815 and 2816.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 through sheet extrusion, pin etc. in preferred check valve; Overcoming the power of closing automatically of check valve, thereby be comprised in the hydrodynamic pressure in the accumulator chamber to allow from the accumulator chamber relief pressure to reduce.

Figure 22 D has described the planimetric map 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 from Beijing-Tianjin like the passing through of the fluid that is formed on substrate 2800 passages, 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 said groove is fabricated in the passage through the sealant of preferred binder film or sealant.

Substrate 2800 machine associated components can be by the polymkeric substance manufacturing, for example polypropylene, tygon, polycarbonate, high density polyethylene, polychlorotrifluoroethylene 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; Can import spiral 2812 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 accumulator chamber 2815 and 2816 interior hydrodynamic pressures.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 around the chamber for hydrating 2850 in elastic material block zone 2807; Said 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 through the for example volatile liquid of water can increase humidity, preferably passes through sorbing material or sponge humidification.Can preferably use polyvinyl alcohol (PVA) (polyvinyl alcohol).The ratio that is preferred for the tygon alcohol and water of humidification sorbing material or sponge through change can obtain humidity control.Moisture control unit through using the for example moistening encapsulation of HUMIDIPAKTM can also be controlled hydration, and said moisture control unit for example uses permeable and sealing bag liquid non-permeate of water vapor to hold to have the salt solusion 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 the specific purpose of moisture control unit and method are combined.Hydration lid 2850 is preferably transparent, with in order in elastic material block, not cover visual situation during use.Likewise, 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 said 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; Through first port 2890, passage 2870 and second port 2892 and then get into the groove of elastomeric layer 2808, said groove is sealed to form passage 2885 by the last end face 2897 of substrate 2800.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, polycarbonate or polypropylene.In one embodiment, sealant 2881 is flexible, splicing tape for example, and can be attached to substrate 2800 through bonding, for example use bonding agent or heated sealant or mechanical attachment, for example through compression.The material that is used for sealant 2881 preferably is easy to form the fluid-tight 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 elastic material block 2808 and the elastic material block zone 2807 of substrate 2800.As waiting at Grossman described in the unexamined patent application US11/043395; Wherein be used for scanning all purposes of stating with the there here and being combined; And be used for disclosing about the purpose of integrated carrier with the further details of the use of automatic starting outfit, 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 likewise blocked in whole or in part, to form the passage in second elastomeric layer, has 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; Said first elastomeric layer 2920 has and is formed on the basal surface that wherein has first groove 2901, and said 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 through 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 and the quilt that use automatic laser ablation system and method with instruction through being used for all purposes.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 said through hole is arranged in microfluidic device described here.Ground floor 2920 that microfluid piece 2921 comprises and have the ground floor groove that is formed on wherein when being bonded to the second layer (or passage) 2907 and ground floor 2923 with 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 be in substrate 2800 in hole 2999 other fluids be communicated with, pass 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 can fragment chamber 2989 be cast in the layer 2920; Wherein after the first photoresist layer is patterned and develops; The second photoresist layer is overlapped 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 application that they are wanted selectively is designed and has 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 the membrane thicknesses section of realizing through the present invention and flow channel cross comprise is rectangle, trapezoidal, circular, oval-shaped, paraboloidal, hyperbola with polygonal, upper shape part in addition.More complicated shape of cross section for example has the embodiment of bump (protrusion) or the embodiment that in flow channel, has depression, also uses the present invention to be implemented.

