CN101613660A - Detect and analyze the method and apparatus of pathogenic agent - Google Patents

Detect and analyze the method and apparatus of pathogenic agent Download PDF

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CN101613660A
CN101613660A CN 200910160476 CN200910160476A CN101613660A CN 101613660 A CN101613660 A CN 101613660A CN 200910160476 CN200910160476 CN 200910160476 CN 200910160476 A CN200910160476 A CN 200910160476A CN 101613660 A CN101613660 A CN 101613660A
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chamber
immunocapture
dna
pathogen detection
pcr
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CN101613660B (en
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R·A·马蒂斯
W·H·格罗弗
B·佩格尔
A·斯克利
C·N·刘
E·拉加利
R·布拉泽
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University of California
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University of California
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Abstract

The invention provides the method and apparatus that is used to realize the microfluidic analysis device.The integral, flexible film spare that accompanies with integrated inflation collector can be provided with and drive the closely spaced array of multiple Flow Control structure, for example is used to isolate, the structure of routing, mixing, shunting and the storage flow scale of construction.This Flow Control structure can be used for implementing the 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 so that carry out further analytical procedure.

Description

Detect and analyze the method and apparatus of 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 permission No.NAG 5-9659, and NIH permission HG01399 and P01 CA 77664 and implement.
Background of invention
The present invention relates to the detection and the analysis of pathogenic agent.In one embodiment, the invention provides specimen preparation, processing, detection that utilizes micro-fluidic structure to realize 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 specimen 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 because the restriction in making processes, wearing quality and/or the design, many microfluid components are difficult to make intensive array.The device of many routines need continue to drive to keep Flow Control.The micro fluidic device that adopts this valve is removed the controllability that can lose afterwards to the composition that flows in the 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 specimen preparation.
Therefore people wish to provide improved method and apparatus to realize micro-fluidic controlling organization, for example valve, pump, selector switch, reactor or the like 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 to realize single channel system but also can be used to realize 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.With integral, flexible film spare that integrated inflation collector accompanies can be provided with and drive multiple Flow Control structure (for example be used to isolate, routing, mix, the structure of shunting and the storage flow scale of construction) closely spaced array.This Flow Control structure can be used for realizing the 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 so that 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 the 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 the single unit system.This system comprises a plurality of immunocapture chambers that are integrated on the 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 that pathogenic agent is analyzed that is used for.By the microfluidic channel that is integrated on the single unit system fluid analysis thing is offered a plurality of immunocapture chambers.The target compound relevant with this fluid analysis thing is hunted down in the immunocapture chamber.Utilize a plurality of DNA analysis mechanism that accompanies with a plurality of immunocapture chambers that target compound is carried out DNA analysis.Described a plurality of DNA analysis mechanism is integrated on the described single unit system.
In following invention description and accompanying drawing, will introduce these and its its feature and advantage of the present invention in further detail, 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 in conjunction with the accompanying drawings, and wherein accompanying drawing has been 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 a diagram of describing a kind of surge pump.
Fig. 3 is the plane diagram of a kind of Flow Control selector switch of expression.
Fig. 4 is a kind of plane diagram of mixing loop of expression.
Fig. 5 A-5D is the diagram of a kind of fluid liquid storage tank of expression.
Fig. 6 is the diagram of expression 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 is the diagram that the expression immunoaffinity is caught 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 integrated PCR of structure and the diagram of CE with immunoaffinity.
Figure 12 is that the expression combined immunization is caught the diagram with the PCR chamber.
Figure 13 A is the diagram of a kind of pathogen detection system.
Figure 13 B is the diagram in a kind of microfabrication stage of expression.
Figure 14 is the radial arrays diagram of a kind of pathogen detection system.
Particular embodiment describes in detail
To introduce some particular embodiment of the present invention in detail now, comprise the embodiment of enforcement the best of the present invention that the inventor thinks.The example of having represented these embodiments in the 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 the scope, such as in the appended claims qualification.For example, will technical scheme of the present invention be described with the glass micro-fluidic device in the 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 the 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 so that complete understanding of the present invention is provided.The present invention can implement under the situation that does not have part or all of these specific details.In other cases, known processing operation is not described in detail in order to avoid with the present invention unnecessary obscuring takes place.
