CN101613660B - Methods and apparatus for pathogen detection and analysis - Google Patents

Abstract

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of dense arrays of a variety of fluid control structures, such as structures for isolating, routing, merging, splitting, and storing volumes of fluid. The fluid control structures can be used to implement a pathogen detection and analysis system including integrated immunoaffinity capture and analysis, such as polymerase chain reaction (PCR) and capillary electrophoresis (CE) analysis. An analyte solution can be input into the device and pumped through a series of immunoaffinity capture matrices in microfabricated chambers having antibodies targeted to the various classes of microbiological organisms such as bacteria, viruses and bacterial spores. The immunoaffinity chambers can capture, purify, and concentrate the target for further analysis steps.

Description

The method and apparatus of determination and analysis pathogenic agent

The application is dividing an application of the application 200380110066.6 that enters the China national stage on August 30th, 2005.

The statement of government concerned's sponsored research

Technology of the present invention and mechanism are under government supports, by the DEFG91ER61125 contract of USDOE, and according to NASA license No.NAG 5-9659, and NIH license HG01399 and P01 CA 77664 and implement.

Background of invention

The present invention relates to the determination and analysis of pathogenic agent.In one embodiment, the invention provides sample preparation, processing, the detection that utilizes micro-fluidic constitution realization and the system that analyzes.In another embodiment, the invention provides the powerful technology of making the fluidic closely spaced array that is used for the high throughput analysis application.

Conventional microfluidic analysis mechanism has limitation.Some existing mechanisms comprise single passage tripping device and hyperchannel tripping device.Other have comprised sample preparations more integrated and the analytical equipment of analysis measures.Yet many microfluidic analysis devices with Flow Control ability do not have chemistry or physical compatibility to many chemistry or biochemical measurement test.And due to the restriction in making processes, wearing quality and/or design, many microfluid components are difficult to make intensive array.The device of many routines need to continue to drive to keep Flow Control.After being removed, the micro fluidic device that adopts this valve can lose the controllability to the composition that flows in device from its Controlling System.In addition, the technology of many microfluidic analysis and mechanism also lack the ability of sensitivity, specificity or quantitative analysis.Especially, for the system of pathogen detection device and analyser for example of system, conventional microfluidic analysis mechanism lacks function and the ability that effectively realizes sample preparation.

Therefore people wish to provide Innovative method and equipment to realize micro-fluidic controlling organization, for example valve, pump, selector switch, reactor etc. so that in micro fluidic device the function of the introducing of integrated sample, preparation, processing and analysis effectively.In an example, micro fluidic device with microfabrication usefulness preferably is provided, it not only can be used for realizing single channel system but also can be used for realizing system based on array, wherein said system can be used as pathogen detection device and analyser with provide false positive results seldom, high-throughput and cheap continuous monitoring.

The invention summary

The method and apparatus of realizing the microfluidic analysis device is provided.Can be provided with and drive the closely spaced array of the multiple Flow Control structure structure of shunting and the storage flow scale of construction (for example be used for isolation, routing, mix) with integral, flexible film spare that integrated inflation collector accompanies.This Flow Control structure can be used for realizing monitoring and the analytical system of pathogenic agent, comprises that the integrated form immunoaffinity catches and analyze, and for example polymerase chain reaction (PCR) and capillary electrophoresis (CE) are analyzed.Analyte solution introducing and pump pressure are caught matrix by a series of immunoaffinities in the microfabrication chamber, wherein have the antibody of all kinds of microbial organisms of target, for example the antibody of bacterium, virus and bacterial spore.This immunoaffinity chamber can be caught, purifying and enrichment target compound be in order to carry out further analytical procedure.

In one embodiment, provide a kind of pathogen detection system.This system comprises the immunocapture chamber that is integrated on micro fluidic device.This immunocapture chamber can be used for catching the target compound that offers the immunocapture chamber by microfluidic channel.This is the logical DNA analysis mechanism that accompanies with the immunocapture chamber that also comprises.This DNA analysis mechanism is integrated on this micro fluidic device.This DNA analysis mechanism can be used for target compound is carried out DNA analysis.

In another embodiment, provide a kind of pathogen detection system that is positioned on single unit system.This system comprises a plurality of immunocapture chambers that are integrated on single unit system.This immunocapture chamber can be used for catching the target compound that offers the immunocapture chamber by microfluidic channel.This system also comprises a plurality of DNA analysis mechanism that accompanies with the immunocapture chamber.Described a plurality of DNA analysis mechanism is integrated on this single unit system.Described a plurality of DNA analysis mechanism can be used for target compound is carried out DNA analysis.

In another embodiment, provide a kind of method for Analysis of pathogens.By the microfluidic channel that is integrated on single unit system, the fluid analysis thing is offered a plurality of immunocapture chambers.The target compound that closes with this fluid analysis phase is hunted down in the immunocapture chamber.Utilize a plurality of DNA analysis mechanism that accompanies with a plurality of immunocapture chambers to carry out DNA analysis to target compound.Described a plurality of DNA analysis mechanism is integrated on described single unit system.

In following invention description and accompanying drawing, will introduce in further detail these and its its feature and advantage of the present invention, wherein understand principle of the present invention by example.

The accompanying drawing summary

With reference to following explanation, the present invention will be more readily understood by reference to the accompanying drawings, and wherein accompanying drawing has represented particular embodiment of the present invention.

Figure 1A-1E represents the mechanism's diagram on a kind of micro fluidic device that is suitable for realizing the technology of the present invention.

Fig. 2 is the diagram of describing a kind of surge pump.

Fig. 3 means a kind of plane diagram of Flow Control selector switch.

Fig. 4 means a kind of plane diagram of mixing loop.

Fig. 5 A-5D means a kind of diagram of fluid liquid storage tank.

Fig. 6 means the diagram of bus valve (bus valve).

Fig. 7 is the diagram of a kind of pathogen detection system.

Fig. 8 describes the diagram that immunoaffinity is caught valve system.

Fig. 9 means that immunoaffinity catches the diagram of valve system.

Figure 10 A and 10B represent the diagram with routing of catching of analyte that immunoaffinity is caught.

Figure 11 represents and can catch the integrated PCR of structure and the diagram of CE with immunoaffinity.

Figure 12 means that combined immunization catches the diagram with the PCR chamber.

Figure 13 A is the diagram of a kind of pathogen detection system.

Figure 13 B means the diagram in a kind of microfabrication stage.

Figure 14 is the radial arrays diagram of a kind of pathogen detection system.

Particular embodiment describes in detail

To introduce in detail some particular embodiment of the present invention now, comprise the embodiment of enforcement the best of the present invention that the inventor thinks.The example that has represented these embodiments in accompanying drawing.Although the present invention describes in conjunction with these special embodiments, be understood that this and do not mean that the present invention only limits to described embodiment.On the contrary, but it comprises that those drop on selection scheme, evolutionary approach and equivalence within essence of the present invention and scope, such as in the appended claims restriction.For example, will technical scheme of the present invention be described with the glass micro-fluidic device in literary composition, although also can adopt other device, for example plastic device.

Should be pointed out that the Flow Control structure that is applicable to the glass micro-fluidic device can be applied in various micro fluidic devices.In a kind of possible application that utilizes Flow Control structure advantage, the pathogen detection system is exactly a good example.In the following description, many special details have been listed in order to complete understanding of the present invention is provided.The present invention can be in the situation that do not have partly or entirely these specific details and implement.In other cases, known processing operation is not described in detail in order to avoid with the present invention, unnecessary obscuring occurs.

