CN105658781B

CN105658781B - Microfluidic device with isolation rail and the micro- mesh calibration method of biology is tested with it

Info

Publication number: CN105658781B
Application number: CN201480058444.9A
Authority: CN
Inventors: E·D·霍布斯; M·P·怀特; J·坦纳·内维尔; D·马莱奥; 史蒂文·W·肖特
Original assignee: Berkeley's Light Life Technology Co
Current assignee: Berkeley's Light Life Technology Co
Priority date: 2013-10-22
Filing date: 2014-10-22
Publication date: 2019-07-02
Anticipated expiration: 2034-10-22
Also published as: CN110437975B; DK3060645T3; DK3473700T3; CN105658781A; CN110437975A; HK1225407A1

Abstract

A kind of microfluidic device may include at least one affected area for being fluidly coupled to bypassed area domain.The stream that can there is no medium to enable diffusion but between affected area and bypassed area domain is fluidly connected between affected area and bypassed area domain.Ability for generating the micro- target of biology of interested analyte can be measured in such microfluidic device.The micro- target of biology in the sample material being loaded into microfluidic device can be selected for special characteristic and be arranged in the domain of bypassed area.Then, sample material can flow out from affected area and can be the measurement material being flowed into affected area.The stream of medium in affected area has no substantial effect on the micro- target of biology in the domain of bypassed area, but it can be never diffused into affected area affected area by any interested analyte that the micro- target of biology generates, wherein analyte can be reacted with measurement material to generate local detectable response.Any this reaction detected can be analyzed to determine the producer which micro- target of biology (if any) is interested analyte.

Description

Microfluidic device with isolation rail and the micro- mesh calibration method of biology is tested with it

Background technique

As microfluidic field is constantly progressive, microfluidic device has become for handling and operation such as biological cell The convenient platform of micro- target.Some embodiments of the present invention are intended to improve microfluidic device and operate the side of the microfluidic device Method.

Summary of the invention

In some embodiments of the invention, a kind of microfluidic device may include flow region and microfluid isolation rail. Flow region can be configured as the stream comprising first fluid medium.Microfluid isolation rail may include isolation structure and bonding pad Domain.Isolation structure may include the area of isolation for being configured to contain second fluid medium.Join domain can be will be isolated on fluid Region is connected to flow region, so that when flow region and microfluid isolation rail are substantially filled with fluid media (medium): second is situated between The component of matter can be diffused into first medium or the component of first medium can be diffused into second medium；And it is basic The upper not first medium from flow region is flowed into area of isolation.

Some embodiments of the present invention include a kind of process of micro- target of biology analyzed in microfluidic devices, the miniflow Body device may include microfluidic channel connected to it at least one microfluid isolation rail fluid.At least one isolation rail It may include fluid isolation structure, which includes that area of isolation is connected to the company in channel on area of isolation and fluid Connect region.The process may include by one or more micro- target loads of biology at least one isolation rail, and culture The micro- target of biology of loading for a period of time, is enough that the micro- target of biology is made to generate interested analyte.The process may also include Setting captures micro- target in the channel of adjacent openings (opening is from the join domain of at least one isolation rail to channel), with And it is monitored to that will capture micro- target and be bonded to interested analyte.Capturing micro- target may include that specific can bond sense The affinity agent of at least one type of the analyte of interest.

Detailed description of the invention

Fig. 1 be according to some embodiments of the present invention can to micro- target in microfluidic devices execute at least two The example of the process of test.

Fig. 2A is the microfluidic device of the process that Fig. 1 can be executed by it according to some embodiments of the present invention Perspective view.

Fig. 2 B is the sectional view of the microfluidic device of Fig. 2A.

Fig. 2 C is the overhead sectional view of the microfluidic device of Fig. 2A.

Fig. 3 A is the micro- of Fig. 2A to Fig. 2 C for lacking barrier (for ease of description) according to some embodiments of the present invention The side partial cross-sectional of fluid means, wherein selector is configured as dielectrophoresis (DEP) device.

Fig. 3 B is the partial cross-sectional top view of Fig. 3 A.

Fig. 4 A is another exemplary perspective view of microfluidic device according to some embodiments of the present invention.

Fig. 4 B is the sectional view of the microfluidic device of Fig. 4 A.

Fig. 4 C is the overhead sectional view of the microfluidic device of Fig. 4 A.

Fig. 5 shows the example of isolation rail according to some embodiments of the present invention, wherein from channel to area of isolation The length of join domain is greater than the penetration depth of the medium flowed in the channel.

Fig. 6 is another example of isolation rail according to some embodiments of the present invention, which includes from logical To the join domain of area of isolation, which is longer than the penetration depth of the medium flowed in the channel in road.

Fig. 7 A to Fig. 7 C shows another example of the configuration of isolation rail according to some embodiments of the present invention.

Fig. 8 shows the microfluid by the micro- target load of biology to Fig. 2A to Fig. 2 C according to some embodiments of the present invention and fills The example in flow path set.

Fig. 9 shows the microfluid dress for making the micro- target of biology be flowed into Fig. 4 A to Fig. 4 C according to some embodiments of the present invention The example in channel set.

Figure 10 shows the microfluid tested for fisrt feature in Fig. 2A to Fig. 2 C according to some embodiments of the present invention The example of the micro- target of biology in the flow path of device.

Figure 11 is that the biology of selection according to some embodiments of the present invention in the microfluidic device of Fig. 2A to Fig. 2 C is micro- The example of target.

Figure 12 shows biology of the selection according to some embodiments of the present invention in the microfluidic device of Fig. 4 A to Fig. 4 C The example of micro- target.

Figure 13 show according to some embodiments of the present invention the selected micro- target of biology is moved to Fig. 2A to Fig. 2 C Microfluidic device in keep fence in example.

Figure 14 is shown in the flow path of the microfluidic device of slave Fig. 2A to Fig. 2 C according to some embodiments of the present invention The example of the micro- target of biology washed away.

Figure 15 shows being moved to the selected micro- target of biology from channel according to some embodiments of the present invention Example in the isolation rail of the microfluidic device of Fig. 4 A to Fig. 4 C.

Figure 16 is washed away in channel in the microfluidic device of slave Fig. 4 A to Fig. 4 C according to some embodiments of the present invention The micro- target of biology example.

Figure 17 is the microfluid dress being supplied to measurement material in Fig. 2A to Fig. 2 C according to some embodiments of the present invention The example for the micro- target of biology kept in fence set.

The holding that Figure 18 shows the microfluidic device for being diffused into Fig. 2A to Fig. 2 C according to some embodiments of the present invention is enclosed Measurement material in column.

Figure 19 shows the survey in the channel of the microfluidic device in Fig. 4 A to Fig. 4 C according to some embodiments of the present invention Determine the example of the micro- target of biology of material and the interested analyte of generation in isolation rail.

Figure 20 shows the area of isolation for diffusing out isolation rail according to some embodiments of the present invention and arrives in Fig. 4 A Showing for the component of the interested analyte of the measurement material reaction of channel proximal openings is adjacent in the microfluidic device of Fig. 4 C Example.

Figure 21 be in the microfluidic device in Fig. 4 A to Fig. 4 C according to some embodiments of the present invention include label The micro- target of capture measurement material example.

Figure 22 is micro- including capturing in the microfluidic device in Fig. 4 A to Fig. 4 C according to some embodiments of the present invention The example of the measurement material of the mixture of target and label object.

Figure 23 shows the micro- target of capture of Figure 22 according to some embodiments of the present invention, the component of label object and interested Analyte example.

Figure 24 shows the example of the micro- target of compound capture including multiple affinity agents according to some embodiments of the present invention.

Figure 25 be show detection local reaction according to some embodiments of the present invention and identification microfluidic device (such as Device shown in Fig. 4 A to Fig. 4 C) in the isolation rail comprising the micro- target of positive organisms exemplary process.

Figure 26 show according to some embodiments of the present invention by the micro- target of negative organisms from keeping fence to be moved to and scheming In flow path in the device of 2A to Fig. 2 C.

Figure 27 show according to some embodiments of the present invention from the flowing road in the microfluidic device in Fig. 2A to Fig. 2 C The micro- target of negative organisms is washed away in diameter.

Figure 28 shows measurement of the cleaning according to some embodiments of the present invention in the microfluidic device of Fig. 4 A to Fig. 4 C The example in the channel of material.

Figure 29 is according to some embodiments of the present invention the negative organisms in the fluid means of Fig. 4 A to Fig. 4 C are micro- The example that target and the micro- target of positive organisms separate.

Figure 30 shows the isolation rail in the microfluidic device in Fig. 4 A to Fig. 4 C according to some embodiments of the present invention The middle example for generating the micro- target of clone biological.

Figure 31 A to Figure 31 C describes the microfluidic device including microchannel and the multiple isolation rails for opening microchannel.Often A isolation rail includes multiple mouse boosting cells.Figure 31 A is the bright field image of a part of micro-channel device.Figure 31 B and figure 31C is the fluorescent image obtained using Texas red optical filter.In 31B, the antigentic specificity described in example 1 is surveyed 5 minutes acquisition images after fixed beginning.In Figure 31 C, the antigentic specificity described in example 1 measurement start after 20 minutes Obtain image.White arrow in Figure 31 C is directed toward the isolation rail for generating positive signal in the assay.

Specific embodiment

Present specification describes exemplary embodiment of the present invention and application.However, that the present invention is not restricted to these is exemplary Embodiment and application are either not limited to that mode or exemplary embodiment and application operation mode is described herein.And And attached drawing can show simplified or partial view, and for clarity, the component size in attached drawing can not to scale (NTS) expand or Person reduces.In addition, when term used herein " ... on ", " being attached to " or when " being connected to ", an element is (for example, material Material, layer, substrate etc.) can " on the other element ", " being attached to another element " or " being connected to another element ", and Regardless of an element directly on the other element, be attached or coupled to another element, still have one or more Intervening element is between an element and another element.In addition, if provide if, direction (for example, above, Below, top, bottom, side, it is upper and lower ... below ... above, above, below, level, vertical, " x ", " y ", " z " Deng) be opposite and be provided by way of example only, in order to illustrating and discuss and without limitation.In addition, to a series of In the case that element (such as element a, b, c) does appended drawing reference, these appended drawing references are intended to include listed element itself Any one, the combination of element listed by any combination and/or whole less than element listed by whole.

Expected purpose is sufficiently achieved as it is used herein, " substantially " referring to.Term " multiple " refers to more than one.

As it is used herein, term " micro- target " may include following one or more: without the micro- target of life, such as Particle, microballon are (for example, polystyrene bead, Luminex^TMPearl etc.), magnetic bead, micron bar, microfilament, quantum dot etc.；The micro- target of biology, Such as cell (for example, the cell obtained from tissue or body fluid sample, haemocyte, hybrid cell, culture cell, from cell line Cell, cancer cell, infected cell, transfection and/or transformed cells, reporter cell etc.), liposome is (for example, the film system of synthesis Agent or as derived from film preparation), nano-lipid raft etc.；Or without the micro- target of life and the micro- target of biology (for example, being attached to cell Microballon, liposomal microballon, liposomal magnetic bead etc.) combination.For example, in " Ritchie et al. (2009) Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,Mehotd Enzymol., (Ritchie et al. (2009), the recombination of the memebrane protein in phospholipid bilayer nanometer disk, the side 464:211-231 Method zymetology, 464:211-231) " in, nano-lipid raft is described.

As it is used herein, term " cell " refers to biological cell, can be plant cell, zooblast (for example, Mammalian cell), bacterial cell, fungal cell etc..Zooblast can be for example from the mankind, mouse, rat, horse, mountain Sheep, Mianyang, ox, primate etc..

" component " of fluid media (medium) is that any chemical or biochemical molecule, the medium presented in the medium includes molten Agent molecule, ion, small molecule, antibiotic, nucleotide and nucleosides, nucleic acid, amino acid, peptide, protein, carbohydrate, carbohydrate, Lipid, fatty acid, cholesterol, metabolite etc..

As herein in regard to used in fluid media (medium), " making ... diffusion " and " diffusion " refers to the component of fluid media (medium) towards concentration The thermodynamic motion in the low direction of gradient.

Phrase " stream of medium " refers to the mass motion as caused by any mechanism in addition to diffusion of fluid media (medium).Example Such as, the stream of medium may include from a point to the movement of another fluid media (medium) put due to the pressure difference between point.It is such Stream may include the continuous of liquid, pulse, period, random, interval or reversing current, or any combination thereof.When a fluid media (medium) When being flowed into another fluid media (medium), the turbulent flow and mixing of medium can lead to.

Phrase " there is no stream " refers to the flow velocity of fluid media (medium), is less than material (for example, interested analysis Object) diffusion of components into fluid media (medium) or the rate that spreads material in fluid media (medium).The expansion of the component of this material Scattered rate may depend on the intensity of the interaction between the size and component and fluid media (medium) of such as temperature, component.

As herein in regard to used in the different zones in microfluidic device, phrase " connecting on fluid " refers to ought not same district When domain is substantially filled with liquid (such as fluid media (medium)), the fluid in each region is connected to form the single sheet of fluid Body.This fluid being not meant in different zones (or fluid media (medium)) must be identical in composition.On the contrary, in microfluid Fluid on the different fluids of device in join domain can have different compositions (for example, the solute of various concentration, such as albumen Matter, carbohydrate, ion or other molecules), since solute is mobile and continuous to the low direction of its respective concentration gradient Variation and/or fluid are flowed by the device.