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 specimen preparation and analytic function on single chip.The device that combines multichannel analysis and integrated specimen preparation simultaneously can reduce and carry out 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 minute and cost reduce and improve flux efficient and the wearing quality of measuring in single assembly.In all cases, 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 in the micro fluidic device.
Some valve technologies that are used for silicon, glass silicon, polymkeric substance and elasticity micro fluidic device have satisfied these needs with 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 keeping mobile is controlled.Adopt the micro fluidic device of this valve from Controlling System, to remove, otherwise can lose controllability the composition that flows in the device.In addition, some typical devices adopt the latex film spare that is provided with separately.Though the pneumatic latex film spare that is provided with develops to some extent separately, it is hyperchannel, high-throughout analytical equipment that this manufacture method has hindered large scale integration.
Other micro fluidic devices are to utilize anode bonded silicon and chip glass to make, and pass through Piezoelectric Driving.Yet, because the electroconductibility of silicon and chemical compatibility cause its application in analytical equipment complicated.In conjunction with or the film that is deposited on the 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 contact with the fluid of surface of elastomer and minimize.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 high-density that is suitable for to be integrated into integral membrane part valve arrangement on the micro fluidic device.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 Control 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 the 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 the wafer in conjunction with reversible and firm.
Before combination, in the wafer etching fluid channel 103 be used to carry fluid.Etch collector path 10 7 and valve zone 109 similarly in order under pressure or vacuum, to transport air or other working fluid to drive this valve.Be typically, this filling channel 107 and 109 is positioned on the wafer 105, be referred to as to inflate wafer here, and the fluid channel is etched on second wafer 101, is called fluid chip here.These etched channel characteristics can directly contact and form hybrid glass/elastomerics passage with film spare, 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 the 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 constitute XY are just made by glass.
The example of four laminar devices comprises the fluid chip 151 that is thermally bonded on the binding wafer 155.The through hole of minor diameter is arranged on the discontinuous point in the fluid channel 153.Elastic film piece 157 sticks to connecting wafer 155 1 sides of this fluid/binding wafer sandwich XZ.Valve bias current chamber 161 is etched in the collector wafer 159 and is bonding with film spare 157, has finished this four laminars device 150.Like this, fluid channel 153 has kept full glass to learn upward useful structure, can realize the Flow Control structure of large scale integration simultaneously.In some embodiments, the three-layer type device of comparing of four laminar devices shown in Fig. 1 C has suitable advantage, because it has reduced the contact between sample and the elastic film piece.
According to numerous embodiments, the various Flow Control compositions in the integral membrane part device are to exert pressure or vacuum drives by the hole on the inflation wafer.Here any one 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 to exerting pressure with the etched channels that accompanies of inflation wafer or the mechanism of vacuum is referred to herein as gas port or inflation inlet.In the three-layer type device, the etched channels of inflation in the 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 the discontinuous point of film spare away from passage, and for fluid flow provides the path of passing this discontinuous point, thereby open valve, 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, open fluid channel 103 by applying vacuum to valve zone 109 with the etched channels that accompanies of inflation wafer 105.When no longer when valve zone 109 applies vacuum or suction, film spare 111 is closed fluid channel 103, shown in Figure 1B.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 the elastic film piece, only at 161 places, valve zone contact is arranged in some cases.
Structure can be towards any direction shown in should be noted that.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, and also keeps closing condition even that is to say this valve when not being connected with the driving pressure source at device.The existing microfluidic valve that is generally opening type need continue to drive to keep installing the controllability of the composition that flows.And unlike the structure of shape memory alloy, the switch temperature of this valve constitution all is to be under the envrionment temperature, and this helps working under the biofluid situation of the aqueous solution.
In many typical embodiment, need many interfaces between the micro fluidic device so that 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 parallel in a large number inflation driving 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 the device.