Since single passage disrupter the earliest, microfluidic analysis technical field just development is very fast.Some devices contain the hyperchannel disrupter that is useful on high throughput analysis and the analyzer of integrated sample preparation and analytic function on one single chip.The device that combines simultaneously multichannel analysis and integrated sample preparation can reduce and carry out the required resource of a plurality of determination tests and cost amount.Can find an illustration in genomics field: the preparation of integrated order-checking sample, purifying and electrophoretic analysis can make flux efficient and the wearing quality that minute and cost and raising are measured in single assembly.In all cases, in micro fluidic device, high-caliber integrated level all needs chip mechanism to have wearing quality so that separation, routing, mixing, shunting and store a large amount of fluids.

Some valve technologies that are used for silicon, glass silicon, polymkeric substance and elasticity micro fluidic device have satisfied these needs in limited mode.Yet many these technology and many chemistry or biochemical measurement test are incompatible on chemistry or physical properties.And concerning existing glass micro-fluidic device, many technology lack the various chemical property that durable surface is modified.In addition, typically making single microfluidic valve is to adopt independent film spare, and it stays open usually.Have and be generally the valve opened and just need to continue to drive to keep the control of flow.Adopt the micro fluidic device of this valve to remove from Controlling System, otherwise can lose the controllability to the composition that flows in device.In addition, some typical devices adopt the latex film spare that arranges separately.Although the pneumatic latex film spare that arranges separately develops to some extent, this manufacture method has hindered large-scale integrated and has turned to hyperchannel, high-throughout analytical equipment.

Other micro fluidic devices are to utilize the silicon of anode combination and chip glass to make, and pass through Piezoelectric Driving.Yet, because electroconductibility and the chemical compatibility of silicon causes its application in analytical equipment more complicated.In conjunction with or the film that is deposited on silicon can only partly reduce its electroconductibility and chemical compatibility.

People have also tested elastic device.Yet the hydrophobicity of resilient material and porousness make elastic device incompatible with the biochemical measurement test with many chemistry.Therefore people wish to minimize with the fluid contact of surface of elastomer.Complicated making, chemical compatibility, insecure fluid operated property and other problem have caused existing fluid operated technology to be not suitable for being integrated on the device of extensive, high-throughout chip type laboratory (lab-on-a-chip).

Therefore, technology of the present invention and mechanism provide a kind of integral membrane part valve arrangement of High Density Integration to the micro fluidic device that be suitable for.Various Flow Control structures based on this integral membrane part valve arrangement also are provided.

Micro fluidic device with integral membrane part is an example that is particularly suitable for realizing the device of pathogen detection system on chip.According to numerous embodiments, the pathogen detection system comprises immunocapture and DNA analysis mechanism, for example polymerase chain reaction (PCR), and capillary electrophoresis (CE) mechanism.In one embodiment, this pathogen detection system can realize on the glass micro-fluidic device with various Flow Controls structures.

Figure 1A-1E is the diagram of the integral membrane part valve that can realize on the glass micro-fluidic device.Figure 1A is the top view diagram of integral membrane part valve.Figure 1B is the side-view diagram with three-layer type device of this valve.Fig. 1 C is the side-view diagram with four laminar devices of this valve.Fig. 1 D is the side-view diagram of a kind of opening type valve of three-layer type device.Fig. 1 E is the side-view diagram of a kind of opening type valve of four laminar devices.According to the various embodiments shown in Figure 1A and 1B, three-layer type glass micro-fluidic device comprises the elastic film piece 111 that is clipped between two chip glasses 101 and 105.In one embodiment, this elastic film piece is polydimethylsiloxane (PDMS) film spare, available from Bisco Silicons of Elk Grove, IL, the HT-6135 that 254um is thick and HT-6240 type film spare.Also can adopt other flexible membrane spare.Elastic film piece 111 make between wafer in conjunction with reversible and firm.

Before combination, in wafer etching fluid channel 103 be used for the carrying fluid.Etch similarly collector path 10 7 and valve zone 109 in order to transport air or other working fluid to drive this valve under pressure or vacuum.Be typically, this filling channel 107 and 109 is positioned on a wafer 105, be referred to as to inflate wafer here, and the fluid channel is etched on the second wafer 101, referred to herein as fluid chip.These etched channel characteristics can directly contact and form hybrid glass/elastomerics passage with film spare, as shown in Figure 1B.

Perhaps, film spare can be positioned between the full glass flow wafer sandwich (XY) and inflation wafer 159 of thermal bonding, as shown in the four laminar devices 150 of Fig. 1 C.Having all-glass passage can allow device benefit from good physical and the chemical property of glass.Any layer with good physical and chemical property is known as the unreactiveness layer.This unreactiveness layer can be used for making XY.In one embodiment, 151 and 155 interlayers that consist of XY are just made by glass.

The example of four laminar devices comprises the fluid chip 151 that is thermally bonded on link wafer 155.The through hole of minor diameter is arranged on discontinuous point in fluid channel 153.Elastic film piece 157 sticks to connecting wafer 155 1 sides of this fluid/link wafer sandwich XZ.Valve bias current chamber 161 is etched in collector wafer 159 and is bonding with film spare 157, has completed this four laminars device 150.Like this, fluid channel 153 has kept full glass to learn upper useful structure, can realize the Flow Control structure of large scale integration simultaneously.In some embodiments, four laminar devices shown in Fig. 1 C compare the three-layer type device have advantages of suitable because it has reduced the contact between sample and elastic film piece.

According to numerous embodiments, the various Flow Control compositions in integral membrane part device are to exert pressure or vacuum drives by the hole on the inflation wafer.Here any single film spare is called the integral membrane part.Any single assembly with integral membrane part is referred to herein as single unit system.Be used for exerting pressure or the mechanism of vacuum is referred to herein as gas port or inflation inlet to the etched channels that accompanies with the inflation wafer.In the three-layer type device, the etched channels of inflation in wafer will drive in the valve zone 109 that vacuum is scattered in elastic film piece 111.The vacuum that collector passage by 109 places, valve zone applies promotes film spare away from the discontinuous point of passage, and the path of passing this discontinuous point is provided for fluid flow, thereby opens valve, as shown in Fig. 1 D.But but the valve that utilizes the inflation pressure switch is referred to herein as switch-valve or pneumatic on-off valve.

Apply the situation that inflation pressure comprises pressurization or applies vacuum.Thereby film spare 157 can regulate near fluid flow in the fluid channel, as shown in Fig. 1 D.In Fig. 1 D, by 109 applying vacuum and open fluid channel 103 to the valve zone with the etched channels that accompanies of inflation wafer 105.To the valve zone 109 when applying vacuum or suction, film spare 111 is closed fluid channel 103, as shown in Figure 1B when no longer.Fig. 1 E has represented a kind of four laminar devices.This four laminars device comprises channel layer 151, passage 153, articulamentum 155, film spare layer 157, and airflow layer 159.As mentioned above, this four laminars device has suitable benefit than three-layer type device, because it minimizes contacting between sample and elastic film piece, only at 161 places, valve zone, contact is arranged in some cases.

Shown in should be noted that, structure can be towards any direction.In certain embodiments, valve can be inverted on device.Airflow layer can be positioned at fluidised bed above or below.Technical scheme of the present invention allows towards various directions, because gravity can't cause adverse influence to this film spare valve.

The Flow Control structure has multiple advantage.For example, integral membrane part valve is generally the valve of cutting out, even that is to say that this valve does not keep closing condition when the driving pressure source is connected at device yet.The existing microfluidic valve that is generally opening type need to continue to drive to keep to installing the controllability of the composition that flows.And unlike the structure of shape memory alloy, the switch temperature of this valve constitution is all to be under envrionment temperature, and this is conducive to work in the biofluid situation of the aqueous solution.

In many typical embodiments, need many interfaces between micro fluidic device in order to control various Flow Control mechanism.Yet according to numerous embodiments of the present invention, parallel drive can be come by the ventilating control passage that connects them in a plurality of zones of film spare.In one embodiment, utilize single inflation inlet just can control series of valves.Therefore, only use the external interface of limited quantity or inflation inlet just can control considerable valve.This problem with regard to having simplified implementation procedure and having minimized device interface.According to various embodiments, control valve can carry out a large amount of parallel inflations drivings to the integral membrane part by this way, in order to other Flow Control structure in driven valve, pump, liquid storage tank, selector switch and device.