In some embodiments, microfluidic device may include " involving " region and " not involving " region.Bypassed area domain can To be fluidly connected to affected area, it is assumed that fluidly connect that be configured such that between affected area and bypassed area domain can It spreads, but there is no the stream of medium between affected area and bypassed area domain.Therefore microfluidic device can be configured to Substantially be isolated bypassed area domain and the stream of the medium in affected area, at the same make affected area and bypassed area domain it Between can only be diffused fluid communication.

Ability for generating the micro- target (for example, biological cell) of biology of particular organisms material can be in such miniflow It is measured in body device.For example, it is directed to the production of interested analyte, the sample material including the micro- target of biology to be determined It can be loaded onto the affected area of microfluidic device.Multiple micro- targets of biology can be selected for special characteristic and by It is arranged in the domain of bypassed area.Then, remaining sample material can be flowed out from affected area, and measured material and can be flowed into In affected area.Since the selected micro- target of biology is in the domain of bypassed area, the selected micro- target of biology substantially not by From the influence of the inflow of the outflow or measurement material of remaining sample material.The selected micro- target of biology can permit generation sense The analyte of interest can be never diffused into affected area affected area, wherein interested analyte can be with measurement To generate local detectable response, each part detectable response can be related to specific bypassed area domain for material reaction.With inspection The associated any bypassed area domain of the reaction measured can be analyzed to determine (if any) in the domain of bypassed area Which micro- target of biology is enough producers of interested analyte.

Fig. 1 shows the process for being used to test micro- target in microfluidic devices according to some embodiments of the present invention 100 example.Fig. 2A to Fig. 2 C is shown can be by it come the example and Fig. 3 A of the microfluidic device 200 of implementation procedure 100 The example of dielectrophoresis (DEP) device of a part that can be microfluidic device 200 is shown with Fig. 3 B.Fig. 4 A to Fig. 4 C was shown Journey 100 can also pass through another example for the microfluidic device 400 that it is performed.However, 200 He of device of Fig. 2A to Fig. 2 C The device 400 of Fig. 4 A to Fig. 4 C is not limited to execute the process 100 of Fig. 1.Process 100 is also not necessarily limited to the quilt on device 200 or 400 It executes.

As shown in Figure 1, the mixture of micro- target can be loaded into microfluidic devices by process 100 at step 102 Flow path in.The mixture loaded at step 102 may include different types of micro- target and fragment and other targets. At step 104, process 100 can test micro- target in flow path for fisrt feature, and at step 106, mistake Journey 100 can will test the micro- target not being positive with fisrt feature is directed to for micro- target of fisrt feature positive test (for example, micro- target that test is negative) separates.As shown, process 100 can repeat step 102 to step 106 any time Number.For example, step 102 can be performed k times to step 106, then, k micro- targets being loaded at step 102 Mixture is divided into the original group of micro- target at step 104,106, and (all micro- targets of the original group are special for first Sign is tested to be positive).Quantity k can be 1 or any integer greater than 1.(hereinafter, for the biology of positive test Micro- target is sometimes referred to as " positive ", and the micro- target of biology (for example, being negative for test) not being positive for being used to test The sometimes referred to as micro- target of " feminine gender " biology.)

Then, process 100 can continue to 108, after wherein process 100 can execute the original group of micro- target Continuous test.The follow-up test executed at step 108 can be different from the first test executed at step 104.For example, subsequent Test can be tested for the subsequent characteristics different from the fisrt feature tested at step 104.As another example, The follow-up test executed at step 108 can be carried out for feature identical with step 104 (fisrt feature above-mentioned) Test, but sensitivity, accuracy, the precision degree etc. that follow-up test is different.For example, being directed to fisrt feature, executed at step 108 Follow-up test can than at step 104 execute first test it is sensitiveer.Anyway, at step 110, process 100 Can by the micro- target being positive for follow-up test at step 108 and the micro- target being negative for follow-up test every It opens.

If the first test and the follow-up test of step 108 of step 104 are directed to identical characteristic test, in step 108 After 110, in response to two different tests, (it is referred to as the first spy in the discussion of above-mentioned steps 104 for this feature Sign) positive test micro- target with a micro- mesh of k for being loaded onto when k time of step 102 executing in microfluidic device Target mixture separates.As shown, step 108 and 110 can be repeated, and in each repeat, process 100 can be in step Using the different follow-up tests tested for identical feature at rapid 108.In fact, step 108 and 110 can be weighed Multiple n times, then, process 100 is from the mixture of the k being loaded onto microfluidic device at step 102 micro- targets Sub-elect the fisrt feature positive test n+1 times micro- target for being directed to and having tested at step 104 and step 108.Quantity n It can be 1 or any integer greater than 1.

As mentioned, alternatively, process 100 can be at step 108 for first with the test at step 104 The different subsequent characteristics of feature are tested.In such embodiments, from being loaded into microfluidic device at step 102 In k micro- target mixture in sub-elect micro- target with both fisrt feature and subsequent characteristics.If step 108 and 110 are repeated, then in each repeat, process 100 can be tested at step 108 for different subsequent characteristics.Example Such as, in each execution of step 108, process 100 can for it is not only different from fisrt feature but also with it is in office how before It is tested by step 108 subsequent characteristics different with any pervious subsequent characteristics of 110 tests.In the every of step 110 When secondary execution, process 100 can be separated micro- target that subsequent characteristics positive test is directed at step 108.

As mentioned, n times step 108 and 110 can be repeated.After executing n times step 108 and 110, process 100 Sub-elected from the mixture of the k being loaded at step 102 in microfluidic device micro- targets have in step 104 and The micro- target for all n+1 features tested at 108.Quantity n can be 1 or any integer greater than 1.

Contemplate the modification of process 100.For example, in some embodiments, the repetition of step 108 can be directed to sometimes not to exist The new feature that do not tested when any preceding execution of step 104 place or step 108 is tested, and it is other when can For at step 104 or identical feature that when preceding execution of step 108 tested is tested.As another A example, at step 106 or the step 110 of any repetition, process 100 can be by micro- target of positive test and test The micro- target being negative separates.As another example, it is more that process 100 can repeat step 104 before continuing step 106 It is secondary.In such an example, process 100 can be tested at each repetition of step 104 for different features, then It is in yin by micro- target of positive test is tested with the place of repeating at least once in step 104 at each repetition of step 104 Micro- target of property separates.Similarly, step 108 can be repeated quickly and easily as many times as required before continuing step 110.

Now discuss for Fig. 2A to Fig. 7 C to the example of microfluidic device 200 and 400.Then, for Fig. 8 to Figure 30 To the operation example of the process 100 (the wherein micro- target of biology that micro- target includes such as biological cell) using device 200 and 400 It is described.

Fig. 2A to Fig. 2 C is shown can be by it come the example of the microfluidic device 200 of implementation procedure 100.As shown, Microfluidic device 200 may include that microfluidic device 200 may include shell 202, selector 222, detector 224, stream controller 226 With control module 230.

As shown, shell 202 may include one or more flow regions 240 for keeping liquid medium 244.Figure 2B, which shows medium 244 thereon, can be set to the inner surface of uniform (for example, flat) and undistinguishable flow region 240 242.Alternatively, however, inner surface 242 can be non-uniform (for example, uneven) and including such as electrode terminal The feature of (not shown).

Shell 202 may include one or more entrances 208, and medium 244 can pass through one or more entrance 208 are input into flow region 240.Entrance 208 can be such as input port, opening, valve, another channel, fluid and connect Connect device etc..Shell 202 may also include one or more outlets 210, and medium 244 can pass through one or more of outlets 210 It is removed.Outlet 210 can be such as output port, opening, valve, channel, fluid connector.As another example, out Mouth 210 may include No. 13/856,781 (attorney docket of the U.S. Patent Application Serial submitted in such as on April 4th, 2013 BL1-US the drop output mechanism of any output mechanism in the output mechanism disclosed in).The all or part of shell 202 can be with Be it is ventilative, to allow gas (for example, surrounding air) to enter and leave flow region 240.

Shell 202 may also include the microfluidic structures 204 being arranged on base portion (for example, substrate) 206.The microfluidic structures 204 may include flexible material, such as rubber, plastics, elastomer, silicone resin (for example, silicone resin of patternable), poly dimethyl Siloxanes (" PDMS ") etc., can be ventilative.Alternatively, microfluidic structures 204 may include include rigid material Other materials.Base portion 206 may include one or more substrates.Although being shown as single structure, base portion 206 may include multiple mutual Structure even, such as multiple substrates.Microfluidic structures 204 equally may include the multiple structures that can be interconnected.For example, microfluid Structure 204 may also include the lid (not shown) made of the material identical or different with the other materials in the structure.

Microfluidic structures 204 and base portion 206 can limit flow region 240.Although showing a flowing in Fig. 2A to Fig. 2 C Region 240, but microfluidic structures 204 and base portion 206 can limit multiple flow regions for medium 244.Flow region 240 can Including the channel (252 in Fig. 2 C and 253) that can be interconnected and chamber to form microfluidic circuit.For including more than one Flow region 240 encloses boundary, each flow region 240 can with one or more entrances 108 and it is one or more go out Mouthfuls 110 associated, for input respectively and from 240 removal medium 244 of flow region.

As shown in fig. 2 b and fig. 2 c, flow region 240 may include one or more channels 252 for medium 244.Example Such as, channel 252 usually can be from entrance 208 to outlet 210.Again as shown, limiting non-current space (or area of isolation) Keep fence 256 that can be arranged in flow region 240.That is, a part of each inside for keeping fence 256 can To be the non-flowing space, other than when empty flow region 240 is by primitively filled media 244, Jie from channel 252 Matter 244 does not flow directly into non-current space.For example, each holding fence 256 may include one or more barriers 254, It forms part and encloses boundary, which may include non-current space.When flow region 240 is filled with medium 244, limits and protect Therefore the barrier 254 for holding fence 256 can prevent medium 244 from being directly flowed into the protected of any holding fence 256 from channel 252 It is internal.For example, the barrier 254 of fence 256 can substantially prevent to come from channel when flow region 240 is filled with medium 244 The bulk flow of 252 medium 244 is flowed into the non-current space of fence 256, on the contrary, substantially only allowing in fence 256 Medium in non-current space is mixed with the diffusion of the medium from channel 252.Therefore, keeping non-current in fence 256 Exchanging for nutriment and waste between space and channel 252 substantially can only pass through diffusion.

It is aforementioned can by by fence 256 be oriented such that in fence 256 opening not directly facing in channel 252 The stream of medium 244 is completed.For example, if the stream of medium be in channel 252 in fig. 2 c from entrance 208 to outlet 210 (simultaneously Therefore from left to right), because left side of the opening of each fence 256 not in Fig. 2 C (otherwise will be directed into such In stream), so each of fence 256 is essentially prevented medium 244 and is directly flowed into fence 256 from channel 252.

Can be there are many such holdings fence 256 in the flow region 240 being arranged with any pattern, and holding is enclosed Column 256 can be any many different size and shape.Although the side wall for the microfluidic structures 204 being shown as in opposite Fig. 2 C Setting, but one or more (including all) fences 256 can be the microfluidic structures 204 being arranged in channel 252 Absolute construction except side wall.As shown in Figure 2 C, the opening of fence 256 is kept to can be set to adjacent channel 252, it can be with Adjacent to the opening of more than one fence 256.Though it is shown that a channel 252 of neighbouring 14 fences 256, but can have More channels 252, and can have more or less fences 256 of neighbouring any special modality 252.

The barrier 254 of fence 256 may include any kind of material discussed above in relation to microfluidic structures 204.Screen Hindering 254 may include and the identical material of microfluidic structures 204 or different materials.As shown in Figure 2 B, barrier 254 can be from base portion 206 surface 242 passes through the upper wall (opposite with surface 242) that entire flow region 240 extends to microfluidic structures 204.It can replace Dai Di, one or more barriers 254 can be extended with only partially through flow region 240, therefore not extend completely to surface 242 or microfluidic structures 204 upper wall.

Selector 222, which can be configured as, is formed selectively electric power to micro- target (not shown) in medium 244.Example Such as, selector 222 can be configured as selectively activation (for example, opening) and deactivate (for example, closing) in flow region Electrode at 240 inner surface 242.Electrode can form attraction or repel the medium of micro- target (not shown) in medium 244 Power in 244, and therefore selector 222 can select and one or more micro- targets in move media 244.Electrode can be with It is such as dielectrophoresis (DEP) electrode.

For example, selector 222 may include one or more optics (for example, laser) forceps device and/or one or more (for example, as disclosed in U.S. Patent No. 7,612,355, (entire contents pass through reference to multiple photoelectricity tweezers (OET) devices Mode be incorporated herein), or as U.S. Patent Application Serial the 14/051st, 004 (attorney docket BL9-US) is disclosed (entire contents are incorporated herein also by the mode of reference).As another example, selector 222 may include for moving One or more device (not shown) of drop of the medium 244 for one or more micro- targets that wherein suspend.Such device (not shown) may include Electrowetting device, and such as photoelectricity wetting (OEW) device is (for example, such as 6,958, No. 132 institutes of U.S. Patent No. It is disclosed).Therefore selector 222 can be characterized as being DEP device in some embodiments.