Film spare valve can be used for constituting 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 the 2B, three valves that series connection is provided with have constituted 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 be full of this pump automatically 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.In 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 is comprised in the diaphragm valve of opening, and this volume depends on the size of inflating cells in the diaphragm valve again 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 automatically and can aspirate fluid forward or aspirate fluid backward by the described driving working cycle of inverted running.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.To illustrate that although should be noted in the discussion above that following structure this structure also can be used for the structure of four layers or more multi-layered formula by the literal of three-layer type structure.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 in this suction working cycle to have driven at what position which I/O valve at.Drive two or more transfer valves simultaneously and can several strands of different fluid streams be mixed into a plume at the delivery valve place.On the contrary, driving two or more delivery valves simultaneously can become several strands of different liquid to flow sub-thread fluid diverting flow 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.The valve 317,309 and 303 of using as described above then 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 being implemented in all types of operations of carrying out on the 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 to lead to or from the path of mixing tank.By passage 413 and 415 fluid of two or more flows is entered in this mixing loop, and as mentioned above, it is drawn in the circulation mix by diffusion up to these fluids.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 with boring discharge opeing chamber and large vol liquid storage tank of the pump that is used for spontaneous filling in/distribution process.This liquid storage tank is included on the inflation wafer 513, and wherein film spare 505 is clipped between this inflation wafer 513 and the 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 the valve zone 503 plays the work of liquid storage tank in order to be used for fluidic storage on the chip.The size of inflation wafer 513 middle chambers has determined to be stored in the fluid volume in the liquid storage tank, and this liquid storage tank fills up when applying vacuum, its emptying when exerting pressure.
According to numerous embodiments, make when being used to store a large amount of fluidic liquid storage tank and can replace the etching inflating cells, and can directly apply driving pressure or vacuum to this boring with boring.Perhaps, liquid storage tank can be connected to make on the 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 the pump.This liquid storage tank is filled with according to the direction of suction or is found time, and it has the advantage 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 aperture 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 to introduce reactant indirectly or discharge product.According to numerous embodiments, because the limitation of size that the volume of reactor is subjected to inflating wafer middle chamber 503 under the situation that changes the design of fluid chip inner structure not significantly, can have any volumetrical reactor at any position of device.And, according to the reaction needed that relates to the different volumes reactant on the chip, can partly fill in this reactor.
Most of elastic film pieces are ventilation property, thereby this character is utilized at present to simplify the fluid injection process of all elastic devices.
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 process 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 the 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 that the control fluid distributes.Typical bus valve has dead volume in bus one side.Dead volume causes the bus that is difficult between the 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 that the bus between the fluid routing operation can be washed up hill and dale, and has prevented mixing or the crossed contamination between the 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 the polysilicon deposition, with positive this wafer of photoresist material spin coating, gentleness is toasted and is made pattern with the contact alignment machine.Remove the ultraviolet exposure zone of photoresist material with the Microposit photographic developer.Remove the exposure area of polysilicon by in the SF6 plasma, carrying out etching.In HF solution (49% HF is used for the Borofloat wafer, and 1: 1: 2 HF: HCl: H2O is used for the D263 wafer) with speed of 7um/min etc. to etched wafer, up to reaching 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.With PRS-3000 and SF plasma remaining photoresist material and polysilicon are peelled off from wafer respectively then.Get out the through hole that runs through fluid and collector wafer and clean this wafer with sulfuric acid/hydrogen peroxide once more.
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 the etch configuration in the wafer of fluid channel, and no matter on the collector boring or etching discharge opeing chamber be how towards, all directly push collector hybrid glass-PDMS fluid channel and cross the PDMS film from valve position with valve.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 vacuum oven (J.M.Ney, Yucaipa, CA) in 570 ℃ of heating 3.5h and bonding.The two-layer structure bond that contains full glazing channel that will obtain then is on PDMS film and collector wafer.The glass of Xing Chenging-PDMS bonding is a reversible like this, but still enough firmly to bear vacuum and the pressure range that is applied on this device.Randomly, before assembling by UV ozone clean device (Jelight Company Inc., Irvine, CA) in cleaning collector wafer and PDMS film can obtain irreversible glass-PDMS bonding.