Film spare valve can be used for consisting of various Flow Control mechanism.Fig. 2 A and 2B are the diagram of the pump of employing film spare valve formation.According to the numerous embodiments shown in Fig. 2 A and 2B, three valves that series connection arranges have consisted of surge pump 210.Drive these valves according to the circulation of five steps and just can realize aspiration procedure.Fig. 2 A has represented a kind of top view of three-layer type integral membrane part surge pump.Fig. 2 B has represented the side-view of this three-layer type integral membrane part surge pump.This surge pump comprises transfer valve 201, diaphragm valve 203 and delivery valve 205.It should be noted that this surge pump can work on both direction, thereby being appointed as arbitrarily of transfer valve and delivery valve.This pump comprises the fluidised bed 209 with etched fluid channel 211, integral membrane part 207, and collector layer 213.The airtight feature of valve can automatically be full of this pump and can also suction air except other gas and fluid.

According to various embodiments, aspiration procedure can be implemented by a series of stages.The first step, delivery valve 205 cuts out, and transfer valve 201 is opened.Second step, diaphragm valve 203 is opened.In the 3rd step, transfer valve 201 cuts out.In the 4th step, delivery valve 205 is opened.In the 5th step, diaphragm valve 203 is closed, by the delivery valve 205 suction analyte fluid of opening.

The amount of each cyclic suction depends on the volume that comprises in the diaphragm valve of opening, and this volume depends on again the size of inflating cells in diaphragm valve successively.Therefore, can to make in order measuring by the size of regulating the diaphragm valve inflating cells and knownly to receive the pump that the Fluid Volume that rises to the microlitre specification designs.This surge pump can be full of and pumping fluid or by the described driving working cycle of inverted running pumping fluid backward forward automatically.The valve position that should also be noted that film spare contact glass capsulation surface can be etched into has convex ridge or other finishing, with the tackiness of controlling diaphragm spare with glass surface.

Integral valve can also be used to form selector switch, mixing tank and splitter.Although should be noted in the discussion above that following structure will illustrate by the word that three-layer type is constructed, should structure also can be used for the structure of four layers or more multi-layered formula.Fig. 3 is a kind of diagram of selector switch 300.This selector switch comprises valve 301,303,305 and 317, filling channel 331,333,335,337 and 339, fluid channel 321,323,325 and 327, and diaphragm valve 309.This selector switch is drawn into arbitrary delivery port with fluid from arbitrary input aperture, and it depends on to have driven at what position which I/O valve in this suction working cycle.Drive simultaneously two or more transfer valves and can several strands of different flows be mixed into a plume at the delivery valve place.On the contrary, drive simultaneously two or more delivery valves and can the sub-thread flow be split into several strands of different liquid streams at the delivery valve place.

For example, be routed to passage 321 for making fluid from passage 327, valve 301 and 305 will keep cutting out.Then the valve 317,309 and 303 of using as described above is as pump.Selector switch comprises the function of mixing and shunting fluid passage.For fluid is mixed into passage 323 from passage 325 and 327, valve 303 will keep cutting out.For fluid is diverted to passage 323 and 327 from passage 321, valve 301 will keep cutting out.And in another embodiment, for being routed to passage 325 through the fluid that passage 327 is introduced, valve 303 and 305 will keep cutting out. Open valve 317 and 301 so that fluid flows to passage 325 by passage 327.Various set-up modes are all possible.

Utilize integral valve can also form the mixing loop.In one embodiment, can realize mixing process by fluid is mobile between two zones of device.Mixing process can be used for realizing all types of operations of carrying out on chip.Fig. 4 is a kind of diagram of mixing loop 400.This mixing loop or mixing tank comprise valve 401,403 and 405, fluid channel 411,413 and 415, and filling channel 421,423 and 425.The passage that valve is arranged that is connecting other on this loop provides to fluid and leads to or from the path of mixing tank.By passage 413 and 415, the fluid of two or more flows is entered in this mixing loop, and as mentioned above, it is drawn in circulation until these fluids mix by diffusion.Again this mixture is aspirated out this and mix loop.Mixing process can also realize by fluid is flowed between two liquid storage tanks back and forth.

Fig. 5 A-5C is the diagram of liquid storage tank 500.Fig. 5 A is the liquid storage tank top view with etched discharge opeing chamber.Fig. 5 B is the side-view of this liquid storage tank.Fig. 5 C is the side-view of the liquid storage tank after expression is filled with.Fig. 5 D is the side-view of large capacity liquid storage tank that has boring discharge opeing chamber and be used for the pump of spontaneous filling in/distribution process.This liquid storage tank is included on inflation wafer 513, and wherein film spare 505 is clipped between this inflation wafer 513 and fluid chip 511.Can fill with or emptying by passage 501 liquid storage tanks.According to numerous embodiments, the integral membrane part valve of opening in valve zone 503 plays the work of liquid storage tank in order to be used for the storage of fluid on chip.The size of inflation wafer 513 middle chambers has determined to be stored in the fluid volume in liquid storage tank, and this liquid storage tank fills up when applying vacuum, and when exerting pressure, it is emptying.

According to numerous embodiments, can replace the etching inflating cells with boring when make being used for storing the liquid storage tank of a large amount of fluids, and can directly apply driving pressure or vacuum to this boring.Perhaps, liquid storage tank can be connected to make on surge pump and not have the liquid storage tank that direct inflation connects.Fig. 5 D has represented a kind of liquid storage tank 503 that is connected on pump.This liquid storage tank is filled with or finds time according to the direction of suction, and it has advantages of variable volume.In one embodiment, pump for example valve 531,533 and 535 can be used for liquid storage tank 503 is filled in or distributing fluids.

Integral membrane part liquid storage tank with one or more fluids input apertures plays the effect of chip reactor.A burst of as liquid storage tank, this reactor can utilize the direct compression or the vacuum that apply by inflation collector wafer directly to introduce reactant or discharge product.Perhaps, this reactor can utilize integrated pump, mixing tank and/or selector switch structure indirectly to introduce reactant or discharge product.According to numerous embodiments, be subject to inflating the limitation of size of wafer middle chamber 503 due to the volume of reactor, in the situation that change not significantly the design of fluid chip inner structure, can have the reactor of any volume at any position of device.And, according to the reaction needed that relates to the different volumes reactant on chip, can partly fill in this reactor.

Most of elastic film pieces are ventilation property, thereby this character is utilized to simplify the fluid injection process of all elastic devices at present.

According to numerous embodiments, the ventilation property of film spare can eliminate bubble or air pocket.When applying to integral membrane part reactor or other fluidal texture when driving vacuum, the bubble that comes from the reaction that produces gas can be eliminated.For example, Breathable films spare can reduce the bubble that forms in the Thermal Cycling of PCR reactant on chip, and this bubble can cause the loss of reaction mixture content.

Complicated micro fluidic device can comprise the standalone module that some are connected with mobile bus.In one embodiment, provide analyte fluid to be very useful to a plurality of different fluid channels.In another embodiment, obtainable plurality of reagents can be introduced in micro fluidic device.Fig. 6 is the diagram that can be used for distributing the bus valve 600 of analyte fluid.This bus valve 600 comprises valve 601,603,605 and 607, and it is made into to make fluid to be routed to fluid channel 621,623,625 and 627 from fluid bus run 611.Filling channel 631,633,635 and 637 is in charge of the valve of controlling the fluid distribution.Typical bus valve has dead volume in bus one side.Dead volume causes the bus that is difficult between cleaning down fluid routing operation.According to numerous embodiments, the bus valve that technology of the present invention provides at the dead volume of bus side seldom or do not have.This makes the bus between fluid routing operation to be rinsed up hill and dale, and has prevented mixing or the crossed contamination between different fluid in the device operating process.