Fig. 3 A and 3B show the example that wherein selector 222 includes DEP device 300.As shown, DEP device 300 can wrap Include first electrode 304, second electrode 310, electrode activation substrate 308, power supply 312 (for example, exchange (AC) power supply) and light source 320.Medium 244 and electrode activation substrate 308 in flow region 240 can separate electrode 304,310.Change and comes from light source The pattern of 320 light 322 selectively activates and deactivates the change at the region 314 of the inner surface 242 of flow region 240 The pattern of DEP electrode.(hereinafter, region 314 is referred to as " electrode zone.")

In the example shown in Fig. 3 B, the light pattern 322' being directed on inner surface 242 illuminate shown in square figure The cross-hatched electrode zone 314a of case.Another electrode zone 314 is not illuminated, thus hereinafter referred to as " dark " electrode district Domain 314.Pass through that relative resistance that electrode activates substrate 308 to second electrode 310 is anti-to be greater than from the from each scotomete region 314 One electrode 304 passes through the relative impedances that the medium 244 in flow region 240 arrives scotomete region 314.However, illuminating electrode district Domain 314a is reduced from the electrode zone 314a being illuminated and is passed through the relative impedances that electrode activates substrate 308 to second electrode 310, The impedance is less than the medium 244 from first electrode 304 in flow region 240 to the opposite of the electrode zone 314a being illuminated Impedance.

It is aforementioned in the electrode zone 314a being illuminated and neighbouring scotomete region in the case where power supply 312 is activated Electric-force gradient is formed in medium 244 between 314, which forms in turn attracts or repel near in medium 244 The local DEP power of micro- target (not shown).It can be therefore by changing from light source 320 (for example, laser source or other kinds of light Source) light pattern 322 that projects microfluidic device 200, flow region 240 inner surface 242 it is many it is different as The DEP electrode for attracting or repelling micro- target in medium 244 is selectively activated or deactivated at electrode zone 314.Whether DEP power Micro- target near attracting or repelling may depend on such as frequency of power supply 312 and medium 244 and/or micro- target (not shown) Dielectric properties parameter.

The square pattern 322' for the electrode zone 314a being illuminated shown in Fig. 3 B is only example.Electrode zone 314 The pattern for the electrode district 322' that any pattern can be illuminated by projecting the pattern of the light 322 in device 200, and be illuminated It can be repeatedly changed by changing light pattern 322.

In some embodiments, electrode activation substrate 308 can be light-guide material, and inner surface 242 can be no spy Sign.In such embodiments, can according to light pattern 322 from anywhere on the inner surface 242 of flow region 240 simultaneously Form DEP electrode 314 with any pattern (referring to Fig. 3 A).The quantity and pattern of electrode zone 314 is not therefore fixed, Corresponding to light pattern 322.Example illustrates in U.S. Patent No. 7,612,355 above-mentioned, wherein the attached drawing institute of aforementioned patent The non-impurity-doped amorphous silicon material 24 shown can be the example that may make up the photoconductive material of electrode activation substrate 308.

In other embodiments, electrode activation substrate 308 may include the circuit substrate of such as semiconductor material, including be formed Multiple doped layers, electric insulation layer and the conductive layer of known semiconductor integrated circuit in such as semiconductor field.Such In embodiment, circuit element may be formed at electrode zone 314 at the inner surface 242 of flow region 240 and second electrode 310 it Between electrical connection, which selectively can be activated and be deactivated by light pattern 322.When inactive, it is each electrically connected High impedance can be had by connecing, so that being greater than from corresponding electrode zone 314 to the relative impedances of second electrode 310 from first electrode 304 arrive the relative impedances of corresponding electrode zone 314 by medium 244.However, when being swashed by the light in light pattern 322 When living, each electrical connection can have Low ESR, so that the relative impedances from corresponding electrode zone 314 to second electrode 310 are small In passing through medium 244 to the relative impedances of corresponding electrode zone 314 from first electrode 304, this is being corresponded to as discussed above Electrode zone 314 at activate DEP electrode.Light pattern 322 can therefore be passed through in many of the inner surface 242 of flow region 240 The DEP for attracting or repelling micro- target (not shown) in medium 244 is selectively activated and deactivated at different electrode zones 314 Electrode.The non-limiting example of such configuration of electrode activation substrate 308 includes U.S. Patent No. 7,956,339 Figure 21 With the OET device 300 shown in Figure 22 based on phototransistor and in U.S. Patent Application Serial the 14/051st above-mentioned, No. 004 all OET devices shown in the drawings.

In some embodiments, first electrode 304 can be a part of the first wall 302 (or lid) of shell 202, and Electrode activation substrate 308 and second electrode 310 can be a part of the second wall 306 (or base portion) of shell 202, usually as schemed Shown in 3A.As shown, flow region 240 can be between the first wall 302 and the second wall 306.However, aforementioned only show Example.In other embodiments, first electrode 304 can be a part of the second wall 306, and electrode activation substrate 308 and/or the One or two of two electrodes 310 can be a part of the first wall 302.As another example, first electrode 304 can be with It is a part of wall 302 or 306 identical with electrode activation substrate 308 and second electrode 310.For example, electrode activates substrate 308 It may include first electrode 304 and/or second electrode 310.In addition, light source 320 is alternatively located at 202 lower section of shell.

It is configured to DEP device 300 shown in Fig. 3 A and Fig. 3 B, selector 222 can be therefore by projecting light pattern 322 The electrode zone of the inner surface 242 of the flow region 240 in the pattern for surrounding and capturing micro- target is activated in device 200 One or more DEP electrodes at 314 carry out micro- target (not shown) in the medium 244 in selective flow region 240. Then, selector 222 can be by mobile come the micro- target of mobile capture relative to device 200 by light pattern 322.Alternatively, it fills Setting 200 can be mobile relative to light pattern 322.

The barrier 254 of fence 256 is kept to show in Fig. 2 B and Fig. 2 C and discuss as above as physical barriers although limiting, But barrier 254 includes alternatively virtual barrier, which includes the DEP power activated by light pattern 322.

Referring again to Fig. 2A to Fig. 2 C, detector 224 can be the mechanism for detecting the event in flow region 240. For example, detector 224 may include one or more radiation feature (examples for the micro- target (not shown) being able to detect in medium Such as, due to fluorescence or shine) photodetector.Such detector 224 can be configured as one in detection such as medium 244 A or more micro- target (not shown) is just in radiating electromagnetic radiation and/or the approximate wavelength of radiation, brightness, intensity etc..Properly The example of photodetector include but is not limited to photomultiplier detector and avalanche photodetector.

It includes micro- target (not shown) in medium 244 that detector 224, which alternately or additionally includes for capturing, The imaging device of the digital picture of flow region 240.The example for the suitable imaging device that detector 224 may include includes number Camera or optical sensor, such as charge-coupled device, complementary metal oxide semiconductor imager.Image can in this way Device is captured and analyzes (for example, by control module 230 and/or operator).

Stream controller 226 can be configured as the stream of the medium 244 in control flow region 240.For example, stream controller 226 can control the direction of stream and/or speed.The non-limiting example of stream controller 226 includes one or more pumps or stream Body actuator.In some embodiments, stream controller 226 may include additional element, such as sensing such as flow region One or more sensor (not shown) of the speed of the stream of medium 244 in 240.

Control module 230 can be configured as from selector 222, detector 224, and/or stream controller 226 and receive signal And control selections device 222, detector 224, and/or stream controller 226.As shown, control module 230 may include controller 232 and memory 234.In some embodiments, controller 232 can be digital electronic controller (for example, microprocessor, micro- Controller, computer etc.), it is configured as according to the machine readable instructions (example for being stored as non-transitory signal in memory 234 Such as, software, firmware, microcode etc.) it operates, which can be digital and electronic, optics or magnetic memory apparatus.It is alternative Ground, controller 232 may include hard-wired digital circuit and/or analog circuit or the number that is operated according to machine readable instructions The combination of word electronic controller and hard-wired digital circuit and/or analog circuit.Controller 232, which can be configured as, executes this paper All or any a part of disclosed process 100,2500.

In some embodiments, fence 256 can shield illumination (for example, by detector 224 and/or selector 222) Or it can only selectively illuminate the of short duration time.After the micro- target of biology is moved in fence 256, the micro- target of biology The further illumination that further illumination or the micro- target of biology can be therefore protected from can be minimized.

Fig. 4 A to Fig. 4 C shows another example of microfluidic device 400.As shown, microfluidic device 400 may include Microfluidic circuit 432, the microfluidic circuit 432 include the fluid circuit element of multiple interconnection.Show shown in Fig. 4 A to Fig. 4 C In example, microfluidic circuit 432 includes flow region/channel 434 connected to it on 436,438,440 fluid of isolation rail.Show Gone out a channel 434 and three isolation rails 436,438,440, but can have more than one channel 434 and with it is any specific It is channel attached to be more or less than three isolation rails 436,438,440.Channel 434 and isolation rail 436,438,440 are stream The example of body loop element.Microfluidic circuit 432 may also include additional or different fluid circuit element, such as fluid cavity Room, liquid reservoir etc..

Each isolation rail 436,438,440 may include limiting to connect area of isolation 444 on area of isolation 444 and fluid To the isolation structure 446 of the join domain 442 in channel 434 (see Fig. 4 C).Join domain 442 may include the proximal end to channel 434 Opening 452 and to area of isolation 444 distal openings 454.Join domain 442 can be arranged such that in channel 434 with most Big speed (V_max) maximum penetration of stream of fluid media (medium) (not shown) of flowing do not extend in area of isolation 444.Setting Micro- target (not shown) or other materials (not shown) in the area of isolation 444 of fence 436,438,440 can therefore with it is logical The stream of medium (not shown) in road 434 is separated and is not influenced by the stream of the medium (not shown) in channel 434 substantially.It is logical Therefore road 434 can be bypassed area domain for the example of affected area and the area of isolation of isolation rail 436,438,440 Example.Before to aforementioned progress more detail discussion, brief description and the relevant control system of microfluidic device 400 are provided The example of system 470.

Microfluidic device 400 may include surrounding microfluidic circuit 432 to enclose boundary 402, and it may include one or more that this, which encloses boundary 402, Multiple fluid media (medium)s.However, device 400 can be physically constructed in different ways, the example shown in Fig. 4 A to Fig. 4 C In, it encloses boundary 402 and is depicted as including support construction 404 (for example, base portion), microfluidic circuit structure 412 and lid 422.Support knot Structure 404, microfluidic circuit structure 412 and lid 422 can be connected to each other.For example, microfluidic circuit structure 412 can be arranged on In support construction 404 and lid 422 can be arranged on 412 top of microfluidic circuit structure.Pass through support construction 404 and lid 422, microfluidic circuit structure 412 can limit microfluidic circuit 432.The inner surface of microfluidic circuit 432 is marked in the accompanying drawings Know is 406.

As shown in Figure 4 A and 4 B shown in FIG., support construction 404 can be located at bottom, and lid 422 can be located at the top of device 400 Portion.Alternatively, support construction 404 and lid 422 can be located at other directions.For example, support construction 404 can be located at top, And lid 422 is located at the bottom of device 400.It is anyway possible to there is one or more ports 424, it is each include enter or from The access 426 of Kai Wei circle 402.The example of access 426 includes valve, door, through-hole etc..Show two ports 424, but device 400 Can only have one or more than two.

Microfluidic circuit structure 412 can limit the loop element of microfluidic circuit 432 or enclose the circuit in boundary 402. In this example, as shown in Fig. 4 A to Fig. 4 C, microfluidic circuit structure 412 includes frame 414 and microfluidic circuit material 416.

Support construction 404 may include the substrate of substrate or multiple interconnection.For example, support construction 404 may include one or more Semiconductor base, printed circuit board of multiple interconnection etc..Frame 414 can partly or wholly surround microfluidic circuit material 416.Frame 414 can be the relative rigid structure for example essentially around microfluidic circuit material 416.For example, frame 414 can Including metal material.

Microfluidic circuit material 416 is patterned as returning with chamber etc. to limit microfluidic circuit element and microfluid The interconnection on road 432.Microfluidic circuit material 416 may include flexible material, such as rubber, plastics, elastomer, silicone resin (for example, The silicone resin of patternable), PDMS etc., can be ventilative.Other of the material of composable microfluidic circuit material 416 show Example includes molding glass, the etchable material of silicon, photoresist (for example, SU8) etc..In some embodiments, in this way Material (and therefore be microfluidic circuit material 416) can be rigid and/or substantially air-locked.Anyway, Microfluidic circuit material 416 can be arranged in support construction 404 and frame 414 inside.

Lid 422 can be the integrated component of frame 414 and/or microfluidic circuit material 416.Alternatively, lid 422 can be with It is element different in structure (as shown in Figure 4 A and 4 B shown in FIG.).Lid 422 may include and frame 414 and/or microfluidic circuit material 416 identical or different materials.Similarly, support construction 404 can be from frame 414 or microfluidic circuit material as shown The independent structure of the integrated component of material 416 or frame 414 or microfluidic circuit material 416.Similarly, frame 414 and miniflow Body return path materials 416 can be the independent structure as shown in Fig. 4 A to Fig. 4 C or mutually isostructural integration section.In some realities It applies in example, lid 422 and/or support construction 404 can be transparent relative to light.

The exemplary simplified frame of control/monitoring system 470 that can be used in combination with microfluidic device 400 is also shown in Fig. 4 A Figure description.As shown, system 470 may include control module 472 and control/monitoring device 480.Control module 472 can be by It is configured to control and monitor device 400 directly or by control/monitoring device 480.