In addition, although the present invention mainly combines the wall of flow channel wherein and ceiling is made up of elastic body 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 possibly be formed in the following substrate, have only ceiling in the flow channel by elastomeric construction.The exciting force that applies of response, this elastic body 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 and be formed on the passage in the substrate, wherein this structure has a plurality of advantages.For example, comprise that the substrate of optical waveguide can be configured, so that optical 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 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 skeleton 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, the type of for example in Figure 22 A-22E, being described.Platen 3207 is suitable for contacting top 3209 of microfluidic device 3205.Platen 3207 has in the port of microfluidic device 3205 alignment therein, with the hydrodynamic pressure with preferred gas pressure hole and accumulator to the microfluidic device 3205 is provided.In one embodiment; The motion of pressure case 3207 through arm 3211 is pushed above microfluidic device 3,205 3221; Said arm 3211 is suspended on the pivot 3213 and through piston 3215 and is energized, and said piston 3215 at one end is connected to 3211 and be connected to platform 3217 at the other end.Along the sensor piston motion of piston 3215 with will go to controller in the information about piston position, the controller under the computing machine (not shown) control of following software scripts (software script) preferably.Platen detecting device 3219 detects microfluidic devices 3205 and is receiving the inboard existence in gulf 3203, preferably can detect into the fluid clavicle 3205 correct location.For example make through the side of light being departed from microfluidic device 3205 and use up existence and the position of detecting microfluidic device 3205, this possibly 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 hydrodynamic pressure offers platen 3207, and the controlled compression fluid is supplied to microfluidic device 3205.Through 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 used gimbal around the axle of vertical micro fluid device 3205 upper surfaces 3221 to platen 3207.

Figure 25 has described the outboard profile of cutting open of platen 3207, and said 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 equipment 32056 parts and the surface fluid-tight between 3227.In one embodiment, platen face 3227 comprises or by the compliant materials manufacturing, elasticity elastic body 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 non-return valve (check valve) purifies actuator 3233; Said non-return valve purifies actuator 3233 and preferably pneumatically is energized and when being energized, will pins and 3231 push non-return valve 2812 downwards; To open and to alleviate hydrodynamic pressure, perhaps allow the introducing of fluid through non-return valve 2718 through overcoming its opening resistance.In one embodiment, platen 3207 has first and second and purifies actuator 3233, and itself and non-return valve 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 between culture period.At interphase 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 said platen 3207 is pushed to the upper surface 3221 of microfluidic device 3205, wherein through 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, hydrodynamic pressure preferred gas pressure is applied to pressure chamber 3255, through fluid is imported cavity 3255 from the pressure line of the arm 3211 of tracing back to loading terminal (charging station) 3200.Through the pressure regulators adjustment pressure relevant, preferably can change the electric controlled variable pressure regulator of output pressure, preferably under computer control through the signal that sends according to the loading terminal controller with loading terminal 3200.Hydrodynamic pressure in the pressure cavity 3255 next drive hole 2805 interior fluids passes the passage in the substrate 2800; And then get 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, with the mode in its a certain zone of fluid-tight.Particularly; Fluid-tight is set in demarcation strip 3520 and carrier 3400 and the chip between one or more zones, for example first protein zone, 3430, second protein zone, 3432, first well area 3420, second well area, 3422 boundary accumulators 3460, non-return valve 3465, container accumulator 3450 and/or non-return valve 3455.In one embodiment, demarcation strip 3520 offers zone 3420,3422,3430,3432 with fluid-tight, and offers accumulator 3450 and 3460.In one embodiment, demarcation strip 3520 provides one or more check valve actuator 3570, sees like the best in Figure 27 B.

In certain embodiments, demarcation strip 3520 fluid-tight that all are desirable offers carrier 3400 and microfluidic device.When doing like this, demarcation strip 3520 can comprise packing washer 3580.Packing washer 3580 can comprise material widely, includes but not limited to silica gel, elastic body etc.In certain embodiments, packing ring 3580 comprises compliant materials, to help going out to form fluid-tight in the position of wanting.By this way, system 3500 can offer the appropriate area in chip and the carrier 3400 with the pressure of wanting.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 said separating plate 3520 is suitable for assembling said 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 in 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 is loaded into carrier hole carrier being placed into reagent and the protein that will want before the receiving station 3510.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 combination carrier 3400.Plate 3520 can be manually, robot ground or be lowered on the contrary to use down the part or all of fluid-tight 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 through use is applied to accumulator 3450,3460 with convenient pressure and drives in chip.In specific embodiment, system 3500 automatically performs this technology, and said 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.