Micro fluidic device mechanism can adopt various technology to make.According to numerous embodiments, channel characteristics is etched advances chip glass, for example, utilizes the wet chemical etch technology of standard.Chip glass (thickness 1.1mm, diameter 100mm) cleans (20: 1) and utilizes LPCVD stove or sputtering system to apply sacrifice layer (200nm) polysilicon etching mask layer with sulfuric acid/hydrogen peroxide (piranha).Borofloat chip glass or Schott D263 borosilicate glass wafer are used for described device with three-layer type or the design of four laminars.After polysilicon deposition, with positive this wafer of photoresist material spin coating, gentleness is toasted and uses the contact alignment machine to make pattern.Remove the ultraviolet exposure zone of photoresist material with the Microposit photographic developer.Remove the exposure area of polysilicon by carry out etching in the SF6 plasma.In HF solution (49% HF is used for the Borofloat wafer, and the HF of 1: 1: 2: HCl: H2O is used for the D263 wafer) with the speed of 7um/min etc. to etched wafer, until reach required etch depth.

According to numerous embodiments, the fluid channel chip etching of three-layer type device becomes 20um dark, and that four laminar devices are etched into 40um is dark.The collector chip etching 70um of three-layer type device is dark, and four laminar devices are holed at valve position.Then with PRS-3000 and SF plasma, remaining photoresist material and polysilicon are peelled off from wafer respectively.Get out the through hole that runs through fluid and collector wafer and again clean this wafer with sulfuric acid/hydrogen peroxide.

In certain embodiments, the assembling of adopting the device of triple layer designs is by with PDMS film spare (HT-6135 that 254um is thick and HT-6240, Bisco Siliconees, Elk Grove, IL) be added on etch configuration in the wafer of fluid channel, and no matter on collector boring or etching discharge opeing chamber be how towards, all directly press collector hybrid glass with valve-PDMS fluid channel and cross the PDMS film from valve position.The assembling of adopting the device of four laminars designs is that to have diameter be on the thick D263 connecting wafer of the 210um of paired drilled via of 254um by at first the fluid channel wafer being thermally bonded to, wherein the position in this lead to the hole site respective channel gap.This fluid channel and connecting wafer are bonding with 570 ℃ of heating 3.5h in vacuum oven (J.M.Ney, Yucaipa, CA).The two-layer structure bond that contains full glazing channel that then will obtain is on PDMS film and collector wafer.Form like this glass-the PDMS bonding is reversible, but still enough firm in to bear vacuum and the pressure range that is applied on this device.Randomly, can obtain irreversible glass-PDMS bonding by cleaning collector wafer and PDMS film in UV ozone cleaner (Jelight Company Inc., Irvine, CA) before assembling.

The mechanism of above-mentioned micro fluidic device can be used for realizing various devices.The structure that comprises valve and pump can be set neatly so that multichannel chip type Laboratory Instruments to be provided, can be on single assembly integrated sample preparation and analytical procedure.This micro-fluidic platform is particularly suitable for the device that can realize integrated pathogen detection system as one.

Conventional quick pathogen detection system's employing enzyme-linked immunosorbent assay (ELISA) or fluorescence immunoassay (FIA) detect.Typically, detection can relate to the analyte specific antibody fixing, with the incubation of sample solution, be connected the identification of the sandwich antibody of enzyme or fluorophore, be then to develop the color and testing process.Also can use immunofluorescence detection assay method.Yet each assay method all has the detection limitation relatively.

Adopt gene test and the type mensuration of various forms of PCR-based also very general, because it has specificity and the acquisition amount of height.Yet although powerful based on the PCR method of DNA, they all are positive to alive and dead pathogenic agent, and this just might produce false-positive result.Thereby can preferably detect the RNA target compound, because it can be degraded soon, just mean that also the target compound that needs are lived can be detected.

People have also proposed the detection method that plurality of optional is selected.Develop mass spectrometry and detected pathogenic agent, gemma and other biological reagent by detecting neutral fat plastid, polarity plasmalogen and gemma specific biological mark.Yet outstanding and its specificity of the speed of mass spectrometry, flux and portability also is not confirmed.

Detection to the gemma (for example anthrax) that comes from soil, air etc. is a difficult problem, because it has the infectivity (can reach the dosage that sucks 10000 gemma under 10 spore/L in 10 minutes) of height.Most of advanced persons' detection principle is to utilize in having the silicon microreactor processed of thin film heater and integrated fluorescence excitation and testing process the PCR product is carried out detecting in real time.This system had been extended to a kind of high order nucleic acid analyser (ANAA) and a kind of portable form of ten passages afterwards.The various forms of this system also is developed in military affairs and post office.Also develop a kind of GeneXpert sample preparation system with integrated (many microlitres) sample preparation process and be used for carrying out the PCR in real time analysis.

Develop can Rapid Implementation automatic and complicated aforementioned chemical reaction and quantitatively the hand-held analyzer of pathogenic agent concentration and antibiotics resistance be a very large progress.Similarly, when a large amount of samples of needs screenings or the infected individual that may exist, can in use, high-throughput, Multi-example screening detect rapidly and measure the type of a large amount of samples and false positive rate is very low also is very useful.The development of this class automatization clinical analysers is also progressive to some extent.In one embodiment, developed the complicated micro-fluidic circuit system of the miniature form that is roughly common automatic analyser for the blood clinical analysis.Also developed a kind of integrated analyser (microlitre volume specification) comprehensively, can be used for the blood sample preparation and analyze in order to carry out HIV on microarray.This system can comprise that the complexity of a plurality of nucleic acid steps measures, and adopted＞the dead volume aerated film part valve of 100nL, and this valve will be done more detailed discussion below.

Having developed the micro-fluidic pipe of transparent plastics (lucite) flows on six different immuno-array sensors for controlling solution, this sensor is with the form control fluid of small portable system, thereby it has the performance that simple depressurized system is conducive to its immunoassay.The system of this form of developing as, utilize integrated form flow system and fibre optics biosensor Raptor hand-held analyzer capillaceous, can be at four kinds of different reagent of operating time inner analysis of ten minutes.Thereby people have been familiar with the lamination microscale experiment chamber that the peculiar property of addressable array is developed a kind of integrated form, and it can realize automatization electric field driven immunocapture and DNA hybridization array analysis.For example, the immunocapture bacterium is released for strand displacement amplification (SDA) subsequently, and the will that then increases is congratulated the hybridization analysis of sample (Shiga like) toxin gene.Yet it can not carry out the Multi-example analysis and also not study the limitation that detects.

Although conventional microfabrication is carried out on silicon, people have found that the glass micro control structure presents preferred chemistry and electrophoretic property, and the scope of plastic construction is also in continuous expansion for chemistry and biochemical analysis.In high throughput applications, technology of the present invention has channels designs radially, and it can analyze 96 to 384 clip size or the separation of checking order abreast fast parallelly.Direct integrated PCR and CE analyze and have the DNA enzyme and cut function with affinity capture on chip.

According to numerous embodiments, micro fluidic device of the present invention mechanism can form complicated channels configuration, and it can make the complex array of chamber, valve and CE analysis channel.These small-sized CE passages help to realize high resolving power electrophoretic separation very fast with intersecting to use together with syringe.Basically all operations that carries out in chromatographic column or kapillary also all has been reduced to the form of chip, and required sample size reduces thereupon, and analysis time and susceptibility are improved.