Control module 472 may include digitial controller 474 and digital storage 476.Controller 474 can be for example digital Processor, computer etc. and digital storage 476 can be for by data and machine-executable instruction (for example, software, Firmware, microcode etc.) it is stored as the data of non-transitory or the non-transitory digital storage of signal.Controller 474 can be matched It is set to according to the such machine-executable instruction operation being stored in memory 476.Alternatively, or in addition, controller 474 It may include hard-wired digital circuit and/or analog circuit.Therefore control module 472 can be configured as executing all or part of any Process (for example, the process 100 of Fig. 1 and/or process 2500 of Figure 25), herein to such process the step of, function, movement etc. It discusses.

Control/monitoring device 480 may include for controlling or monitoring microfluidic device 400 and by the microfluidic device Any amount of different types of device of 400 processes executed.For example, equipment 480 may include power supply (not shown), being used for will Electric power is supplied to microfluidic device 400；Fluid medium source (is not shown, but may include 226 stream controller of similar Fig. 2A), uses In fluid media (medium) is supplied to microfluidic device 400 or removes medium from microfluidic device 400；Power plant module (it is not shown, But may include 222 selector of similar Fig. 2A), for controlling the selection of micro- target (not shown) in microfluidic circuit 432 And movement；Image capture mechanism (is not shown, but can be 224 detector of similar Fig. 2A), returns for capturing in microfluid Inside road 432 (for example, micro- target) image；Excitation mechanism (not shown), for energy to be directed to microfluidic circuit 432 In with provocative reaction etc..

As mentioned, control/monitoring device 480 may include for selecting and moving micro- mesh in microfluidic circuit 432 Mark the power plant module of (not shown).Various power mechanisms can be utilized.For example, dielectrophoresis (DEP) mechanism is (for example, similar figure The selector 222 of 2A) it can be utilized to select and move micro- target (not shown) in microfluidic circuit.Microfluidic device 400 base portion 404 and/or lid 422 may include DEP configuration, for selectively DEP power to be directed in microfluidic circuit 432 Fluid media (medium) (not shown) in micro- target (not shown), with selection, capture and/or mobile each micro- target.Control/monitoring Equipment 480 may include one or more control modules for such DEP configuration.

The exemplary of this DEP of support construction 404 or lid 422 configuration configures for photoelectricity tweezers (OET).Support construction 404 or lid 422 suitable OET configuration and it is relevant be monitored and controlled equipment example show in following american documentation literature Out: U.S. Patent No. 7,612,355, U.S. Patent No. 7,956,339, U.S. Patent Application Publication No. 2012/0325665 Number, U.S. Patent Application Publication No. 2014/0124370, U.S. Patent Application Serial the 14/262,140th (not yet adjudicate) It (is not yet adjudicated) with U.S. Patent Application Serial the 14/262nd, 200, entire contents are incorporated herein by reference. Micro- target (not shown) can be therefore using DEP device and such as OET technology in the microfluidic circuit 432 of microfluidic device 400 It is individually chosen, captures and moves.

As mentioned, channel 434 and fence 436,438,440 can be configured as is situated between comprising one or more fluids Matter (not shown).In the example shown in Fig. 4 A to Fig. 4 C, port 424 is connected to channel 434 and allows fluid media (medium) (not Show) it is introduced into microfluidic circuit 432 or is removed from microfluidic circuit 432.Once microfluidic circuit 432 is situated between comprising fluid Matter (not shown) then can selectively generate in channel 434 and stop the stream of fluid media (medium) (not shown).For example, as schemed Shown, port 424 can be arranged at the different positions (for example, opposite end) in channel 434, and medium (not shown) Stream can be formed from a port 424 for playing the role of entrance to another port 424 for playing the role of outlet.

As discussed above, each isolation rail 436,438,440 may include join domain 442 and area of isolation 444.Even Connecing region 442 may include the proximal openings 452 to channel 434 and the distal openings 454 to area of isolation 444.Channel 434 and every A isolation rail 436,438,440 can be arranged such that the maximum of the stream of the medium (not shown) flowed in channel 434 is worn Saturating depth extends to join domain 442 but does not extend in area of isolation 444.

Fig. 5 shows the exemplary detailed view of isolation rail 436.Fence 438,440 can be similarly configured.It is also shown The example of micro- target 522 in fence 436.It is well known that the microfluidic channel 434 of the proximal openings 452 by fence 436 In the stream 512 of fluid media (medium) 502 can lead to the Secondary Flow 514 of medium 502 and flow into or out fence.In order to by fence 436 Area of isolation 444 in micro- target 522 separated with Secondary Flow 514, proximally be open 452 to distal openings 454 isolation enclose The length L of the join domain 442 on column 436_conIt can be greater than when the speed of the stream 512 in channel 434 is in maximum (V_max) when flow Enter the maximum penetration D to the Secondary Flow 514 in join domain 442_p.As long as the stream 512 in channel 434 is no more than maximum speed Spend V_max, stream 512 and generate Secondary Flow 514 can therefore be limited in channel 434 and join domain 442 and be maintained at every Except region 444.Therefore stream 512 in channel 434 will not will make micro- target 522 leave area of isolation 444.Area of isolation Micro- target 522 in 444 will therefore stay in area of isolation 444 without the stream 512 in tube passage 432 how.

In addition, miscellaneous particles (for example, particle and/or nanoparticle) will not be moved to fence from channel 434 by stream 512 In 436 area of isolation 444, miscellaneous particles will not be brought into channel 434 by stream 512 from area of isolation 444.Therefore, make The length L of join domain 442_conGreater than maximum penetration D_pCan prevent a fence 436 by from channel 434 or another The miscellaneous particles of fence 438,440 pollute.

Since the join domain 442 of channel 434 and fence 436,438,440 can be by the medium 502 in channel 434 The influence of stream 512, therefore, what channel 434 and join domain 442 can be considered as microfluidic circuit 432 involves (or flowing) area Domain.On the other hand, the area of isolation 444 of fence 436,438,440 can be considered as not involving (or non-current) region.For example, First medium 502 (for example, component (not shown) in first medium 502) in channel 434 can be substantially only through making One medium 504 be diffused into from channel 434 by join domain 442 in the second medium 504 in area of isolation 444 come with isolated area Second medium 504 (for example, component (not shown) in second medium 504) mixing in domain 444.Similarly, area of isolation 444 In second medium 504 (for example, component (not shown) in second medium 504 can be substantially only through making second medium 502 Be diffused into from area of isolation 444 by join domain 442 in the first medium 502 in channel 434 come with first in channel 434 Medium 504 (for example, component (not shown) in first medium 502) mixing.First medium 502 can be and second medium 504 Identical medium or different media.In addition, first medium 502 and second medium 504 can be initially it is identical, then become Different (for example, by the micro- target adjusting second medium of one or more biologies in area of isolation 444, or by changing Medium of the unsteady flow through channel 434).

The maximum penetration D of the Secondary Flow 514 as caused by stream 512 in channel 434_pIt may depend on multiple parameters.In this way The example of parameter include: the shape in channel 434 (for example, medium can be directed in join domain 442, by medium by channel Join domain 442 is offset from or simply flowed through from join domain 442)；The width in the channel 434 at proximal openings 452 Spend W_ch(or cross-sectional area)；The width W of join domain 442 at proximal openings 452_con(or cross-sectional area)；Channel The maximum speed V of stream 512 in 434_max；First medium 502 and/or the viscosity of second medium 504 etc..

In some embodiments, the size of channel 434 and isolation rail 436,438,440 can be relative in channel 434 Stream 512 orient it is as follows: channel width W_ch(or the cross-sectional area in channel 434) may be substantially perpendicular to stream 512, in proximal end The width W for the join domain 442 being open at 552_con(or cross-sectional area) can be substantially parallel to stream 512, and join domain Length L_conIt may be substantially perpendicular to stream 512.Aforementioned is only example, and channel 434 and isolation rail 436,438,440 Size can relative to each other in the other direction.

In some embodiments, the width W in the channel 434 at proximal openings 452_chIt in office can how descend in range: 50 to 1000 microns, 50 to 500 microns, 50 to 400 microns, 50 to 300 microns, 50 to 250 microns, 50 to 200 microns, 50 arrive 150 microns, 50 to 100 microns, 70 to 500 microns, 70 to 400 microns, 70 to 300 microns, 70 to 250 microns, it is 70 to 200 micro- Rice, 70 to 150 microns, 90 to 400 microns, 90 to 300 microns, 90 to 250 microns, 90 to 200 microns, 90 to 150 microns, 100 to 300 microns, 100 to 250 microns, 100 to 200 microns, 100 to 150 microns and 100 to 120 microns.It is aforementioned to be only Example, and the width W in channel 434_chIt can be within the scope of other (for example, the model limited by the above-mentioned any endpoint listed It encloses).

In some embodiments, the height H in the channel 134 at proximal openings 152_chIn office how about can descend in range: 20 to 100 microns, 20 to 90 microns, 20 to 80 microns, 20 to 70 microns, 20 to 60 microns, 20 to 50 microns, it is 30 to 100 micro- Rice, 30 to 90 microns, 30 to 80 microns, 30 to 70 microns, 30 to 60 microns, 30 to 50 microns, 40 to 100 microns, 40 to 90 Micron, 40 to 80 microns, 40 to 70 microns, 40 to 60 microns or 40 to 50 microns.Aforementioned is only example, and channel 434 Height H_chIt can be within the scope of other (for example, the range limited by the above-mentioned any endpoint listed).

In some embodiments, how about the cross-sectional area in the channel 434 at proximal openings 452 in office can descend range It is interior: 500 to 50000 square microns, 500 to 40000 square microns, 500 to 30000 square microns, 500 to 25000 squares it is micro- Rice, 500 to 20000 square microns, 500 to 15000 square microns, 500 to 10000 square microns, 500 to 7500 squares it is micro- Rice, 500 to 5000 square microns, 1000 to 25000 square microns, 1000 to 20000 square microns, 1000 to 15000 squares It is micron, 1000 to 10000 square microns, 1000 to 7500 square microns, 1000 to 5000 square microns, 2000 to 20000 flat Square micron, 2000 to 15000 square microns, 2000 to 10000 square microns, 2000 to 7500 square microns, 2000 to 6000 Square micron, 3000 to 20000 square microns, 3000 to 15000 square microns, 3000 to 10000 square microns, 3000 are arrived 7500 square microns or 3000 to 6000 square microns.Aforementioned is only example, and the channel 434 at proximal openings 452 Cross-sectional area can be within the scope of other (for example, the range limited by the above-mentioned any endpoint listed).

In some embodiments, join domain L_conLength in office how about can descend in range: 1 to 200 microns, 5 arrive 150 microns, 10 to 100 microns, 15 to 80 microns, 20 to 60 microns, 20 to 500 microns, 40 to 400 microns, it is 60 to 300 micro- Rice, 80 to 200 microns and 100 to 150 microns.Aforementioned is only example, and the length L of join domain 442_conCan with In the different range of aforementioned exemplary (for example, the range limited by the above-mentioned any endpoint listed).

In some embodiments, the width W of the join domain 443 at proximal openings 452_conIn office how about can descend model In enclosing: 20 to 500 microns, 20 to 400 microns, 20 to 300 microns, 20 to 200 microns, 20 to 150 microns, 20 to 100 microns, 20 to 80 microns, 20 to 60 microns, 30 to 400 microns, 30 to 300 microns, 30 to 200 microns, 30 to 150 microns, 30 to 100 Micron, 30 to 80 microns, 30 to 60 microns, 40 to 300 microns, 40 to 200 microns, 40 to 150 microns, 40 to 100 microns, 40 To 80 microns, 40 to 60 microns, 50 to 250 microns, 50 to 200 microns, 50 to 150 microns, 50 to 100 microns, it is 50 to 80 micro- Rice, 60 to 200 microns, 60 to 150 microns, 60 to 100 microns, 60 to 80 microns, 70 to 150 microns, 70 to 100 microns and 80 to 100 microns.Aforementioned is only example, and the width W of the join domain 442 at proximal openings 452_conCan with it is preceding (for example, the range limited by the above-mentioned any endpoint listed) is stated in the different range of example.

In other embodiments, the width W of the join domain 442 at proximal openings 452_conIn office how about can descend model In enclosing: 2 to 35 microns, 2 to 25 microns, 2 to 20 microns, 2 to 15 microns, 2 to 10 microns, 2 to 7 microns, 2 to 5 microns, 2 to 3 Micron, 3 to 25 microns, 3 to 20 microns, 3 to 15 microns, 3 to 10 microns, 3 to 7 microns, 3 to 5 microns, 3 to 4 microns, 4 to 20 Micron, 4 to 15 microns, 4 to 10 microns, 4 to 7 microns, 4 to 5 microns, 5 to 15 microns, 5 to 10 microns, 5 to 7 microns, 6 to 15 Micron, 6 to 10 microns, 6 to 7 microns, 7 to 15 microns, 7 to 10 microns, 8 to 15 microns and 8 to 10 microns.It is aforementioned only to show Example, and the width W of the join domain 442 at proximal openings 452_conCan in the range different from aforementioned exemplary (example Such as, the range limited by the above-mentioned any endpoint listed).

In some embodiments, the length L of the join domain 442 at proximal openings 452_conWith the width of join domain 442 Spend W_conRatio can be greater than or equal to any following range: 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0, 6.0,7.0,8.0,9.0,10.0 or more.Aforementioned is only example, and the length of the join domain 442 at proximal openings 452 Spend L_conWith the width W of join domain 442_conRatio can be different from aforementioned exemplary.