Through 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 to drive reagent entering 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 get into 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, is accumulated storehouse or the like, opens the boundary valve in the chip through the non-return valve 3465 that discharges in the boundary accumulator 3460.In specific embodiment, non-return valve 3465 is released, and to open the dividing valve in the chip, this moment, system's 3500 excitations combined the check valve actuator 3570 of non-return valve 3465.In certain embodiments, check valve actuator 3570 comprises the pin that is suitable for combining non-return valve 3465, binding post etc., to discharge the pressure in the boundary accumulator 3460.In alternative embodiment, non-return valve 3465 is by manual releasing 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 several 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 closed.In one embodiment, actuator 3450 and 3460 keep-ups pressure and on threshold pressure, continued 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 is suitable for carrying out the experiment of wanting according to the embodiment of the invention, the system of the application of the invention with microfluidic device.More specifically, 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 in the embodiment of selection of the present invention, provide still less 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 combines the upper surface and the microfluidic device thereof of carrier 3400.In certain embodiments, stand 3510 with platen 3520 be similar to the station 3200 with 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 pressure two lines wanting six lines in zone and be used to provide the machinery of excitation non-return valve 3455 and 3465 to carrier 3400 being provided.

Shown in Figure 26 A, system 3500 further comprises processor, and said in one embodiment processor is the processor that is associated with notebook or other computer equipment 3530.Computer equipment 3530 comprises the storer 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 appear 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 said 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, with the fluid-tight mode in a certain zone wherein.Particularly; Fluid-tight is set between the one or more zones in demarcation strip 3520 and carrier 3400 and the chip, for example first protein zone, 3430, second protein zone, 3432, first well area 3420, second well area 3422, boundary accumulator 3460, non-return valve 3465, container accumulator 3450 and/or non-return valve 3455.In one embodiment, demarcation strip 3520 offers zone 3420,3422,3430,3432 with fluid-tight, and offers accumulator 3450 and 3460.In one embodiment, demarcation strip 3520 provides one or more non-return valve accumulations 3570, is seen like the best in Figure 27 B.

In certain embodiments, demarcation strip 3520 is sealed to carrier 3400 and microfluidic device with whole ideal fluids.Do like this, demarcation strip 3520 can comprise packing washer 3580.Packing washer 3580 can comprise material widely, includes but not limited to silica gel, elastic body etc.In certain embodiments, packing ring 3580 comprises compliant materials, to help going out to form fluid-tight in the position of wanting.By this way, system 3500 can offer the appropriate area in chip and the carrier 3400 with the pressure of wanting.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 said separating plate 3520 is suitable for assembling said 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 in 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 is loaded into carrier hole carrier being placed into reagent and the protein that will want before the receiving station 3510.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 to combine carrier 3400.Plate 3520 can be manually, robot ground or be lowered on the contrary with the part or all of fluid-tight 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 through use is applied to accumulator 3450,3460 with convenient pressure and drives in chip.In specific embodiment, system 3500 automatically performs this technology, in about 20 (20) hours, takes place 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.

Through 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 get into 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 gets into 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 chip interior well of wanting, chamber, accumulates storehouse or the like, open the boundary valve in the chip through the non-return valve 3465 that discharges in the boundary accumulator 3460.In specific embodiment, non-return valve 3465 is released, and to open the dividing valve in the chip, this moment, system's 3500 excitations combined the check valve actuator 3570 of non-return valve 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 for combining non-return valve 3465, binding post etc.In alternative embodiment, non-return valve 3465 is by manual releasing 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 several 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 closed.In one embodiment, actuator 3450 and 3460 keep-ups pressure and on threshold pressure, continued 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 elastic body 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 possibly be not enough in hidden reaction array in the elasticity chip, produce uniform heat-field.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 possibly require its reservation is constrained on the platen.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 to combine the described ICC of Figure 28 B that improved uniform heat-field is provided, and improved ICC reservation method is depicted among Figure 29 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, can heat energy (for example, from PCR equipment) be passed to elastomer block.In certain embodiments, Heat Conduction Material comprises silicon (Si).In specific embodiment, use from the silicon of polishing, with employed similar or identical in the semi-conductor industry with 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 increase can be by the quantity of the light of the collected of using 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 through conversion zone and the heat control source that makes elastomer block.The heat 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), 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 through 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 shown in Figure 29 B, vacuum chuck 3950 comprises or by being one or more made of good conductor of heat, said good conductor of heat is incorporated in to the heat control source, for example one or more Peltier (amber ear card) device.In one embodiment, use a plurality of heat controls to be created in the hot tonsure in the reaction array.