According to numerous embodiments, pathogen detection of the present invention system has the feature that susceptibility combines with specificity and quantitation capabilities, thereby a kind of useful especially measuring method is provided.Many pathogenic agent are being taken in amount of bacteria＞10 ³The time have infectivity, and V.cholera (cholera virus) in the per os intake lower than 10 ⁵The time can not cause symptom, perhaps B.anthracis (anthrax bacillus) also is considered to have influence power on the much lower level of amount.The identification bacterial strain is in order to make a distinction pathogenic strains and non pathogenic strain, and the existence of evaluation specificity toxin or antibiotics resistance gene is also very crucial for identifying dangerous and definite methods for the treatment of.In addition, can determine the concentration of bacterium or dosage and along with described qualification result carries out the determination test that excites that quantitatively report is different from background technology together, can provide the important determination test result that excites.

Fig. 7 is an embodiment diagram of a kind of pathogen detection system 700.Analyte is incorporated in immunoaffinity capture chamber 703,713 and 723 by passage 701, passage 731 places collection resistates.According to numerous embodiments, immunoaffinity reagent is used for catching, concentrate also layering to the immune chamber 703,713 and 723 of a series of separation the bacterial mixture of input.This easy process is small interface with important large-scale interface processing, and originally they were to use the obstacle that microfluidic system is carried out the trace pathogen detection.The fs of immunocapture is also playing an important role aspect the specificity that improves mensuration.For reaching higher sensitivity, the user of pathogen detection system can be then to existing reagent to carry out the duplicate acknowledgment of PCR-based, also developed the method based on the method for specific primer or more common mensuration gene type of utilizing that DNA analysis mechanism 705,715 and 725 carries out, PCR for example is in order to the existence of identifying specific bacterial strain, toxin gene and the existence of antibiotin resistance marker.In one embodiment, DNA analysis mechanism 705,715 and 725 comprises PCR and CE.

According to numerous embodiments, immunoaffinity capture chamber 703,713 and 723 and the PCR chamber integrated, but CE mechanism keeps independent.Combined immunization is caught sensitivity and the specificity that has greatly improved individual determination test with foranalysis of nucleic acids.

Very important many application from genetics differentiation pathogenic agent and non-pathogenic agent.The upstream extremity that combined immunization is caught as pcr analysis has important purification to the bacterial population of inputting, and can process the PCR inhibition that usually is present in impure complexity " reality " sample.According to numerous embodiments, the pathogen detection system will set up the PCR that implements low-circulation number (non-asymptotic) scheme, can keep and report like this quantity of the target population of inputting.In many examples, the sample of processing can then be provided with the CE analysis.Use modern microflow control technique can make cheapness, quick and durable mensuration system, its volume is little, be easy to carry and only need seldom energy, resource and operating skill.

Comprised integrated form immunity affinity capture chamber in the Analysis of pathogens instrument.Can use various capture mechanismss, for example frit, microballon, gel, single piece (monoliths) and polymkeric substance.Fig. 8 and Fig. 9 mean the immunocapture chamber diagram of utilizing silicon-dioxide frit and microballon to realize.According to numerous embodiments, the immunocapture chamber comprises a series of silicon-dioxide frits made from the mixtures filling wafer hole of silicon dioxide powder and sodium silicate binder.Through dehydration and flushing, this silicate condenses into silica gel and has formed insoluble silicon-dioxide frit at 801,803,805 and 807 places.

According to numerous embodiments, each the silicon frit diameter that forms in the thick chip glass of 1.1mm is 1mm.The immunocapture chamber accompanies with the passage 821 that is used for introducing and discharge analyte.Frit in wafer be easy to be integrated into contain film spare 811 and 813 and the device of valve and pump configuration on.In Fig. 8, four silicon frits 801,803,805 and 807 close by film spare 811 and 813.The larger silicon face of each frit is adapted to pass through various organosilane reagent and chemically derives.Be the further manufacturing of simplification device, PDMS is non-thermally bonded can chemically derived described overall chip before to all the other devices.

In one embodiment, be added in capture chamber in order to can catch and permitted great substance classes such as the microballon of frit or 1.5um such mechanism, for example gemma and bacterium.It is known for a person skilled in the art that solid-phase capture is permitted great substance classes, and its feature once was described in Weimer, B.C., M.K.Walsh, C.Beer, R.Koka, and X.Wang, 2001 Solid Phase Capture Of Proteins, Spores, and Bacter ia.Appl Environ.Microbiology, 67:1300-1307.In certain embodiments, catch for adopting microballon reagent, modify chamber in order to provide the retainer of microballon and the introducing passage of microballon with cofferdam structure.It is known to those skilled in the art that the filling of electrodynamictype microballon bed is caught with the cofferdam microballon.Perhaps, the magnetic micro-beads introducing with immunologic function can not had in the chamber in cofferdam.

Fig. 9 means the unitarily formed valve diagram of opening with no longer sealing.According to numerous embodiments, 901,903,905,907 and 909 places apply Pneumatic vacuum so that analyte flows through frit 931,933,935 and 937 along passage 921 in the zone.Can contain any amount of frit in the device of manufacturing.

Figure 10 A means the diagram that analyte is caught.According to numerous embodiments, comprise that three film spare valves 1001,1003 and 1005 pump 1000 are used for suction and contain the analyte solution of oligonucleotide, protein, cell etc. by a series of immunocapture chambers.

Chamber can be caught interested target compound with various mechanisms.Any interested material of catching in the immunocapture chamber is referred to herein as target compound.The fluid or the material that carry target compound are referred to herein as analyte.In one embodiment, target compound is Salmonellas or the Listera (Listeria) that carries in the fluid analysis thing.

In another embodiment, each capture chamber has been filled the sticky polymers matrix that contains oligonucleotide probe, so that combining target thing molecule optionally.When DNA analysis, Sanger DNA sequence dna extension products comprises primer and polysaccharase reagent in high salt concentration, and electrophoresis has wherein comprised by the immunocapture chamber immobilization acrylamide matrix that contains the covalency oligonucleotide probe in this immunocapture chamber.The sequence of catching is chosen such that to make to only have the DNA cloning product to be caught by probe, and primer and polysaccharase reagent flow through this device together in company with salt.This is just as purification of target thing molecule the complexity that need to run into when analyzing, impure mixture.

But preparation has a system of selection of little capture chamber that functional polymer catches matrix comprises that preparation has the single piece in the hole of 10-20um scope, and the preparation chamber of the microfabrication element of functional thin crosslinked polymer layer (approximately 100um) that had larger, finishing.The method of great use because sometimes microballon is difficult to be filled in capture chamber and the microballon bed usually lacks enough mechanical stabilities for routine operation.According to numerous embodiments, the forming blocks of the surface-functional polyalcohol integral spare of porous (10-20um) is to be formed directly in capture chamber by the precursor mixture photopolymerization reaction that contains monomer and Kong Sheng (porogenic) solvent.

Because polymerization process is utilized UV light and realized, can form porous polymer in any zone that needs of micro fluidic device by photolithography.the dynamics of this " miniature carving method " polymerization process that utilization is filled with the glass-chip of precursor mixture is known for a person skilled in the art, for example be described in Yu, C., F.Svec, with the Towards stationary phases forchromatography on a microchip:Molded porous polymer monoliths preparedin capillaries by photoinitiated in situ polymerization as separationmedia for electrochromatography. (the solid phase chromatographic technique that is carrying out on microchip: prepare the porous polymer whole spare of moulding as the electrochromatography spacer medium in capillary by the initial home position polymerization reaction of light) in J.M.J.Frechet 2000, Electrophoresis, 21:120-127, and Yu, C., M.Xu, F.Svec, with the Preparation of monolithic polymers withcontrolled porous properties for microfluidic chip applications usingphotoinitated free radial polymerization. (the single piece polymer that utilizes the initial Raolical polymerizable preparation of light to have controlled porous feature is applied for micro-fluidic chip) in J.M.J.Frechet 2002, J.Polymer Sci., 40:755.similarly, on can control device the exact position of integral piece of material with and surface chemical property, this is also known for those skilled in the art, as be described in Rohr, T.C, C.Yu, H.M.Davey, F.Svec, with J.M.J.Frechet 2001.Simple and efficient mixers preparedby direct polymerization in the channels of microfluidic chips. (preparing simple and effective mixture by the reaction of the direct polymerization in the passage of micro-fluidic chip), Electrophoresis, 22:3959.The porosity characteristic of single piece polymkeric substance can realize by the component of regulating porogenic solvents.