As shown in figure 5, the width W of join domain 442_conProximally opening 452 can be uniformly to distal openings 454. The width W of join domain 442 at distal openings 454_conIt can be therefore as above for the bonding pad at proximal openings 452 The width W in domain 442_conIn identified any range.Alternatively, the width of the join domain 442 at distal openings 454 W_conIt can be greater than (for example, as shown in Figure 6) or be less than the connection of (for example, as shown in Fig. 7 A to Fig. 7 C) at proximal openings 452 The width W in region 442_con。

As also shown in fig. 5, the width of the area of isolation 444 at distal openings 454 can be substantially and in proximal openings The width W of join domain 442 at 452_conIt is identical.The width of area of isolation 444 at distal openings 454 can be therefore such as The upper width W for the join domain 442 at proximal openings 452_conIn identified any range.Alternatively, in distal end The width for the area of isolation 444 being open at 454 can be greater than (for example, as shown in Figure 6) or be less than (not shown) in proximal openings The width W of join domain 442 at 452_con。

In some embodiments, the maximum speed V of the stream 512 in channel 434_maxIt is that channel can hold without and cause The maximum speed of the structural damage in microfluidic device where channel.The maximum speed that channel can be kept depends on various Factor, the cross-sectional area of structural intergrity and channel including microfluidic device.Exemplary micro fluid of the invention is filled It sets, the maximum fluidity speed V in the channel of the cross-sectional area with about 3000 to 4000 square microns_maxIt is about 10 μ L/ Second.Alternatively, the maximum speed V of the stream 512 in channel 434_maxIt can be set to ensure area of isolation 444 and channel Stream 512 in 434 separates.Particularly, the width of the proximal openings 452 of the join domain 442 based on isolation rail 436,438,440 Spend W_con, V_maxThe penetration depth D of the Secondary Flow 514 entered in join domain can be set to ensure_pLess than L_con.For example, For having the width W with about 30 to 40 microns_conProximal openings 452 join domain isolation rail, V_maxIt can be with It is arranged to about 0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4 or 1.5 μ L/ seconds.

In some embodiments, the length L of the join domain 442 of isolation rail 436,438,440_conWith area of isolation 444 The summation of correspondence length can be short enough so that the component of the second medium 504 in area of isolation 444 is relatively quickly spread To the first medium 502 in channel 434.For example, in some embodiments, the length L of (1) join domain 442_con(2) in place The micro- target of biology in the area of isolation 444 of isolation rail 436,438,440 and between the distal openings 454 of join domain The summation of distance can be in the following range: 40 microns to 300 microns, 50 microns to 550 microns, 60 microns to 500 microns, 70 Micron is to 180 microns, 80 microns to 160 microns, 90 microns to 140 microns, 100 microns to 120 microns or including aforementioned end One any range in point.The diffusion rate of molecule (for example, interested analyte, such as antibody) depend on it is many because Element, the diffusion coefficient D including temperature, the viscosity of medium and molecule₀.The D of IgG antibody in the aqueous solution at 20 DEG C₀For About 4.4x10^-7Square centimeter/second (cm²/ sec), and the viscosity of the micro- target culture medium of biology is about 9x10^-4Square metre/ Second (m²/sec).Thus, for example, the antibody in the micro- target culture medium of biology at 20 DEG C may have about 0.5 micro- meter per second Diffusion rate.Therefore, in some embodiments, the micro- target of biology for that will be located in area of isolation 444 is diffused into channel Period in 434 can be about 10 minutes or less (for example, 9,8,7,6,5 minutes or less).It can be by changing shadow The parameter of diffusion rate is rung to manipulate the period for diffusion.For example, the temperature of medium can be increased (for example, to such as 37 DEG C of physiological temp) or (for example, to 15 DEG C, 10 DEG C or 4 DEG C) are reduced, to be respectively increased or reduce diffusion rate.

The configuration of isolation rail 436 shown in fig. 5 is example, and many modifications are possible.For example, area of isolation 444 can be adjusted to comprising multiple micro- targets 522, but area of isolation 444 can be adjusted to only comprising one, two, three A, four, micro- target 522 of five or similar relatively small amount.Therefore, the capacity of area of isolation 444 can be for for example, at least 3x10³、6x10³、9x10³、1x10⁴、2x10⁴、4x10⁴、8x10⁴、1x10⁵、2x10⁵、4x10⁵、8x10⁵、1x10⁶、2x10⁶Cube Micron or more.

As another example, show isolation rail 436 generally perpendicularly from channel 434 extend, and therefore with channel 434 Substantially form 90 ° of angles.Isolation rail 436 can alternatively (any angle such as between 30 ° and 150 ° at other angles Degree) extend from channel 434.

As another example, join domain 442 and area of isolation 444 are substantially shown as rectangle in Fig. 5, but even Connecing one or two of region 442 and area of isolation 444 can be other shapes.The example of this shape include ellipse, Triangle, circle, hourglass shape etc..

As another example, join domain 442 and area of isolation 444 are shown as having substantially uniform in Fig. 5 Width.That is, in Fig. 5, the width W of join domain 442_conIt is shown as proximally being open 452 to distal openings 454 It is uniform；The correspondence width of area of isolation 444 is similarly uniform；And the width W of join domain 442_conAnd isolated area The correspondence width in domain 444 is considered to be equal.Aforementioned any aspect can be different from shown in Fig. 5.For example, join domain 442 Width W_conCan proximally be open 452 to distal openings 454 change (for example, in a manner of trapezoidal or hourglass)；Area of isolation 444 width can change (for example, in a manner of triangle or flask)；And the width W of join domain 442_conIt can not It is same as the correspondence width of area of isolation 444.

Fig. 6 shows the example for showing some exemplary isolation rails in aforementioned variant.Fence shown in fig. 6 can be with Substitute any fence 436,438,440 in any figure or content discussed in this article.

The isolation rail of Fig. 6 may include join domain 642 and the isolation structure 646 including area of isolation 644.Join domain 642 may include the proximal openings 652 to channel 434 and the distal openings 654 to area of isolation 644.Example shown in Fig. 6 In, join domain 642 expands so that its width W_conProximally opening 652 increases to distal openings 654.However, in addition to shape it Outside, join domain 642, isolation structure 646 and area of isolation 644 can with the join domain 442 of Fig. 5 as discussed above, every It is roughly the same from structure 446 and area of isolation 444.

For example, the channel 434 of Fig. 6 and isolation rail can be arranged such that the maximum penetration D of Secondary Flow 514_pProlong It reaches join domain 642 and does not extend in area of isolation 644.The length L of join domain 642_conMaximum penetration can be therefore greater than Depth D_p, usually as above discussed for Fig. 5.It is again as discussed above, as long as the speed of the stream 512 in channel 434 does not surpass Cross maximum fluidity speed V_max, therefore micro- target 522 in area of isolation 644 will stay in area of isolation 644.Channel 434 and company Connecing region 642 therefore is the example for involving (or flowing) region, and area of isolation 644 is not involve showing for (or non-current) region Example.

Fig. 7 A to Fig. 7 C shows the example of the microfluidic circuit 432 of Fig. 4 A to Fig. 4 C and the modification in channel 434, and isolation The other example of the modification of fence 436,438,440.Isolation rail 736 shown in Fig. 7 A to Fig. 7 C can be substituted any attached Any fence 436,438,440 in figure and content discussed in this article.Similarly, microfluidic device 700 can substitute in office Microfluidic device 400 in what attached drawing and content discussed in this article.

The microfluidic device 700 of Fig. 7 A to Fig. 7 C may include that support construction is (invisible, but can be similar to Fig. 4 A to Fig. 4 C 404), microfluidic circuit structure 712 and lid (it is invisible, but can be similar to 422).Microfluidic circuit structure 712 can wrap Frame 714 and microfluidic circuit material 716 are included, it can be with 416 phase of frame 414 and microfluidic circuit material of Fig. 4 A to Fig. 4 C Same or frame 414 and microfluidic circuit material 416 generally similar to Fig. 4 A to Fig. 4 C.As shown in Figure 7 A, it is returned by microfluid The microfluidic circuit 732 that pipeline material 716 limits may include multiple channels 734 connected to it on multiple 736 fluids of isolation rail (showing two channels 734, but it can have more).

Each isolation rail 736 may include isolation structure 746, area of isolation 744, Yi Jilian in isolation structure 746 Connect region 742.From the proximal openings 772 at channel 734 to the distal openings 774 at isolation structure 736, join domain 742 can be to be connected to area of isolation 744 for channel 734 on fluid.In general, according to the discussed above of Fig. 5, in channel 734 The stream 782 of one fluid media (medium) 702, which can be formed, enters from channel 734 and/or leaves the company for being connected to the fence 736 in channel 734 Connect the Secondary Flow 784 of the first medium 702 in region 742.

As shown in Figure 7 B, join domain 742 may include that the proximal openings 772 to channel 734 are remote with what it is to isolation structure 746 Region between end opening 774.The length L of join domain 742_conThe maximum penetration D of Secondary Flow 784 can be greater than_p, In this case, Secondary Flow 784 may extend in join domain 742 without being redirected towards area of isolation 744 (as schemed Shown in 7A).Alternatively, as seen in figure 7 c, join domain 742 can have less than maximum penetration D_pLength L_con, at this In the case of kind, Secondary Flow 784 will extend through join domain 742 and can be redirected towards area of isolation 744.Rear In a kind of situation, the length L of join domain 742_c1And L_c2Summation can be greater than maximum penetration D_p.In this way, two Secondary stream 784 will not extend in area of isolation 744.Whether the length L of join domain 742_conGreater than penetration depth D_pStill The length L of join domain 742_c1And L_c2Summation be greater than penetration depth D_p, it is less than maximum speed V_maxChannel 734 in The stream 782 of one medium 702, which will generate, has penetration depth D_pSecondary Flow, and micro- mesh in the area of isolation 744 of fence 736 Mark (being not shown, but can be similar to 522 in Fig. 5) will not pass through the stream 782 of the first medium 702 in channel 734 and leave Area of isolation 744.Stream 782 in channel 734 will not make miscellaneous material (not shown) leaving channel 734 enter fence 736 Area of isolation 744 in or leave area of isolation 744 and enter in channel 734.Diffusion is the first medium in channel 734 Component in 702 can by its from the second medium 704 in the area of isolation 744 that channel 734 is moved to fence 736 only One mode.Similarly, diffusion be component in the second medium 704 in the area of isolation 744 of fence 736 can by its from Area of isolation 744 is moved to unique mode of the first medium 702 in channel 734.First medium 702 can be to be situated between with second The identical medium of matter 704 or first medium 702 can be the medium different from second medium 704.Alternatively, first is situated between Matter 702 and second medium 704 can be initially it is identical, then become different (for example, by one in area of isolation 744 A or more micro- target of biology adjusts second medium, or the medium in channel 734 is flowed through by changing).

As shown in Figure 7 B, perpendicular to the direction of the stream 782 (referring to Fig. 7 A) in channel 734 channel 734 width W_chIt can To be substantially perpendicular to the width W of proximal openings 772_con1, and therefore it is arranged essentially parallel to the width W of distal openings 774_con2.So And the width W of proximal openings 772_con1With the width W of distal openings 774_con2It does not need substantially perpendicular to each other.For example, in proximal end The width W of opening 772_con1The width W of the axis (not shown) and distal openings 774 that are directed on it_con2It is directed on it Angle between another axis can be non-perpendicular, therefore not be 90 °.The example of alternative angle is included in any as follows Angle in range: between 30 ° and 90 °, between 45 ° and 90 °, between 60 ° and 90 ° etc..

For the discussion previously with regard to the microfluidic device with channel and one or more isolation rails, fluid media (medium) (for example, first medium and/or second medium), which can be, can be such that the micro- target of biology is substantially able to maintain can to measure state Any fluid.The measurement that state will depend on the micro- target of biology and be performed can be measured.For example, if the micro- target of biology is to sense The micro- target of biology that the secretion of the protein of interest is measured, it is assumed that the micro- target of biology is feasible and can express (exprese) and secretory protein, then biological micro- target will be substantially measurable.

Fig. 8 to Figure 30 shows the microfluidic device 200 of test chart 2A to Fig. 2 C or the microfluidic device 400 of Fig. 4 A to Fig. 4 C In the micro- target (such as biological cell) of biology Fig. 1 process 100 example.However, process 100 is not limited to the micro- mesh of biology Mark classify or operate to microfluidic device 200,400.Microfluidic device 200,400 is also not necessarily limited to process 100.This Outside, when the various aspects when process 100 the step of can be discussed with coupling apparatus 200 rather than device 400 (or vice versa), this It can be used for other devices or any other similar microfluidic device in terms of sample.

In step 102, process 100 can be by the micro- target load of biology into microfluidic device.Fig. 8 shows wherein biological micro- Target 802 (for example, biological cell) is loaded onto showing in the flow region 240 (for example, channel 252) of microfluidic device 200 Example.Fig. 9 shows the sample material 902 including the micro- target 904 of biology and is flowed into the channel 434 of microfluidic device 400 Example.