The mechanism of above-mentioned micro fluidic device can be used for realizing various devices.Can be provided with neatly comprise valve and pump structure so that multichannel chip type Laboratory Instruments to be provided, make its can be on single assembly integrated specimen 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 to develop the color and testing process then.Also can use immunofluorescence detection assay method.Yet each assay method all has the detection limitation relatively.
Adopt the gene test and the type mensuration of various forms of PCR-based also very general, because it has the 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.Developed 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 of 10000 gemma of suction under 10 spore/L in 10 minutes) of height.Most of advanced persons' detection principle is to utilize in the silicon system microreactor with thin film heater and integrated fluorescence excitation and testing process the PCR product to be detected 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 and is used for military going up and the post office.Also developed a kind of GeneXpert sample preparation system and be used to carry out the PCR in real time analysis with integrated (many microlitres) sample preparation process.
Developing the hand-held analyzer that can implement automatic and complicated aforementioned chemical reaction and quantitative pathogenic agent concentration and antibiotics resistance fast is a very big progress.Similarly, when needs screen a large amount of samples or during the infected individual that may exist, can in use, high-throughput, the screening of various product detect and measure the type of a large amount of samples apace 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, the complicated micro-fluidic circuit system of having developed the miniature form that is roughly common automatic analyser is used 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 on microarray, to carry out HIV.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 argumentation below.
Having developed the micro-fluidic pipe of transparent plastics (lucite) is used to control solution and flows on six different immuno-array transmitters, this transmitter is with the form control fluid of small portable system, thereby it has the performance that simple depressurized system helps its immunoassay.The system of this form of being developed 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 out and is used for strand displacement amplification (SDA) subsequently, and the will that increases is then congratulated the hybridization analysis of sample (Shiga like) toxin gene.Yet it can not carry out various product 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 the radial channels designs, 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 use with the intersection syringe and help to realize high resolving power electrophoretic separation very fast.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) is lower than 10 in the per os intake 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 so that make a distinction pathogenic strains and non-pathogenic bacteria strain, and the existence of evaluation specificity toxin or antibiotics resistance gene is also very crucial for identifying dangerous and definite methods of treatment.In addition, can determine the concentration of bacterium or dosage and, can provide the important determination test result that excites along with described qualification result is quantitatively reported the determination test that excites that is different from background technology together.
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, and resistates is collected at passage 731 places.According to numerous embodiments, immunoaffinity reagent is used for catching, concentrate also layering to a series of isolating immune chambers 703,713 and 723 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 for utilizing that DNA analysis mechanism 705,715 and 725 carries out based on the method for specific primer or more common mensuration gene type, 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 improved individual determination test with foranalysis of nucleic acids greatly.
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 importing, and can handle the PCR inhibition that is present in usually 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 the quantity of the target population of input like this.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 pathogenic agent analyser.Can use various capture mechanismss, for example frit, microballon, gel, single piece (monoliths) and polymkeric substance.Fig. 8 and Fig. 9 are the immunocapture chamber diagrams that expression utilizes 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 to introduce and discharge analyte.Frit in the 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 big 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 the capture chamber so that can catch and permitted great substance classes such as the such mechanism of the microballon of frit or 1.5um, 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, modify chamber so that provide the retainer of microballon and the introducing passage of microballon with cofferdam structure for adopting microballon reagent.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 is the unitarily formed valve diagram of opening that expression has 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.In one mounting arrangement between mounting, can contain any amount of frit.
Figure 10 A is the diagram that the expression analyte is caught.According to numerous embodiments, the pump 1000 that comprises three film spare valves 1001,1003 and 1005 be used to aspirate contain oligonucleotide, protein, cell etc. analyte solution by a series of immunocapture chambers.
Chamber can use various mechanisms to catch interested target compound.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 the high salt concentration, and electrophoresis has wherein comprised the immobilization acrylamide matrix that contains the covalency oligonucleotide probe by the immunocapture chamber in this immunocapture chamber.The sequence of catching is chosen such that to make to have only 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 run into when analyzing, the 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 bigger, finishing.This method of great use because sometimes microballon is difficult to be filled in the 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 the capture chamber by the precursor mixture photopolymerization reaction that contains monomer and Kong Sheng (porogenic) solvent.