In the use, ICC is positioned in vacuum chuck 3950 tops, and reduces downwards or be transferred, so that integrated chip 3910 contacts with vacuum chuck 3950 with hot part 3920 among the ICC3930.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 cementing agent in certain embodiments.As shown in, piece 3910 combines 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, the gap 3940 in these embodiment helps to form sealing when chuck 3950 is applied vacuum, through one or more vacuum ports 3960 so that hot part 3920 is pulled to chuck 3950.Can monitor the vacuum quantity that obtains, to change the good thermal contact that between chuck 3950 and hot part 3920, need make veritably.In certain embodiments, the one or more ports in the vacuum chuck are used and are used to monitor vacuum tightness.In the specific embodiment shown in Figure 29 D and Figure 30, the one or more ports that are used to monitor vacuum tightness will be positioned in the far-end of vacuum ports and through the path fluid communication with each other, the preferred tortuous and narrow path of said 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 confirm 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 said zigzag channel is used to obtain the accurate measurement to different vacuum tightnesss on the hot subregion of vacuum chuck, and promotes vacuum evenly or relatively equably using ICC heat part.Through 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 meaning are open 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 characteristics more of the present invention will be used.For example, except the above-mentioned brake system based on pressure, the moving system of optional static and mangneto also is implemented.Also possible is, encourages said equipment, produce fluid in the control channel and flow through being applied in based on heat energy, or through thermal expansion or through the gas generation from liquid.In addition, in another embodiment, use centrifugal force that protein and reagent are driven the entering 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 to the present invention's instruction.Its purpose is, the invention is not restricted to be used to carry out specific embodiment of the present invention as 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 through " blind filling " sample loading passage and control line-PDMS, drive gas out of passage, be left complete by 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 (0.01MEDTA, 60nMPassive Reference 1, pH 8.3 for 50nMKCL, 10nM Tris-Hcl); 4mM MgCl2,200nMdATP, dCTP, dTTP; 400nMdUTP, 300nM forward direction primer, the reverse primer of 300nM, 200nM probe; 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, various changes of wherein setting forth or change and will give those skilled in the art suggestion, and in the scope of the aim that is comprised in the application and clause and claims of adding.Here whole open, patent of enumerating and patented claim are used for all purposes and are combined through using in full with it, on same degree, with each single open, patent and patented claim by indication is the same specially and individually, combined through application.

Claims (44)

1. method that realizes aspect ratio features in the elastic material block, said method comprises step:

First elastomeric layer is provided;

The photoresist layer is applied on the said first resilient material laminar surface;

Light pattern is applied to said photoresist layer, to form the photo anti-corrosion agent material pattern that has reacted;

Remove unreacted photo anti-corrosion agent material, the remaining photo anti-corrosion agent material pattern that has reacted on the said surface of said first elastomeric layer;

Etchant is applied to said first resilient material surface; There is not said surface with etching by said first elastomeric layer that said pattern was covered of the photo anti-corrosion agent material that has reacted; Thereby remove the zone that does not have by said first elastomeric layer that said pattern covered of the photoresist that has reacted; And then the pattern of the said elastomeric layer of remaining said pattern corresponding to the photo anti-corrosion agent material that has reacted

When wherein looking from the side, aspect ratio is equal to or greater than: the twice at least that highly is the length of characteristic width.

2. the method described in claim 1 comprises that further removal reacted the step of the said pattern of photo anti-corrosion agent material.

3. the method described in claim 2; The said pattern that wherein is applied to the said surface of said elastomeric layer and has reacted photo anti-corrosion agent material through the band that will bond carries out said removal; Then said bonding band is separated with said elastomeric layer, the part or all of said surface from said elastomeric layer of having reacted the said pattern of photo anti-corrosion agent material simultaneously is removed.

4. the method described in claim 1, wherein said photoresist is SU8.

5. the method described in claim 1, wherein said etchant comprises tetrabutylammonium fluoride-trihydrate.

6. the method described in claim 1, the wherein said through hole that is characterized as.

7. the method described in claim 6; Wherein said elastic material block comprises a plurality of elastomeric layers that bond together; Wherein two or more elastomeric layers have the groove that is formed on wherein, and groove in groove in elastomeric layer and another elastomeric layer is in through said through hole and is communicated with.