(it shows and enters similar to Figure 10, Figure 11, Figure 13, Figure 14, Figure 17, Figure 18, Figure 26 and Figure 27 as shown in Figure 8 Partial cross-sectional top view in the flow region 240 of device 200), the mixture of the micro- target 802 of biology can be loaded onto miniflow In the channel 252 of body device 200.For example, the micro- target 802 of biology can be entered by entrance 208 (A to Fig. 2 C referring to fig. 2) Into device 200, and the micro- target 802 of biology can be moved in channel 252 by the stream 804 of medium 244.Stream 804 can be with It is convection current.Once the micro- target 802 of biology is in channel 252 and neighbouring fence 256, stream 804 can be stopped or slow down so that raw The micro- target 802 of object keeps a period of time for being enough to execute step 104 and 106 in the flow channel 252 of neighbouring fence 256.Dress The mixture for the micro- target 802 of biology being loaded in channel 252 may include the micro- target of different types of biology and other components, such as Fragment, protein, pollution, particle etc..

Fig. 9 shows the channel 434 that microfluidic device 400 is flowed into including the sample material 902 of the micro- target 904 of biology In example.In addition to the micro- target 904 of biology, sample material 902 may include that other micro- target (not shown) or material (do not show Out).In some embodiments, channel 434 can have cross-sectional area disclosed herein, for example, about 3000 to 6000 is flat Square micron or about 2500 to 4000 square microns.Sample material 902 can be with rate disclosed herein, for example, about 0.05 to 0.25 μ L/ seconds (for example, about 0.1 to 0.2 μ L/ seconds or about 0.14 to 0.15 μ L/ seconds), is flowed into channel 434 In.In some embodiments, the control module 472 of Fig. 4 A can cause control/monitoring device 480 to make comprising sample material 902 First fluid medium (not shown) be flowed into channel 434 by port 424.Once sample material 902 in channel 434, The stream of medium (not shown) in channel 434 can be slowed or substantially stop.Start and stop the medium in channel 434 The stream of (not shown) may include the valve (not shown) for opening and closing the access 426 including port 424.

The micro- target 802,904 of biology can be times for being used to generate specific analyte or interested analyte to be determined What micro- target 802,904 of biology.The example of the micro- target 802,904 of biology includes the micro- target of biology, and such as mammalian biological is micro- Target, the micro- target of human biological, the immune micro- target of biology (for example, the micro- target of T biology, the micro- target of B biology, macrophage etc.), B The micro- target hybridoma of biology, the dry micro- target of biology (for example, dry micro- target of biology of the micro- target of marrow stem biology, fat etc.), The micro- score of biology (for example, the micro- target of CHO biology of conversion, the micro- target of HeLa biology, the micro- target of HEK biology etc.) of conversion, The micro- target (for example, Sf9, Sf21, HighFive etc.) of insect biology, the micro- target of protozoan biology are (for example, lizard Li Shiman is former Worm), the micro- target (for example, S. saccharomycete, P. saccharomycete etc.) of saccharomycete biology, the micro- target of bacterium living beings is (for example, E. large intestine bar Bacterium, B. bacillus subtilis, B. bacillus thuringiensis etc.), aforementioned any combination etc..The example of the micro- target 904 of biology is also wrapped Include embryo, mammal embryo (for example, the mankind, primate, dog, cat, bear, ox, sheep, goat, horse, pig etc.) Deng.The example of interested analyte includes, protein, carbohydrate, lipid, nucleic acid, metabolin etc..Interested analysis Other examples of object include the material comprising antibody, such as IgG (for example, IgG1, IgG2, IgG3 or IgG4 subclass), IgM, IgA, IgD or IgE class antibody.

At step 104, process 100 can be held the micro- target of biology being loaded into microfluidic device at step 102 Row first is tested.Step 104 may include selecting multiple micro- targets of biology according to the first test.Alternatively, step 104 can wrap One in the micro- target of selection biology is included without executing the first test.Figure 10 is shown in the channel 252 to microfluidic device 200 The micro- target 802 of biology execute the example of the first test and Figure 11 shows according to the first test and selects the micro- target 802 of biology Example.(in Figure 11 and subsequent drawings by the selected micro- target label of biology be 1002).Figure 12 is shown wherein from miniflow The example of the micro- target 1202,1204,1206 of biology is selected in micro- target 904 in the channel 434 of body device 400.

First test may include any amount of possible test.For example, either in microfluidic device 200 or 400 The first test is executed, the first test can be surveyed for the fisrt feature of the micro- target 802 of biology or the micro- target 904 of biology Examination.The first test executed at step 104 can be any test tested for any desired feature.For example, Desired feature can be related to the form of size, shape and/or the micro- target 802 of biology or the micro- target 904 of biology.First test can Image including the capture micro- target 802 of biology or the micro- target 904 of biology and analyze image with determine which micro- target 802 of biology or The micro- target 904 of biology has desired feature.As another example, the first test executed at step 104 can determine Which micro- target 802 of biology or the micro- target 904 of biology show the specific detectable condition for showing fisrt feature.For example, the One feature can indicate one or more cell surface markers and the first test executed at step 104 can detecte Exist in the micro- target 802,904 of biology or there is no such cell surface markers.By to suitable cell surface The combination of marker or cell surface marker is tested, and particular cell types can be identified and select at step 104. The example of this particular cell types may include healthy cell, cancer cell, and infected cell is (for example, virus infection or parasitism Worm), immunocyte (for example, B cell, T cell, macrophage), stem cell etc..

In the example depicted in fig. 10, the detectable condition of the micro- target of biology in microfluidic device 200 is energy 1006 Radiation, which can be such as electromagnetic radiation.By the micro- target load of biology into microfluidic device 200 or channel 252 It before, can be with the measurement material (not shown) for micro- 802 radiation energy 1006 of target of biology for making to have fisrt feature to biology Micro- target 802 is pre-processed.

The example for the fisrt feature tested at step 104 may include and be not limited to the biological aspect of the micro- target 802 of biology (for example, cell type) or specific bioactivity.For example, fisrt feature can be observable physical features, such as ruler Very little, shape, color, texture, surface topography, certifiable sub-component or other feature marker.Alternatively, fisrt feature Can be measurable feature, such as permeability, electric conductivity, capacitor, in response in environment variation or generation (for example, table Reach, secrete) specific interested biomaterial.Specific interested biomaterial can be cell surface marker (for example, annexin matter, glycoprotein etc.).Another example of specific interested biomaterial is human cytokines, all As specificity is bonded to the antibody (for example, IgG type antibody) of interested antigen.Therefore, the micro- target 1002 of selected biology It can be one to be positive for the particular organisms testing of materials for generating (for example, expression) such as cell surface marker or more Multiple micro- targets 802 of biology, and the non-selected micro- target 1004 of biology can be for the aforementioned biology tested and be not positive Micro- target 802.The suitable measurement material that the micro- target 802 of biology can be pretreated by it includes reactant, the reactant It is bonded to specific interested biomaterial and the label including radiation energy 1006.

As shown in figure 11, micro- target 1002 can be trapped by using ligh trap 1102 to select the micro- target 1002 of biology.It is logical It, can be in microfluidic device by the way that the change pattern of light is directed to channel 252 often as referring to discussing Fig. 3 A and Fig. 3 B It is generated in 200 channel 252, mobile and closing ligh trap 1102.It is 1004 by the micro- target label of biology non-selected in Figure 11.? In example shown in Figure 11, ligh trap 1102 is not generated for the non-selected micro- target 1004 of biology.

Figure 12, which is shown, selects life from the micro- target 904 of biology in the channel of microfluidic device 400 434 at step 104 The micro- target 1202,1204,1206 of object.The selection can be in response to the result of the first test executed at step 104.It is alternative The selection on ground, micro- target 1202,1204,1206 can be random selection, thus be in the case where not executing the first test into Capable.If step 104 for example may include that can be observed for one or more as discussed above based on the first test Physical features or measurable feature select the micro- target 1202,1204,1206 of biology.For example, multiple possibility can be based on Detectable feature in any detectable feature, such as biological micro- object type specific characteristics and/or deposited with the micro- target of biology Vigor and the related feature of health, select the micro- target 1202,1204,1206 of biology from micro- target 904 in sample material 90. The example of this feature includes size, shape, color, texture, permeability, electric conductivity, capacitor, the micro- target type specificity mark of biology The expression of will object, in response to variation in environment etc..In a particular embodiment, tool can be selected from sample material 602 There is the micro- target 904 of biology that cross section is circular shape, the cross section is with the diameter in office how about descended in range: 0.5 to 2.5 Micron, 1 to 5 microns, 2.5 to 7.5 microns, 5 to 10 microns, 5 to 15 microns, 5 to 20 microns, 5 to 25 microns, it is 10 to 15 micro- Rice, 10 to 20 microns, 10 to 25 microns, 10 to 30 microns, 15 to 20 microns, 15 to 25 microns, 15 to 30 microns, it is 15 to 35 micro- Rice, 20 to 25 microns, 20 to 30 microns, 20 to 35 microns or 20 to 40 microns.It as another example, can be from sample material Select the micro- target 604 of biology in 902, the size of the micro- target 604 of the biology between 100 and 500 microns (for example, 100 and Between 200 microns, between 150 and 300 microns, 200 to 400 microns or 250 to 500 microns).

Although example shown in Figure 12 is shown selects micro- target 1202,1204,1206, sample in channel 434 Material 902 is alternatively at least partially in the join domain 442 of fence 436,438,440.Micro- target 1202,1204, Therefore 1206 can be selected in join domain 442 simultaneously.

In some embodiments, by making control/monitoring device 480 capture the micro- target 904 of biology in sample material 902 Image, control module 472 can be executed at step 104 first test.It may be configured with the control of known image analysis algorithm Molding block 472 can analyze image and identify the micro- target 904 of multiple biologies with desired feature.Alternatively, Ren Leiyong Family can analyze the image captured.

In order to measure the micro- clarification of objective of biology, human user and/or control module 472 can control measurement.For example, can (to be directed to the micro- target surface egg of biology for example, using for magnetic conductivity, conductivity or the micro- object type Specific marker of biology The antibody of white matter) measure the micro- target of biology, such as biological micro- target.

At step 106, process 100 can separate the micro- target of selected biology or be selected as the one of step 104 The partial micro- target of biology.However, if the micro- target of biology executes first step, step by selection and not at step 104 106 can be skipped or may include simply (also, optionally, the non-selected micro- target of biology being flushed out channel 252 Also flow region 240 is flushed out).Figure 13 and Figure 14, which is shown, is wherein moved to microfluid dress for the micro- target 1002 of selected biology Set the example that channel 252 is flushed out in the holding fence 256 in 200 and by the non-selected micro- target 1004 of biology.Figure 15 and Figure 16 show wherein by the micro- target 1202,1204,1206 of selected biology be moved to microfluidic device 400 fence 436, 438, in 440 area of isolation 444, then non-selected micro- target 904 is flushed out to the example of flow channel 434.

As it is above-mentioned be previously mentioned for Figure 11, the micro- target 1002 of each biology can be selected by ligh trap 1102.For example, by The selector 222 (A to Fig. 2 C referring to fig. 2) for being configured to the DEP device of Fig. 3 A and Fig. 3 B can produce selected by trapping individually The micro- target 1002 of biology ligh trap 1102.As shown in figure 13, then, ligh trap 1102 can be moved to fence by DEP device 300 In 256, the micro- target 1002 of selected biology trapped is moved in fence 256.As shown, each selected The micro- target 1002 of biology can be trapped individually and be moved to and be kept in fence 256.Alternatively, more than one selected The micro- target 1002 of biology can be captured by single trap and/or the more than one micro- target 1002 of selected biology can be by It is moved in any fence 256.Anyway, two or more selected micro- targets 1002 of biology can be in channel 252 It is middle to be selected and be moved in fence 256 parallel.

It is discussed above with reference to Fig. 3 A and Fig. 3 B, ligh trap 1102 can be the flow region for projecting microfluidic device 200 A part of the change pattern 322 of light on the inner surface 242 in domain 240.As shown in figure 14, once the selected micro- target of biology 1002 in fence 256, and the ligh trap corresponding to the micro- target 1002 of biology can be closed.Detector 224 can capture flow region All or part of images in domain 240, including the micro- target of selected and non-selected biology 1002,1004, channel 252 and enclose The image on column 256, and these images can help to the micro- identification of target 1002 of individually selected biology, trapping and movement Specific fence 256.Therefore detector 224 and/or selector 222 (for example, the DEP device for being configured as Fig. 3 A and Fig. 3 B) can be For micro- target (for example, selected micro- target 1002 of biology) of fisrt feature positive test will to be directed to and for the first spy Sign tests the one or more of the separator that the micro- target (for example, non-selected micro- target 1004 of biology) being negative separates Example.

As shown in figure 14, in the case where the micro- target 1002 of selected biology is in fence 256, the stream 804 of medium 244 The non-selected micro- target 1004 of biology can be flushed out channel 252 by (for example, overall flow).As mentioned, in step 102 After the micro- target 904 of biology is loaded into channel 252 by place, the stream 804 of medium 252 can be stopped or slow down.As step 106 A part, stream 804 can be resumed or increase so that the non-selected micro- target 1004 of biology is flushed out channel 252, and In some examples, microfluidic device 200 (for example, by outlet 210) is flushed out.

The selected micro- target 1202,1204,1206 of biology can be moved to isolation in a manner of any amount of possibility In the isolation rail 436,438,440 of 444 microfluidic device 400 of region.For example, as discussed above, microfluidic device encloses Boundary 402 may include the DEP configuration that can be used for capturing and moving specific multiple micro- targets 904 of biology in sample material 902.