Because polymerization process utilizes UV light to realize, can form porous polymer in any zone that needs of micro fluidic device by photolithography.The kinetics 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 the microchip: in kapillary, prepare the porous polymer whole spare of moulding as the electrochromatography spacer medium) among the J.M.J.Frechet 2000 by the initial home position polymerization reaction of light, 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 polymkeric substance that utilizes the initial Raolical polymerizable preparation of light to have controlled porous feature is used for micro-fluidic chip and uses) among the 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 also is 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. (by the simple and effective mixture of the direct polymerization prepared in reaction 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 the single piece still surface of use with microfabrication element can use same method of joining to introduce required joint element.Because purpose is antibody is fixed on these unitarily formed hole surfaces, therefore the chemical substance that 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 on the porous polymer whole spare of moulding in the micro fluidic device passage fixed Regular Insulin to make protein pattern), Anal.Chem., 74:4081:4088.The surface that can modify (porous integral spare, perhaps microfabrication element) is immersed in the monomer solution, thereby and is to realize on the selection area engaging process with the UV light irradiation apparatus.Surface-functionalized degree is by monomeric concentration, radiated time and the control of UV light intensity in the reaction soln.
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 constitute.The function of azlactone is that the amine and the thiol group reaction of easy and enzyme forms stable covalent linkage.In certain embodiments, the porousness feature of single piece optimization has formed low-down back pressure, makes it possible to use simple mechanism to aspirate not only to realize from solution immobilized enzyme but also realizes 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 the solution, and the reaction times is to effect and process variable such as the substrate flow velocity and the effect of the time of arrheaing in reactor of enzymic activity.On the chip 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 quick flow rate confirms that wherein the residence time only is 11.7s.Utilize MALDI-TOFMS to characterize digest then, discerned 102 in 153 possible peptide segments, the sequence fraction of coverage 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-area of these passages and volume ratio are 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 have only conduit wall can be used to the interaction that provides required, described micro device can only be handled the compound of trace.
Figure 10 B is the diagram that the two-dimension analysis system is used in expression.After single piece 1027 has been provided by the target compound that is provided by the pump with valve 1001,1003 and 1005, single piece 1027 sealings.In one embodiment, each chamber is heated with the fusing double-stranded DNA subsequently and isolates the single stranded DNA product.According to numerous embodiments, purification occurred in 120 seconds, can reach only 200 times of enriched materials of 20nL from initial 3uL.Every pipeline 1011,1013,1015,1017,1019 and 1021 all comprises being used to control or aspirate and is hunted down the valve of target compound to be used for further analytical procedure.
In one embodiment, provide the captured object thing to be used for PCR and CE analysis on the 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 to the DNA that increases and obtained from the target cell cracking; And 3) capillary electrophoresis microchannel, its etching advances to be used to separate and detect the glass substrate of 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, before injecting the CE microchannel, be used for the DNA that pre-concentration increases if necessary.Although previous studies show that, for carrying out the analysis of success, with pcr amplification directly on the injection CE microchannel, can avoid need 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.Purified genomic dna can be filled the silicon-dioxide microballon of carboxylation in purification chamber if desired, and is used as the matrix of comprehensively catching DNA of bacteria before PCR.
Integrated a kind of method is only to make the separate modular of immunocapture, masterplate purifying, PCR, amplicon purifying and CE on glass-chip.Utilize microchannel and various PDMS valve arrangement that these modules are joined each other again.Provided a kind of synoptic diagram that is made with the pathogenic agent analysis chip of independent immunocapture and PCR reactor among 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 also receives by these immunoaffinity capture chamber 1101 captured object things with these immunoaffinity capture chamber 1101 couplings.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 one and this immunocapture chamber and/or PCR chamber link coupled well heater (not shown).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 reasonable plan, but suggestion is used and is simpler device according to numerous embodiments, separates and detection so that help the highly sensitive of capture rate, PCR efficient and dna fragmentation.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 is that the expression combined immunization is caught 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 of making (RTD) 1205 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 the chamber 1201, and waste liquid is collected at outlet 1213 places by valve 1223.After pathogenic agent is fixing, wash 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 primary sample inlet 1211, or enter the mouth by independent special use and to introduce.According to the target pathogenic agent in the chamber 1201, the chemical cracking agent can directly be included in this PCR damping fluid.Introduce after lytic reagent and/or the PCR damping fluid, with catch/ integrated form well heater 1203. in the PCR chamber is elevated to sample temperature under one temperature, make pathogenic agent from catch matrix, discharge simultaneously and bacteriolyze take place according to reagent type.