8. microfluidic device that is used to make the different samples of M kind and the different reagent reactings of N kind, it comprises:

A plurality of reaction members; Each reaction member comprises sample chamber and reagent chamber; Said sample chamber is in the fluid connection through the boundary passage with related dividing valve with said reagent chamber, and the fluid that said dividing valve is used to control between said sample chamber and the said reagent chamber is communicated with;

A plurality of samples inlet, each sample inlet and said sample chamber are in during fluid is communicated with;

A plurality of reagent inlet, each reagent inlet and said reagent chamber are in during fluid is communicated with;

In the wherein said chamber at least one comprises oligonucleotide or protein polymer and internal contrast (intercollating) dyestuff.

9. microfluidic device described in claim 8, wherein said reaction member is formed in the elastomer block, and said elastomer block is made up of the multilayer that bonds together, and said dividing valve is deflectable barrier film.

10. the microfluidic device described in claim 8; Wherein said sample inlet and said sample chamber are in through sample channel and are communicated with; And said reagent inlet and said reagent chamber are in through reagent passage and are communicated with; The part of the part of said sample channel and said reagent passage approximately is positioned parallel to each other and each passage has the container valve that is associated, and the fluid that is used to control through passage is communicated with.

11. the microfluidic device described in claim 10, the wherein said said valve that is associated in said sample channel operationally is in the connection mutually through common container control channel with the said valve that is associated in said reagent passage.

12. the microfluidic device described in claim 11, wherein said common container control channel is along approximately being positioned perpendicular to the line one of in said sample channel and the said reagent passage.

13. the equipment described in claim 8, wherein the internal contrast dyestuff is SYBR Green (TM).

14. a method that is used for confirming protein or oligonucleotide denaturation temperature, said method comprises the step that the equipment in the claim 8 is provided, the temperature in the change equipment, and then detect the change in the dyestuff.

15. vacuum use that the PCR reaction vessel is remained on the heat control surface of thermal control equipment.

16. the use described in claim 15, wherein container is a microfluidic device.

17. the use described in claim 16, wherein container is the resilient material microfluidic device.

18. one kind is used the heat conduction embolus so that the use of the heat conduction embolus of heat conduction localization in the microfluid support base.

19. the use described in claim 18, wherein substrate is an integrated carrier.

20. the use described in claim 18, wherein the heat conduction embolus is a silicon.

21. the use described in claim 20, wherein silicon polishes.

22. the use described in claim 15 wherein through being formed on the passage in the heat control surface that contacts with microfluidic device, applies vacuum pressure.

23. the use described in claim 22, wherein microfluidic device is the resilient material microfluidic device.

24. the use described in claim 22, wherein microfluidic device comprises the heat conduction part in addition.

25. the use described in claim 24, wherein heat conduction partly is silicon.

26. carry out the use of the microfluidic device of PCR with microfluidic device for one kind, wherein PCR equipment have every square of cm being in fluid isolation (centimetre) area is greater than the reaction density of 100 reactions.

27. the use described in claim 26, wherein reaction density is greater than 250.

28. the use described in claim 26, wherein reaction density is greater than 500.

29. the use described in claim 26, wherein reaction density is greater than 750.

30. the use described in claim 26, wherein reaction density is greater than 1000.

31. the use described in claim 26, wherein reaction density is greater than 1250

32. the use described in claim 26, wherein reaction density is greater than 1500.

33. having, the use of a symmetric optical system, said symmetric optical system be lower than 1.5 NA, to comprising the microfluidic device imaging of PCR reaction.

34. the use described in claim 33, wherein NA is lower than 1.0.

35. a glycerine is as the use of control line fluid.

36. the use described in claim 35, wherein glycerine is the part of solution.

37. a PEG solution is as the use of control line fluid.

38. the use of an automatically exciting equipment is with the PCR reaction in the control microfluidic device.

39. the use described in claim 38, wherein excitation set by-pass valve control.

40. the use described in claim 38, wherein microfluidic device comprises elastomer block.

41. the automatic valve excitation set is wherein used in the use described in claim 38 in the system that adopts thermal control equipment.

42. the use described in claim 41, wherein thermal control equipment adopts vacuum, to assist the thermo-contact of realization and microfluidic device.

43. the use described in claim 38, wherein automatic valve excitation set, computer control are used for the excitation of the microfluidic device of PCR.

44. the use described in claim 39, wherein valve comprises deflectable barrier film.

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