For example, as shown in figure 15, control module 472 can be in the micro- target 1202,1204,1206 of selected biology Each drafting from channel 434 to the path 1512 of one area of isolation 444 in isolation rail 436,438,440, 1514,1516.Then, control module 472 can make the variation of the DEP module (not shown) generation light of control/monitoring device 480 The change pattern of light is simultaneously directed in microfluidic circuit 432 by pattern, with capture the micro- target of selected biology 1202,1204, 1206 and the micro- target 1202,1204,1206 of selected biology is moved to isolation rail along path 1512,1514,1516 436, in 438,440 area of isolation 444.Control module 472 can also store the selected biology of mark in memory 476 It is each in micro- target and the specific isolation rail 436,438,440 for moving into each selected micro- target of biology A data.

Although showing a selected micro- target of biology in each fence 436,438,444 in the example of fig. 15 1202,1204,1206, but the micro- target 1202,1204,1206 of more than one biology can be moved in single fence.It can incite somebody to action The example of the quantity of its micro- target of biology being moved in single fence 136,138,140 from sample material 902 includes the following: 1, 2, it 3,4,5,1 to 50,1 to 40,1 to 30,1 to 20,1 to 10,2 to 50,2 to 40,2 to 30,2 to 20,2 to 10,3 to 50,3 arrives 40,3 to 30,3 to 20,3 to 10,4 to 50,4 to 40,4 to 30,4 to 20,4 to 10,5 to 50,5 to 40,5 to 30,5 to 20 with And 5 to 10.Aforementioned is only example, and the micro- target 904 of biology of other quantity can be moved to individually from sample material 902 In fence 436,438,440.

In some embodiments, at least part of sample material 902 can be loaded at step 104 fence 436, 438, in 440 area of isolation 444.In addition, a part as step 104, can select micro- target in area of isolation 144 1202,1204,1206.It in such embodiments, can be at step 106 by the sample including non-selected micro- target 904 Material 902 is removed from area of isolation 444, only leaves selected micro- target 1202,1204,1206 in area of isolation 444.

As shown in figure 16, as a part of step 106, channel 434 can be by being rinsed with scouring media (not shown) The sample material 902 including non-selected micro- target 904 is removed in channel 434.In Figure 16, the flushing in channel 134 will be passed through Failing to be sold at auction for medium is denoted as 1602.The stream 1602 of scouring media can be controlled such that the speed of stream 1602 is maintained at and correspond to such as On the maximum penetration D that is discussed_pMaximum fluidity speed V_maxUnder.For another example discussed above, this will be maintained at it The micro- target 1202,1204,1206 of selected biology in the area of isolation 444 of each fence 436,438,440, and prevent from coming From another in one material contamination fence in channel 434 or fence 436,438,440.In some embodiments, it rushes Wash media flow to cross-sectional area disclosed herein channel 434, for example, about 3000 to 6000 square microns or About 2500 to 4000 square microns.Scouring media can be flowed into channel with rate disclosed herein, for example, about 0.05 to 5.0 μ L/ seconds (for example, about 0.1 to 2.0,0.2 to 1.5,0.5 to 1.0 μ L/ seconds, or about 1.0 to 2.0 μ L/ seconds). As a part of step 106, being purged to channel 434 may include multiple irrigation channel 434

In some embodiments, control module 472 can cause control/monitoring device 480 to be purged channel 434. For example, control module 472 can cause control/monitoring device 480 to be flowed into scouring media in channel 434 by port 424 And flow out another port 424.Control module 472 can keep the speed of stream 1602 lower than maximum fluidity speed V_max.For example, For the channel of the cross-sectional area with about 3000 to 6000 square microns (or about 2500 to 4000 square microns) 434, the speed for flowing 1602 can be maintained below 5.0 L/ seconds V of μ by control module 472_max(for example, 4.0,3.0 or 2.0 μ L/ Second).

After step 102 to 106, process 100 is by the micro- mesh of biology in microfluidic device (for example, 200,400) The mixture of mark (for example, 802,904) be divided into the selected micro- target of biology (for example, 1004,1202,1204,1206) and not The micro- target (for example, 1004,904) of biology of selection.The selected micro- target of biology is also placed on microfluidic device by process 100 In holding fence (for example, 256,436,438,440) in and rinse and remove the non-selected micro- target of biology.As discussed above , step 102 to 106 can be repeated and therefore execute k times, and wherein k is 1 (in this case, step 102 to 106 execution Once but do not repeat) or greater than 1.Result may be to have many selected biologies micro- in holding fence in microfluidic devices Target.

It should be noted also that step 104 can execute l for up to l different features before executing step 106 Secondary test, wherein l be positive integer 1 or be greater than 1.For example, step 104 can be for fisrt feature (such as ruler of the micro- target of biology Very little, shape, form, quality, visible mark object etc.) it is tested, later, step 104 can be repeated to be directed to subsequent characteristics (such as measurable feature) is tested.Therefore, the selected micro- target of biology may include from the loading at step 102 The micro- target of biology of (more) groups of the micro- target of biology being positive for up to l different characteristic tests.

As mentioned, the selected micro- target of biology is moved in fence from channel (for example, 252,434) and from logical Road, which rinses, removes the example how non-selected micro- target of biology only executes step 106.Other examples include, will be unselected The micro- target of biology selected, which is moved in fence from channel and rinses from channel, removes the selected micro- target of biology.For example, selected The micro- target of biology selected is flushed channel and otherwise is collected or is sent to other equipment in microfluidic devices (not shown), wherein the selected micro- target of biology can be further processed.The non-selected micro- target of biology then can be from Fence is kept to remove and abandon.

At step 108, process 100 can execute test to the micro- target of selected biology or the micro- target of biology.If First test is performed as a part of step 104, then the test can be follow-up test (for example, second test).(under Wen Zhong, the test executed at step 108 are referred to as " follow-up test " to distinguish above-mentioned " the first survey discussed at step 104 Examination ").As mentioned, the follow-up test executed at step 108 can be for spy identical with the first of step 104 the test Sign (that is, fisrt feature) or different features are tested.Again as mentioned above, if what is executed at step 108 is subsequent Test is for fisrt feature (and therefore for the identical feature tested at step 104), then follow-up test can also be with First test is different.For example, the detection for fisrt feature, follow-up test may be more more sensitive than the first test.

Figure 17 and Figure 18, which is shown, wherein measures feature micro- for different from the fisrt feature tested at step 104 The example of the follow-up test executed at step 108 is executed in fluid means 200.Figure 19 to Figure 25, which is shown, wherein to be filled in microfluid Set the example that the test of step 108 is executed in 400.

As shown in figure 17, measurement material 1702, which can be, is flowed into channel 252 with sufficient amount with by the institute in fence 256 The micro- target 1002 of biology of selection is exposed to the stream 804 of measurement material 1702.For example, although barrier 254 can prevent measurement material Material 1702 directly from the inner space that channel 252 is flowed into fence 256, but measure material 1702 can in a manner of diffusion into Enter the inside of fence 256 and therefore reaches the micro- target 1002 of selected biology in fence.Measurement material 1702 may include with The micro- target 1002 of selected biology with subsequent characteristics is reacted to generate the material of different detectable conditions.Measure material 1702 can be different from times as above discussed for the first test at step 104 with the different detectable conditions generated What measurement material and condition.Cleaning buffer (not shown) can also be flowed into channel 252 and allow to be diffused into fence 256 To clean the micro- target 1002 of selected biology.

Detectable condition can be with one or more standards (frequency etc. in threshold intensity, special frequency band) Energy radiation.The color of the micro- target 1002 of biology is the example of the radiating electromagnetic radiation in special frequency band.Shown in Figure 18 Example in, continue to be encoded with for the micro- target 1002 of selected biology of the subsequent characteristics positive test at step 18 Label 1002, but for the micro- target of biology that the subsequent characteristics test at step 108 is negative and (is not positive for example, testing) It is encoded with label 1802.

The example for the subsequent characteristics tested at step 410 can be the viability of the micro- target 1002 of biology.For example, subsequent Feature can be the micro- target 1002 of biology and live or die, and measures material and can be reactive dye, such as 7- amino Actinomycin D.Such dyestuff can make the micro- target 1002 of biology to live become particular color and/or keep the biology died micro- Target becomes different colors.Detector 224 (A to Fig. 2 C referring to fig. 2) can capture the micro- target of biology kept in fence 256 1002 image and control module 230 can be configured as analysis image to determine which micro- target of biology shows to correspond to Which show to correspond to the micro- target of biology died in the color of the micro- target 1002 of biology to live and/or the micro- target of biology 1002 color.Alternatively, human operator can analyze the image for carrying out self-detector 224.Configured in this way detector 224 And/or therefore control module 230 can be filled for special characteristic (for example, fisrt feature or subsequent characteristics) for testing microfluid One or more examples of the test device for the micro- target in the liquid medium in flow path set.

Figure 19 show wherein at step 108 execute test be for by microfluidic device 400 isolation rail 236, 238, the example for the interested analyte that the micro- target 1202,1204,1206 of selected biology in 240 generates.In Figure 19 In, the component of interested analyte 1902 is encoded with label 1904.Interested analyte can be for example, protein, core Acid, carbohydrate, lipid, metabolin or by particular cell types (for example, healthy cell, cancer cell, virus or helminth Infection cell, inflammatory response cell etc.) secretion or other molecules for otherwise discharging.Specific interested analyte can To be that such as growth factor, cell factor (for example, inflammation or other), viral antigen, parasite antigen, cancer cell specificity are anti- Former or curative drug (for example, therapeutic agent, such as hormone or therapeutic antibodies).

In the example shown in Figure 19, step 108 may include that will measure material 1910 to be loaded into microfluidic device 400, And if any, test and analyze the local reaction of object component 1904.Step 108, which may additionally include, fills measurement material 1910 Cultivation cycle is provided after being downloaded in channel 434.

As shown in figure 19, measurement material 1910 can be substantially filled with channel 434 or at least fill up neighbouring fence 436, 438, the region of 440 proximal openings 442.In addition, measurement material 110 extend at least some isolation rails 436,438, In 440 join domain 442.In some embodiments, measurement material is flowed into cross-sectional area (example disclosed herein Such as, about 3000 to 6000 square microns or about 2500 to 4000 square microns) channel 434 in.Measuring material can be with Rate disclosed herein (for example, about 0.02 to 0.25 μ L/ seconds (for example, about 0.03 to 0.2 μ L/ seconds, or about 0.05 By 0.15 μ L/ seconds, have biological cell measurement material compared with low velocity, and there is higher speed for acellular measurement material Degree)) it is flowed into channel.Once measurement material 1910 is loaded onto the position in channel 434, the stream in channel 434 can be by Slow down or is substantially stopped.

Measurement material 1910 can be flowed into fast enough in channel 434, to produce in any fence 436,438,440 Before raw analyte component 1904 can be diffused into channel 434, make to measure material 1910 be located at neighbouring fence 436,438, In the position of 440 proximal openings 452.This can be arranged on to avoid in the micro- target 1202,1204,1206 of selected biology Time in fence 436,438,440 and measurement material 1910 was loaded between the time completed in channel 434, comes from one The problem of analyte component pollution channels 434 and/or other fences of fence 436,438,440.

Measurement material 1910, which is loaded into the speed in channel 434, to be therefore at least minimum flow velocity V_min, for the moment Between section T_loadMaterial 1910 will inside be measured completely to be loaded into the position of located adjacent proximal end opening 452, the period T_loadLess than use In making the analyte component 1904 of sufficient amount be diffused into from the area of isolation 444 of fence 436,438,440 minimum in channel 434 Period T_diff." sufficient amount " used herein refers to the amount of detectable analyte component, this is enough to influence to carry out self-isolation The accurate detection of the analyte component of fence).Minimal flow speed V_minIt can be the function of various different parameters.This parameter Example include the length in channel 434, fence 436,438,440 join domain 442 length L_con, analyte component 1904 Diffusivity, dielectric viscosity, environment temperature etc..Minimal flow speed V_minExample include at least about 0.04 μ L/ seconds (for example, At least about 0.10,0.11,0.12,0.13,0.14 μ L/ seconds or higher).

The minimal flow speed V being loaded into channel 434 for material 1910 will to be measured_minIt can be less than to correspond to and be less than The length L of the join domain 442 of fence 436,438,440 as discussed above_conPenetration depth D_pMaximum fluidity speed V_max.For example, V_max/V_minRatio in office how about can descend in range: about 1,2,3,4,5,6,7,8,9,10,15,20,25, 30,40,50,100 or more.

It can make the micro- target 1202,1204,1206 of biology enough loading the culture period for measuring and providing after material 1910 It generates interested analyte 1902 and is enough the area of isolation 444 for making analyte component 1904 from fence 436,438,440 It is diffused into corresponding join domain 442 or proximal openings 452.For example, culture period can provide time enough so that analyte Component 1904 is diffused into channel 434.

Culture period may include only passively making the micro- target 1202,1204,1206 of biology in isolation rail 436,438,440 Naturally interested analyte 1902 is generated.Alternatively, culture period may include initiatively the micro- target 1202 of stimulating organism, 1204,1206 interested analyte 1902 is generated, for example, by providing nutrients, growth factor, and/or inducible factor Give biology micro- target 1202,1204,1206；Control the temperature of the medium in the area of isolation 444 of isolation rail 436,438,440 Degree, chemical constituent, pH etc.；It is medium that the excitation energy of such as light is directed to area of isolation 444.