The simplest but normally the most effective cleavage method is only to carry out heating.When only heating or heating under the chemical cracking solvent of lower concentration, the eukaryotic cell that gram negative bacterium and some have thin outside cytolemma is easier to cracking.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, N,O-Diacetylmuramidase, Proteinase K, lysostaphin and mutanolysin need be added separately or successively usually, 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 after lysis, carrying out DNA before the 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 to by electrophoresis and be used for amplification in the PCR chamber.Appear in the chamber with PCR damping fluid in case come from the DNA of lysing cell, utilize the well heater and the temperature sensor of microfabrication, just can on the genetic stew of this release, directly carry out PCR.
It should be noted that in some cases, because its complicacy or to the inhibition of PCR, having the single chamber of catching with the PCR purposes concurrently can go wrong.Under these special situations, only need these two stages are separately carried out.In certain embodiments, if, just should do like this if introduced can not flush away or during neutral PCR inhibitor for catching of the existing sample that matrix or microballon suppressed PCR reaction or input.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 finished PCR, amplicon directly can be injected into and be used in the CE microchannel separating 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 required target compound complementary capture oligo), has realized purge process.In conjunction with after wash and utilize micro-heater to carry out the release of temperature correlation, by injecting the cross of CE microchannel, carry out electrophoresis in order to separate and to detect with pcr amplification of desalinization then to concentrating.
The device construction that adopts integral membrane part valve to set up pathogen detection and analytical system can have very big variation.Figure 13 A is an embodiment diagram of a kind of pathogen detection of expression 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 the 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 the layer 1301, have the collector chuck layer on the 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 with contain thin chip glass 1305 thermal bondings that glass drilling is connected as valve.PDMS valve/pumping diaphragm spare 1307 reversible or irreversibly with this multilayer laminated structure bonding mutually.The collector layer 1309 of bottom etching is given valve and pump on the device with vacuum or pressure transmission.
Utilize existing thin film technique to make temperature control element the first kind of feasible way that makes up 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 celebrated with its low-resistivity, optional transparency and with the compatibility of glass substrate.These well heaters can be deposited on the same wafer as temperature sensor, to avoid needing back side processing and plating to form well heater.Well heater can be set directly at be used in the chamber to choose wantonly heat passage, and perhaps it can be arranged to be resisted against on the chamber so that by chip glass conduction heat energy.The also feasible route that can above the fluid microchannel, select the electric heater lead-in wire of the optional transparency of ITO, and can not hinder the visual of sample or pcr amplification to put really or detect.
Figure 13 B is the microfabrication process diagram of expression according to numerous embodiments.1381 and 1383 have represented the microfabrication process.In certain embodiments, (the thick D263 of 550 μ m available from Schott of Yonkers, NY), passes through DC magnetron sputtering system and (available from UHV Sputteringof San Jose, CA) will first cleaning glass wafer then
Figure G2009101604760D00191
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, utilize the St.Petersburg available from Plasma Thermof then, 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 ").With the wafer scribe machine wafer is cut into the slide plate of two 20mm * 75mm then.Form RTD and electrode, at first can with
Figure G2009101604760D00192
Ti and
Figure G2009101604760D00193
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, under 70 ℃, photoresist material was dried 2 hours.Utilize hot chloroazotic acid (3: 1 HCl: HNO3,90 ℃) etching metal can form the RTD element.The formation of integrated form well heater be by: at first utilize available from Perkin Elmer of Wellesley, the RF sputtering system of MA will
Figure G2009101604760D00194
Ti and
Figure G2009101604760D00195
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, dry then.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 last 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 to form pattern overleaf again.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 one immunocapture, PCR and CE system on the substrate, technology of the present invention recognizes that parallel immunocapture, PCR and CE system that exploitation is used for clinical diagnosis also are effective.In one embodiment, a kind of portable pathogenic agent analyser comprises three successive immunocapture/PCR systems, and target is three kinds of different pathogenic agent in the 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 enough surface-area and makes three parallel fully systems.