No matter use has single piece or the surface of microfabrication element, can introduce required joint element with same method of joining.Because purpose is antibody is fixed on these unitarily formed hole surfaces, the chemical substance that therefore engages is easy to react with biological polymer especially.In one embodiment, add the 2-ethene-4 of faced joint thing, 4-dimethyl azlactone can react rapidly with protein.such mechanism is known to those skilled in the art, as be described in Peterson, D.S., T.Rohr, F.Svec, and J.M.J.Frechet, described in 2002, Enzymatic microreactor-on-a-chip:protein Mapping using tryps in immobilized on porous polymer monolithsmolded in channels of microflui dic devices. (enzyme chip microreactor: utilize the Regular Insulin of fixing on the porous polymer whole spare of moulding in the micro fluidic device passage to make protein pattern), Anal.Chem., 74:4081:4088.The surface that can modify (porous integral spare, perhaps microfabrication element) is immersed in monomer solution, thereby and is to realize on selection area engaging process with the UV light irradiation apparatus.Surface-functionalized degree is by concentration, the radiated time of monomer in reaction soln, and the UV light intensity is controlled.

In another embodiment, Regular Insulin is fixed on by 2-ethene-4, on the porous polymer whole spare that 4-dimethyl azlactone, dimethyl ethyl and acrylamide or 2-hydroxyethyl methylacrylate consist of.The function of azlactone is that amine and the thiol group reaction of easy and enzyme forms stable covalent linkage.In certain embodiments, the porousness Characteristics creation that single piece is optimized low-down back pressure, make it possible to aspirate with simple mechanism and not only realize from solution immobilized enzyme but also realize subsequently substrate solution analysis.The Michealls-Menten dynamic characteristic of reactor can utilize low-molecular-weight substrate, detects as N-a-benzoyl-L-arginine ethyl ester.

People have studied the immobilization variable in great detail, the for example functionality effect of the per-cent of insulin concentration and azlactone in single piece in solution, and the reaction times to the effect of enzymic activity, and process variable such as substrate flow velocity and the effect of the time of arrheaing in reactor.On chip the proteolytic activity of enzyme microreactor under different in flow rate along with being confirmed as the caseic cracking of the fluorescent mark of substrate.Myohaemoglobin digestion when the good characteristic of this integral body microreactor can also be 0.5uL/min by rapid flow speed confirms, wherein the residence time is only 11.7s.Then utilize MALDI-TOFMS to characterize digest, identified 102 in 153 possible peptide segments, the sequential covering rate reaches 67%.

Made huge effort and come the microfabrication analytical equipment of development of new and integrated, to make miniature analysis system (Ptas).These systems provide than life size instrument has high-throughput more, still less sample and reagent consumption, smaller szie and the possibility of lower operational cost more.In the various application of micro fluidic device, analytical technology, for example electrophoresis, electrochromatography, relate to the assay method of enzyme, and immunoassay has all obtained confirmation in this way.Obtain success although the micro-fluidic chip technology is undeniable, but still had some problems.For example, most micro-fluidic chip feature is open channels configuration.Therefore, the surface-to-volume ratio of these passages is quite little.In depending on the application that reacts with solid phase surface, for example in chromatographic separation, solid phase extractions and heterogeneous catalyst, it may become serious problem.Owing to only having conduit wall can be used for providing required interaction, described micro device can only process the compound of trace.

Figure 10 B means the diagram of using the two-dimension analysis system.Single piece 1027 sealings have been caught by after having the target compound that valve 1001,1003 and 1005 pump provide in single piece 1027.In one embodiment, each chamber is heated to melt double-stranded DNA and isolate the single stranded DNA product subsequently.According to numerous embodiments, purification occurred in 120 seconds, can reach from initial 3uL only 200 times of enriched materials of 20nL.Every pipeline 1011,1013,1015,1017,1019 and 1021 all comprises for controlling or suction is hunted down the valve of target compound to be used for further analytical procedure.

In one embodiment, provide the target compound of catching to be used for PCR and CE analysis on test set.Utilization can discharge captive target compound such as the mechanism of heating or change change pH values and be used for DNA analysis.The essential characteristic of this integrated test set comprises: 1) immunocapture chamber, its etching advance to have the well heater of microfabrication and the glass substrate of temperature sensor; 2) the polymerase chain reaction chamber of 100-300nL is used for the DNA that amplification obtains from the target cell cracking; And 3) capillary electrophoresis microchannel, its etching advance for separating of with the glass substrate that detects pcr amplification.

The 4th optional feature, integrated DNA pre-concentration/purification chamber also can be added on this device, be used for pathogen gene group DNA that purifying discharges or, be used for if necessary the DNA that pre-concentration increases before injecting the CE microchannel.Although previous studies show that, for carrying out successful analysis, with pcr amplification directly on injection CE microchannel, can avoid need to extra like this complicacy, and it is necessary obtaining this purge process of high-quality electrophoretogram.The chemical property of utilizing oligonucleotide to catch matrix can realize the purge process of amplicon.If need purified genomic dna, can fill the silicon-dioxide microballon of carboxylation in purification chamber, and before PCR as catch the matrix of DNA of bacteria comprehensively.

A kind of integrated method is only to make the separate modular of immunocapture, masterplate purifying, PCR, amplicon purifying and CE on glass-chip.Recycling microchannel and various PDMS valve arrangement join these modules each other.Provided a kind of schematic diagram that is made with the Analysis of pathogens chip of independent immunocapture and PCR reactor in Figure 11.This integrated pathogen detection system comprises immunoaffinity capture chamber 1101.Analyte is incorporated in this pathogen detection system by this immunoaffinity capture chamber 1101.PCR chamber 1103 is with these immunoaffinity capture chamber 1101 couplings and receive the target compound of being caught by this immunoaffinity capture chamber 1101.CE passage 1105 and these PCR chamber 1103 couplings are in order to further to analyze.The electrode 1113 of microfabrication can be used for providing potential difference.Can also have a well heater (not shown) with this immunocapture chamber and/or the coupling of PCR chamber.Various valves have been controlled analyte flow by this integrated system.According to numerous embodiments, these valves are integral valve.

Although providing immunocapture, PCR, CE and purifying separate modular on device is a rational scheme, but suggestion is used for more simply installing, in order to be conducive to the highly sensitive separation and detection of capture rate, PCR efficient and DNA fragmentation according to numerous embodiments.Although immunocapture and PCR can carry out in independent chamber, in one embodiment, immunocapture and PCR can combine so that simplification device and treating processes.In this embodiment, PCR can successfully cause from solid substrate and solid-phase immunity capture agent.In one embodiment, PCR can utilize immune labeled microballon to carry out.

Figure 12 means that combined immunization catches the diagram with PCR chamber 1201.According to numerous embodiments, this associating chamber has integrated resistor heating arrangements (not shown) and the resistance temperature detector (RTD) 1205 of making in this receives the upgrading chamber.In certain embodiments, analyte is introduced by input aperture 1211 and by film spare valve 1221.Utilize the pressure-driven fluid to flow the target pathogenic agent is fixed in chamber 1201, and waste liquid is collected at outlet 1213 places by valve 1223.After pathogenic agent is fixing, rinse this chamber 1201 to remove the lax cell that adheres to or the reagent of non-specific binding with damping fluid.