In another embodiment, provide the parallel immunocapture of the integral body that is used for clinical diagnosis/PCR system.This ability that can analyze from a plurality of individualities or individual in groups multiple different reagent provides 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/PCR chamber 1423 of CE analyzer.Sample makes and catches plurality of reagents continuously continuously by all chambers in the given junior unit of device.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 the negative electrode liquid storage tank and the waste liquid liquid storage tank of the capillary electrophoresis of routine.
Chamber interconnected be used 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.Owing to, use simple ring heater just more than sufficient with well heater and the temperature sensor multiple operation on analysis channel that chamber 1423 accompanies.Thereby no longer need one well heater and temperature sensor, thereby provide economic and effective parallel pathogen detection system.
Although for having described many elements and working method with singulative for simplicity, it will be appreciated by those skilled in the art that above, also can use a plurality of elements and multiple process to put into practice technology of the present invention.
Although the present invention draws and illustrates that those skilled in the art worker should understand under the situation that does not depart from invention essence and scope, can make change to the form and the details of disclosed embodiment according to its specific embodiment.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 (37)

1. pathogen detection system, this system comprises:
Be integrated in the immunocapture chamber on the micro fluidic device, this immunocapture chamber can be used for catching the target compound that offers described immunocapture chamber by microfluidic channel;
With the DNA analysis mechanism that described immunocapture chamber accompanies, this DNA analysis mechanism is integrated on the described micro fluidic device, and described DNA analysis mechanism can be used for target compound is carried out DNA analysis.
2. the described pathogen detection of claim 1 system, wherein said DNA analysis mechanism comprises PCR and CE.
3. the described pathogen detection of claim 2 system, wherein PCR is included in the chamber that is separated with described immunocapture chamber.
4. the described pathogen detection of claim 2 system, wherein PCR is included in the described immunocapture chamber.
5. the described pathogen detection of claim 2 system, the chamber of wherein said PCR is used for amplification from the resulting DNA of required target compound cracking.
6. the described pathogen detection of claim 3 system further comprises etched capillary electrophoresis microchannel, is used for separating and detecting pcr amplification.
7. the described pathogen detection of claim 6 system further comprises DNA pre-concentration and purification chamber, is used for the pathogen gene group DNA that purifying discharges, and perhaps is used for the DNA of desalination and pre-concentration amplification before injecting on the CE microchannel.
8. pathogen detection system, this system comprises:
Be integrated in the immunocapture device on the micro fluidic device, this immunocapture device can be used for catching the target compound that provides by microfluidic channel;
With the DNA analysis device that described immunocapture device accompanies, this DNA analysis device is integrated on the described micro fluidic device, and described DNA analysis device can be used for target compound is carried out DNA analysis.
9. the described pathogen detection of claim 8 system, wherein said DNA analysis device comprises the PCR chamber that is separated with described immunocapture device.
10. the described pathogen detection of claim 9 system, the chamber of described PCR is used for amplification from the resulting DNA of required target compound cracking.
11. be positioned at the pathogen detection system on the 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 described immunocapture chamber accompanies, described a plurality of DNA analysis mechanism is integrated on this single unit system, and described a plurality of DNA analysis mechanism can be used for target compound is carried out DNA analysis.
12. the described pathogen detection of claim 11 system, wherein said a plurality of DNA analysis mechanism comprises PCR and CE.
13. the described pathogen detection of claim 11 system, wherein PCR carries out in the chamber that is separated with described a plurality of immunocapture chambers.
14. the described pathogen detection of claim 13 system further comprises a plurality of etched capillary electrophoresis microchannel, is used for separating and detecting pcr amplification.
15. the described pathogen detection of claim 14 system further comprises a plurality of integrated DNA pre-concentration and purification chamber, is used for the pathogen gene group DNA that purifying discharges, and perhaps is used for the DNA of desalination and pre-concentration amplification before injecting on the CE microchannel.
16. the described pathogen detection of claim 11 system, wherein said immunocapture chamber further can be used for purifying and concentrated target compound.
17. the described pathogen detection of claim 11 system, the immunocapture chamber of wherein said a plurality of microfabrication is made into the selected antibody of Yi Baote.
18. the described pathogen detection of claim 17 system, wherein selected antibody is kept by microballon, frit, collosol and gel, gel or polyalcohol integral spare.
19. the described pathogen detection of claim 17 system, wherein selected antibody by be formed directly into the porous in the capture chamber, surface-functionalized forming polymer piece is kept.
20. being the photopolymerization reactions by the precursor mixture that contains monomer and porogenic solvents, the described pathogen detection of claim 19 system, wherein said forming blocks form.
21. the described pathogen detection of claim 17 system, wherein said a plurality of immunocapture chambers are made into the mode of radial parallel.
22. the described pathogen detection of claim 21 system further comprises and described a plurality of immunocapture chamber link coupled ring heaters, this ring heater can be used for heating described a plurality of immunocapture chamber to discharge institute's captured object thing.
23. the described pathogen detection of claim 17 system, wherein said a plurality of immunocapture chambers are made on glass coating.
24. the described pathogen detection of claim 23 system, wherein said glass coating and the coupling of integral membrane part layer.
25. the described pathogen detection of claim 23 system, wherein said glass coating comprises a plurality of etched channels, and this etched channels can be used for providing the path of fluid flow.
26. the described pathogen detection of claim 25 system, wherein said glass coating and airflow layer clip described film spare layer.
27. one kind is used for the method that pathogenic agent is analyzed, this method comprises:
By the microfluidic channel that is integrated on the single unit system fluid analysis thing is offered a plurality of immunocapture chambers;
In the immunocapture chamber, catch and the relevant target compound of described fluid analysis thing; And
Utilize a plurality of DNA analysis mechanism that accompanies with a plurality of immunocapture chambers that described target compound is carried out DNA analysis, described a plurality of DNA analysis mechanism is integrated on the described single unit system.
28. the described method of claim 27, wherein said a plurality of DNA analysis mechanism comprises PCR and CE.
29. the described method of claim 27, wherein PCR mechanism is included in the chamber that is separated with described a plurality of immunocapture chambers.
30. the described method of claim 29, wherein said a plurality of DNA analysis mechanism comprises the chamber of PCR, is used for amplification from the resulting DNA of required target compound cracking.
31. the described method of claim 29 further comprises a plurality of etched capillary electrophoresis microchannel, is used for separating and detecting pcr amplification.
32. the described method of claim 31 further comprises a plurality of integrated DNA pre-concentration/purification chamber, is used for the pathogen gene group DNA that purifying discharges, and perhaps is used for the DNA of desalination and pre-concentration amplification before injecting on the CE microchannel.
33. the described method of claim 27, wherein said immunocapture chamber further can be used for purifying and concentrated target compound.
34. the described method of claim 27, the immunocapture chamber of wherein said a plurality of microfabrication is made into to keep selected antibody.
35. the described method of claim 34, wherein selected antibody is kept by microballon, frit, collosol and gel, gel or polyalcohol integral spare.
36. the described method of claim 34, wherein selected antibody by be formed directly into the porous in the capture chamber, surface-functionalized forming polymer piece is kept.
37. an equipment that is used to detect pathogenic agent, this equipment comprises:
The fluid analysis thing is offered the device of a plurality of immunocapture chambers by being integrated in microfluidic channel on the single unit system;
Be used to catch the device of the target compound relevant with described fluid analysis thing; And
Utilize a plurality of DNA analysis mechanism that accompanies with a plurality of immunocapture chambers that described target compound is carried out the device of DNA analysis, described a plurality of DNA analysis mechanism is integrated on the described single unit system.
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