PCR damping fluid or introduce by original sample inlet 1211, or introduce by independent special-purpose entrance.According to the target pathogenic agent in chamber 1201, the chemical cracking agent can directly be included in this PCR damping fluid.After introducing lytic reagent and/or PCR damping fluid, with catch/integrated heater 1203. in the PCR chamber is elevated to sample temperature at one temperature, makes pathogenic agent discharge simultaneously from catch matrix and according to reagent type, bacteriolyze occur.

The simplest but normally the most effective cleavage method is only to carry out heating/cooling cycle.When only heating or heating under the chemical cracking solvent of lower concentration, gram negative bacterium is easier to cracking with the eukaryotic cell that some have thinner outside cytolemma.In some cases, for example for gemma or gram positive bacterium, just may need to use the stronger lytic reagent that can disturb the PCR reaction.For example, usually N,O-Diacetylmuramidase, Proteinase K, lysostaphin and mutanolysin need to be added separately or in succession, with cracking some obstinate gram-positive staphylococcus and ﹠amp; Aureus strains.In these cases, use independent immunocapture chamber to add purifying/pre-concentration chamber and can make the intermediate capture process of carrying out DNA after lysis before pcr amplification.

This scheme catch with cracking after, the DNA of extraction can drive by electrophoresis and enter purification chamber, is absorbed and is stored by the carboxyl microballon.Utilize heating or change ionic strength and the DNA of purifying can be discharged from this purification chamber, and be transported in the PCR chamber for amplification by electrophoresis.Appear in the chamber with PCR damping fluid in case come from the DNA of lysing cell, utilize well heater and the temperature sensor of microfabrication, just can directly carry out PCR on the genetic stew of this release.

It should be noted that in some cases, due to its complicacy or to the inhibition of PCR, having the single chamber of catching with the PCR purposes concurrently can go wrong.In the situation that these are special, only need these two stages are separately carried out.In certain embodiments, if if when catching of the existing sample that matrix or microballon suppressed PCR reaction or input has been introduced the PCR inhibitor that can not wash away or neutralize, just should do like this.Like this, the DNA of release is sucked in can the cracking bacterium from capture chamber or electrophoresis is used for analyzing in independent PCR reactor.

In case completed PCR, amplicon can be directly injected in the CE microchannel for separating of and detect, according to required resolving power, can adopt intercalative dye or fluorescently-labeled primer and sex change isolation medium in isolation medium.In some cases, before injecting the CE microchannel, can introduce the DNA purification chamber so that the DNA of desalination and concentrated amplification.DNA electrophoresis by will amplification enters purification chamber, and wherein it is attached to the carboxylation microballon or oligonucleotide is caught matrix (with the capture oligo of required target compound complementation), has realized purge process.In conjunction with after wash and utilize micro-heater to carry out the release of temperature correlation, then by injecting the cross of CE microchannel, pcr amplification of concentrated and desalinization is carried out electrophoresis in order to separation and detection.

The device construction that adopts integral membrane part valve to set up pathogen detection and analytical system can vary widely.Figure 13 A means an embodiment diagram of a kind of pathogen detection and Analytical System Design.This design comprises three glass coatings, comprises channel layer 1303, articulamentum 1305, and collector layer 1309.Be provided with a PDMS film spare layer between articulamentum 1305 and collector layer 1309.Collector layer 1309 has comprised and can apply the mechanism of vacuum in order to control valve mechanism to film spare 1307.

Be provided with power connection on layer 1301, have the collector chuck layer on layer 1311.Channel layer 1303 comprises immunocapture/PCR/ purification chamber and CE microchannel, and the well heater that is positioned at the upper surface of this wafer.According to numerous embodiments, channel layer 1303 and thin chip glass 1305 thermal bondings that contain glass drilling and be connected as valve.PDMS valve/pumping diaphragm spare 1307 reversible or irreversibly with this multilayer laminated structure phase bonding.The collector layer 1309 of bottom etching passes to valve and pump on device with vacuum or pressure.

Utilize existing thin film technique to make temperature control element the first feasible way that builds test set is provided.Yet, can reduce the difficulty of processing of this device by adopting tin indium oxide (ITO) well heater.The ITO well heater is with its low-resistivity, optional transparency and famous with the compatibility of glass substrate.These well heaters can be deposited on same wafer as temperature sensor, to avoid needing back side processing and plating to form well heater.It is interior for optional heat transmission that well heater can be set directly at chamber, and perhaps it can be arranged to be resisted against on chamber in order to conduct heat energy by chip glass.The optional transparency of ITO also makes and can select the route of electric heater lead-in wire above the fluid microchannel, and can not hinder the visual of sample or pcr amplification put fruit or detect.

Figure 13 B means the microfabrication process graphical according to numerous embodiments.1381 and 1383 have represented the microfabrication process.In certain embodiments, first cleaning glass wafer (the 550 thick D263 of μ m are available from Schott of Yonkers, NY), then passing through DC magnetron sputtering system (available from UHV Sputteringof San Jose, CA) will

Amorphous silicon be deposited upon the one side of this wafer.Utilization is available from KarlSuss of Waterbury Center, the contact alignment machine of VT will be available from Shipley 1818 ofMarlborough, in the photoresist material spin coating of MA and form photoengraving pattern, then utilize the St.Petersburg available from Plasma Thermof, the SF6 in parallel plate reactive ion etching (RIE) system of FL gets rid of the bottom silicon etch mask selectively.

In certain embodiments, fluid channel, electrophoresis path and PCR chamber are etched into the degree of depth of 36 μ m in 49% hydrofluoric acid.Use the Park available from Flashcut CNC of Menlo, CA, the CNC grinding machine with diamond head get out the liquid storage tank hand-hole (diameter 1.5mm) of PDMS valve and mobile through hole (diameter 0.020 ").Then with the wafer scribe machine, wafer is cut into the slide plate of two 20mm * 75mm.Form RTD and electrode, at first can with

Ti and Pt (UHV) dash coat on the thick D263 wafer of 550 μ m.Utilization is available from Suss Microtec of Waterbury Center, and the contact alignment machine of VT will be available from Shipley (SJR 5740) of Marlborough, in the thick photoresist spin coating of MA and form pattern.According to numerous embodiments, photoresist material is dried 2 hours under 70 ℃.Utilize hot chloroazotic acid (HCl of 3: 1: HNO3,90 ℃) etching metal can form the RTD element.The formation of integrated heater be by: at first utilize available from Perkin Elmer of Wellesley, the RF sputtering system of MA will

Ti and

The multilayer film of Pt be deposited on the back side of RTD wafer.Thick photoresist is spin-coated on this face, utilizes back side contact alignment machine (Suss) to make this wafer form pattern, then dry.Utilization is available from Technic (TG 25 E) of Anaheim, and the sulfurous acid gold plating bath of CA is electroplated gold in described Ti/Pt kind layer upper 23 minute with the current density of 4.3mA/cm2, and thickness reaches 5 μ m, thereby makes heater conductor.

According to numerous embodiments, remove photoresist material and utilize thick photoresist again to form overleaf pattern.Utilization is available from Veeco Instruments of Plainview, and the ion beam etching system of NY is etched into Ti/Pt kind layer with heating unit.The RTD/ heater wafer is cut into the slide plate (Disco) of two 25mm * 75mm.In certain embodiments, utilize available from Centurion VPM, J.M.Ney, of Yucaipa, the vacuum oven able to programme of CA is with drilled tunnel wafer and RTD/ heater wafer thermal bonding.

Although can comprise single immunocapture, PCR and CE system on substrate, technology of the present invention recognizes, exploitation is used for that the Parallel Immune of clinical diagnosis is caught, PCR and CE system are also effective.In one embodiment, a kind of Portable disease substance analyser comprises three continuous immunocaptures/PCR systems, and target is three kinds of different pathogenic agent in test sample.Directly parallel arranged flow control system, heater circuit, temperature sensor and the electrophoresis apparatus that is used for three systems, single micro-slide glass has the system that enough surface-area are made three complete parallels.

In another embodiment, provide the whole Parallel Immune that is used for clinical diagnosis to catch/the PCR system.This can analyze from a plurality of individualities or in groups the ability of individual multiple different reagent provide a kind of strong method to differentiate and follow the trail of infectious agent on lemology.Figure 14 is a kind of part diagram of immunocapture/PCR device 1400 of radial parallel.Any system or device with a plurality of immunocaptures and DNA analysis mechanism of arranging around the annulus axle is referred to herein as the radial parallel device.

According to numerous embodiments, this design comprises paired analyzer array, each analyzer contain one with the integrated immunocapture of CE analyzer/PCR chamber 1423.Sample by all chambers in the given junior unit of device, makes and catches continuously plurality of reagents continuously.The single junior unit 1401,1403,1405,1407,1409,1411 of device can be analyzed different materials concurrently. Liquid storage tank 1447 and 1445 provides the input of microballon and the discharge of microballon. Liquid storage tank 1443 and 1441 is respectively negative electrode liquid storage tank and the waste liquid liquid storage tank of the capillary electrophoresis of routine.

Chamber is interconnected for compact cascade type immunity affinity capture.Valve 1431 and 1433 has sealed described chamber on cascade loop.Valve 1435 and 1437 has sealed chamber from microballon introducing and waste fluid channel.The CE microchannel is connecting conventional center anode, detects to utilize existing rotation confocal fluorescent scanner (not shown).The parallel array and the well heater with lead-in wire 1451 of joint acquisition chamber 1423 are provided, and the durable array of improved valve and pump.Due to well heater and temperature sensor multiple operation on analysis channel of accompanying with chamber 1423, use simple ring heater just more than sufficient.Thereby no longer need single well heater and temperature sensor, thereby provide economic and effective parallel pathogen detection system.

Although the above has described many elements and working method with singulative for simplicity, it will be appreciated by those skilled in the art that, also can put into practice technology of the present invention with the process of a plurality of elements and repetition.

Although the present invention's embodiment specific according to it drawn and illustrate, those skilled in the art's work should be understood can make change to form and the details of disclosed embodiment in the situation that do not depart from invention essence and scope.For example, above-mentioned embodiment can utilize various materials to realize.Therefore, scope of the present invention should be determined with reference to appended claim.

Claims (18)

1. micro-fluidic structure that comprises micro-fluidic circuit, wherein said circuit is included in the first microfluidic channel and the second microfluidic channel that diaphragm valve is met, wherein said diaphragm valve is built into when described valve opens or closes fluid and flows along first channel, and fluid only flows between the first and second passages when described valve is opened, wherein said diaphragm valve is driven by filling channel, and wherein said microfluidic structures comprises the elastic film piece that is clipped between airflow layer and Flow Control layer, described airflow layer is included in the filling channel on this airflow layer surface, and described Flow Control layer comprises described microfluidic channel, and wherein apply inflation pressure by described filling channel to described film spare and drive described valve.

2. micro-fluidic structure claimed in claim 1, wherein said circuit comprises a plurality of second passages, it is met at diaphragm valve with first channel separately, wherein each valve is built into when described valve opens or closes fluid and flows along first channel, and fluid is only mobile between the first and second passages when described valve is opened.

3. micro-fluidic structure as claimed in claim 1, it comprises a plurality of micro-fluidic circuits.

4. micro-fluidic structure as claimed in claim 2, it comprises a plurality of micro-fluidic circuits.

5. micro-fluidic structure claimed in claim 1, wherein the diaphragm valve in different circuits is controlled by the filling channel that is connected.

6. one of as any in aforementioned claim described micro-fluidic structure, wherein apply vacuum and open described valve.

7. method, it comprises that right to use requires one of any device of 2-6 that the analyte in first channel is assigned to a plurality of second passages.

8. micro-fluidic structure that comprises selector switch, described selector switch comprises at least three microfluidic channels, it intersects at public diaphragm valve, and wherein fluid is controlled by the diaphragm valve of independent operation along each channel flow, wherein said diaphragm valve is driven by filling channel, and wherein said micro-fluidic structure comprises the elastic film piece that is clipped between airflow layer and Flow Control layer, described airflow layer is included in the filling channel on this airflow layer surface, and described Flow Control layer comprises described microfluidic channel, and wherein apply inflation pressure by described filling channel to described film spare and drive described valve.

9. micro-fluidic structure claimed in claim 8, it comprises a plurality of selector switches.

10. micro-fluidic structure claimed in claim 9, the valve in wherein said a plurality of selector switches in each is common the driving.

11. the micro-fluidic structure that one of claim 8-10 is any wherein applies vacuum and opens described valve.

12. a method, it comprises that the valve that drives simultaneously in one of any selector switch of two or more claim 8-11 comes at delivery valve, different flows to be merged.

13. a method, it comprises that the valve that drives simultaneously in one of any selector switch of two or more claim 8-11 is divided into several different flows at delivery valve with single flow.

14. comprise the micro-fluidic structure of a plurality of diaphragm valves of usually opening, described diaphragm valve is controlled fluid and is flowed along each in a plurality of microfluidic channels, wherein each valve is driven by filling channel, and wherein said micro-fluidic structure comprises the elastic film piece that is clipped between airflow layer and Flow Control layer, described airflow layer is included in the filling channel on this airflow layer surface, and described Flow Control layer comprises described microfluidic channel, and wherein applies inflation pressure by described filling channel to described film spare and drive described valve.

15. the described micro-fluidic structure of claim 14, wherein said filling channel are the filling channels that is connected, it drives a plurality of valves.

16. be positioned at the pathogen detection system on single unit system, this system comprises:

Be integrated in a plurality of immunocapture chambers on this single unit system, this immunocapture chamber can be used for catching the target compound that offers described immunocapture chamber by microfluidic channel; With

a plurality of DNA analysis mechanism that is associated with described immunocapture chamber, described a plurality of DNA analysis mechanism is integrated on this single unit system, described a plurality of DNA analysis mechanism can be used for target compound is carried out DNA analysis, wherein said single unit system comprises Flow Control layer and airflow layer, described Flow Control layer comprises the microfluidic channel that the immunocapture chamber is connected with described immunocapture chamber, described airflow layer comprise filling channel and be clipped in airflow layer and the Flow Control layer between elastic layer, and wherein said single unit system comprises a plurality of diaphragm valves of usually cutting out, it is regulated fluid and flows in described Flow Control passage, and wherein the malleation in described airflow layer or negative pressure drive described diaphragm valve.

17. the described pathogen detection of claim 16 system, wherein each passage comprises at least three diaphragm valves, and each free different filling channel drives, and wherein said three valves play the effect of pump.

18. a method that is used for Analysis of pathogens, the method comprises:

By the microfluidic channel that is integrated on single unit system, the fluid analysis thing is offered a plurality of immunocapture chambers;

Catch the target compound related with described fluid analysis phase in the immunocapture chamber; And

utilize a plurality of DNA analysis mechanism be associated with a plurality of immunocapture chambers to carry out DNA analysis to described target compound, described a plurality of DNA analysis mechanism is integrated on described single unit system, wherein said micro fluidic device comprises Flow Control layer and airflow layer, described Flow Control layer comprises the microfluidic channel that the immunocapture chamber is connected with described immunocapture chamber, described airflow layer comprise filling channel and be clipped in airflow layer and the Flow Control layer between elastic layer, and wherein said single unit system comprises a plurality of diaphragm valves of usually cutting out, it is regulated fluid and flows in described Flow Control passage, and wherein the malleation in described airflow layer or negative pressure drive described diaphragm valve.

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