As used herein term " culture " covering it is previously described from only passively make biological micro- target 1202, 1204,1206 analyte 1902 is generated naturally in isolation rail 436,438,440 to the generation for initiatively stimulating analyte Range.The generation of stimulation analyte 1902 may also include the growth of the micro- target 1202,1204,1206 of stimulating organism.Therefore, example Such as, the micro- target 1202,1204,1206 of biology can be stimulated at it with generate before interested analyte 1902 and/or Growth is stimulated when it is stimulated to generate interested analyte 1902.If the micro- target 1202,1204,1206 of biology is It is loaded onto isolation rail 436,438,440 as the single micro- target of biology, then growth stimulation can cause to express and/or divide Secrete the generation of the micro- target population of clone biological of (or can be stimulated to express and/or secrete) interested analyte.

In some embodiments, control module 472 can make control/monitoring device 480 execute one during culture period 150 A or more movement.For example, control module 472 can make control/monitoring device 480 provide life periodically or with continuous flow Long medium and/or induction medium.Alternatively, control module 472 can make control/monitoring device 480 cultivate the micro- target of biology For a period of time, it is enough to be diffused into interested analyte in channel 434.For example, in the protein analyte of such as antibody In the case where, for the micro- target of biology and channel 434 separate at a distance from every 1 micron, control module 472, which can provide, to be equal to greatly About 2 seconds diffusion times.For the protein for being significantly less than antibody and other analytes, the time for spreading required can With less, such as every 1 micron 1.5 seconds or less (for example, 1.25s/ μm, 1.0s/ μm, 0.75s/ μm, 0.5s/ μm or less). For protein or other analytes conversely, for significantly greater than antibody, the time for distributing to diffusion can be bigger, such as often Micron 2.0 seconds or more (for example, 2.25s/ μm, 2.5s/ μm, 2.75s/ μm, 3.0s/ μm or more).

It should be pointed out that culture period can continue during the subsequent step of implementation procedure 100.In addition, culture period can be with Start before completing step 106 (such as during any step 102 to 106).

Measurement material 1910 can be configured as with the interaction of the analyte component 1904 of interested analyte 902 simultaneously Detectable reaction is generated by interaction.As shown in figure 20, the micro- target 1202 of biology in isolation rail 436,438, The measurement material 1910 of the proximal openings 452 of 1204 analyte component 1904 and neighbouring isolation rail 436,438 interacts To generate the detectable reaction in part.However, the micro- target 1206 of biology in isolation rail 440 does not generate interested analyte 1902.Therefore, at the proximal openings 452 of neighbouring isolation rail 440, without such local reaction (for example, similar 2002) Occur.

Local reaction 2002 can be detectable reaction.For example, reaction 2002 can be it is local luminous (for example, glimmering Light).In addition, local reaction 2002 can localize enough or disintegration, so as to pass through the control of human observer, Fig. 4 A Camera etc. in system/monitoring device 480 is detected separately.For example, channel 434 can be sufficiently filled with measurement material 1910, It reacts (for example, similar 2002) and is localized, that is to say, that opens the neighbouring proximal end corresponding to isolation rail 436,438 of limitation The space of mouth 452.As can be seen, reaction 2002 can come from the one or more of neighbouring isolation rail 436,438,440 The aggregation of the component of multiple measurement materials 1910 of proximal openings 452.

The proximal openings 452 of continuous isolation rail 436,438,440 can separate at least distance D_s(C referring to fig. 4), Its such as by human viewer, by cameras capture to image be enough to distinguish in the adjacent offer of proximal openings 452 Local reaction (for example, similar 2002).Between the proximal openings 452 of continuous isolation rail 436,438,440 suitably away from From D_sExample include at least 20,25,30,35,40,45,50,55,60 microns or more.Alternatively, or in addition, measurement material The component of 910 (for example, capturing micro- target, biological micro- target, microballon etc.) of material can be organized before isolation rail.Example Such as, using DEP power etc., capturing micro- target can be grouped together and concentrate on the close of neighbouring isolation rail 436,438,440 In the region in the channel 434 of end opening 452.

As mentioned, including capturing the measurement material of the component of micro- target (for example, the micro- target of biology, microballon etc.) 1910 can enter and therefore be partially disposed in the join domain 442 of isolation rail 436,438,440.In such case Under, relative to substantially completely in channel 434, reaction 2002,2004 can completely, substantially completely or partially exist in generation In join domain 442.In addition, the micro- target of capture (for example, the micro- target of biology, microballon etc.) in measurement material 1910 can be set It sets in area of isolation 444.For example, DEP power etc., which can be used for selecting to capture micro- target and will capture micro- target, is moved to isolated area In domain 444.Target micro- for the capture being arranged in the area of isolation of isolation rail, capture micro- target can be set it is close The micro- target of (multiple) biologies and/or a part in the area of isolation different from the part occupied by the micro- target of (multiple) biologies In (for example, seed cell).

Measurement material 1910 can be specificity and directly or indirectly interact with interested analyte 1902 to generate Any material of detectable reaction (for example, 2002).It includes having two bond sites that Figure 19 to Figure 23, which shows wherein analyte, The example of antigen.It will be understood by those skilled in the art that identical example, which can be easily adaptable to wherein interested analyte, to be had When be different from two bond sites antigen the case where.

Figure 21 (a part of its proximal openings 452 for showing channel 434 and isolation rail 436) is shown with the capture of label The example of the measurement material 1910 of micro- target 2112.Each having the micro- target 2112 of the capture of label may include that specific can bond The bonding material and label substance of analyte component 1904.As the proximal end of analyte component 1904 towards isolation rail 436 is opened Mouth 452 is spread, and has the micro- target 2112 of capture of label that can bond analyte component close to 452 (or in isolation rails) of opening 1904, this can cause the local reaction 2002 of adjacent proximal ends 452 (or inside proximal openings 452) of opening (for example, there is label The micro- target 2112 of capture aggregation).

When micro- 2112 adjacent proximal ends of the target opening 452 of the capture for having label or at 452 inside of proximal openings, analyte group Divide 1904 and has the bonding of the micro- target 2112 of capture of label maximum.This is because in area of isolation 444 and join domain 442 The concentration highest of analyte component 1904, to be conducive to analyte component 1904 and have the viscous of the micro- target 2112 of capture of label It ties and facilitates them and assemble in that region.As analyte component 1904 diffuses out channel 234 and in proximal openings Except 252, their concentration declines.Therefore, less analyte component 1904 is bonded to having except proximal openings 252 The micro- target 2112 of the capture of label.Analyte component 1904 and the reduction of the bonding for the micro- target 2112 of capture for having label are led in turn The aggregation of the micro- target 2112 of capture for having label except proximal openings 452 is caused to reduce.Not close to fence 436,438,440 The micro- target 2112 of the capture for having label of proximal openings 452 (or not in the proximal openings 452 of fence 436,438,440) because This does not generate detectable local reaction 2002 and (or generates detectably to be less than in size and occur to be open in adjacent proximal ends 452 or the local reaction 2002 inside proximal openings 452).

For the analysis for not having two bond sites for the bonding material in the micro- target 2112 of capture for having label Object component, having the micro- target of the capture of label may include two different bonding materials (as discussed in Figure 23 and shown below ), each of which being capable of analyte component specificity bonding.Alternatively, if analyte component aggregation is (for example, form Homodimer, tripolymer etc.), then measurement is feasible.

The example for having the micro- target 2112 of capture of label includes both the micro- target of no life and the micro- target of biology.It is micro- without life The example of target includes micro-structure, microballon (for example, polystyrene bead), micron bar, magnetic bead, quantum dot etc..Micro-structure can With big (for example, 10 to 15 microns of diameter, or bigger) or small (for example, diameter is micro- less than 10,9,8,7,6,5,4,3,2 or 1 Rice, or smaller).The example of the micro- target of biology includes the micro- target (for example, the micro- target of report biological) of biology, liposome (for example, closing At film preparation or as derived from film preparation), the microballon coated with liposome, nano-lipid raft is (see, for example, " Ritchie et al.(2009)Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs, Mehotd Enzymol., 464:211-231 (Ritchie et al. (2009), in phospholipid bilayer nanometer disk Memebrane protein recombination, method zymetology, 464:211-231) ") etc..

Figure 22 shows the example of the measurement material 1910 of the mixture including capturing micro- target 2212 and label object, the label The component of object is identified as 2222 and is hereinafter referred to as " label 2222 ".Figure 23, which is shown, captures micro- target 2212, analyte The example of the configuration of component 1904 and label 2222.Capturing micro- target 2212 may include specificity bonding analyte component 1904 First affinity agent 2312 of first area 2302.Label 2222 may include the second area of specificity bonding analyte component 1904 2304 the second affinity agent 2322.As shown in figure 22, reaction 2002 occurs viscous in the first area of analyte component 1,904 2302 The second area 2304 for tying the first affinity agent 2312 and analyte component 1904 that capture micro- target 2212 is bonded to label When 2222 the second affinity agent 2322.

Due to the analyte component generated by the micro- target 1202 of biology in the area of isolation 444 of isolation rail 436 1904 proximally facing 452 diffusions of opening, analyte component 1904 can be bonded to close to opening 452 (or in opening 452) Micro- target 2212 and label 2222 are captured, is accumulated on the surface for capturing micro- target 2212 so as to cause label 2112.Work as capture When micro- 2212 adjacent proximal ends of target opening 452 (or inside proximal openings 452), analyte component 1904 and there is catching for label The bonding for catching micro- target 2212 is maximum.Similar to as discussed above, this is because in area of isolation 444 and join domain 442 The rather high concentration of analyte component 1904 facilitates analyte component 1904 and captures the bonding and label of micro- target 2212 The 2222 corresponding association at the surface for capturing micro- target 2212.With analyte component 1904 diffuse out channel 434 and Except proximal openings 452, concentration declines and less analyte component 1904 is bonded to the capture except proximal openings 452 Micro- target 2212.The reduction of the bonding of analyte component 1904 and the micro- target 2212 of capture causes except proximal openings 452 The accumulation for capturing the label 2222 at the surface of micro- target 2112 is reduced.Therefore, it is not opened close to the proximal end of fence 436,438,440 The micro- target 2212 of capture of 452 (or inside proximal openings 452 of fence 436,438,440) of mouth is not by detectable terrestrial reference Note, or labeled degree are not up to their detectably labeled degree, which is detectably lower than in size Occur in adjacent proximal ends opening 452 or the label inside proximal openings 452.

The example for capturing micro- target 2212 includes as above all showing for what the micro- target 2112 of capture for having label was proposed Example.The example of first affinity agent 2312 includes the label of specific recognition analyte component 1904 or special by analyte component 1904 The ligand of opposite sex identification.For example, the first affinity agent 2312 can be interested antigen in the case where antibody analysis object.

The example of label 2222 includes the label for having the label object of Luminous label (for example, fluorescence labels) and having Object, the enzyme can crack the signaling molecule to fluoresce in cracking.

The example of measurement material 1910 includes such measurement material, which includes the compound micro- target of capture, should The compound micro- target of capture includes multiple affinity agents.Figure 24 shows compound including the first affinity agent 2402 and the second affinity agent 2404 Capture the example of micro- target 2412.First affinity agent 2402 is capable of the first area 2302 of specificity bonding analyte component 1904 (referring to fig. 2 3) and the second affinity agent 2404 being capable of the specificity identical analyte component 1904 of bonding or different analytes The second area 2304 of component.In addition, the first affinity agent 2402 and the second affinity agent 2404 optionally bond analyte group simultaneously Divide 1904 first area 2302 and second area 2304.

The example of first affinity agent 2402 includes those of as discussed above.The example of second affinity agent 2404 includes special Property discriminance analysis object component 1904 second area 2304 receptor or special by the second area 2304 of analyte component 1904 The ligand of opposite sex identification.For example, the second affinity agent 2404 can be bonded to the constant region of antibody in the case where antibody analysis object Domain.Example above-mentioned includes Fc molecule, antibody (for example, anti-igg antibody), a-protein, protein G etc..

Another example for measuring material 1910 is to measure material as one comprising multiple to capture micro- target.Example Such as, measurement material 1910 may include the first micro- target (not shown) of capture with the first affinity agent 2402, and have second The second of affinity agent 2404 captures micro- target (not shown).First captures micro- target can be different from the second micro- target of capture.Example Such as, first capture micro- target can have distinguish first capture micro- target and second capture micro- target size, color, shape or Other features.Alternatively, other than the type of each affinity agent for including, first, which captures micro- target and second, captures micro- mesh Mark can be the micro- target of capture of substantially the same type.

Another example for measuring material 1910 is to measure material as one comprising a plurality of types of to capture micro- mesh Mark, each of which is designed to be bonded to different interested analytes.For example, measurement material 1910 may include having first The first of affinity agent captures micro- target (not shown) and the second micro- target (not shown) of capture with the second affinity agent, wherein First and second affinity agents are not bonded to identical interested analyte.First, which captures micro- target, can have differentiation first to capture Size, color, shape, label or other features of micro- target and the second micro- target of capture.It in this way, can be simultaneously to more A interested analyte is screened.

Regardless of the particular content of measurement material 1910, in some embodiments, control module 472 can make control/monitoring Equipment 480 is loaded into material 1910 is measured in channel 434.Control module 472 can be by the measurement material 1910 in channel 434 Stream be maintained at minimal flow speed V as discussed above_minWith maximum fluidity speed V_maxBetween.Once measurement material 1910 exists In the position of the proximal openings 452 of neighbouring fence 436,438,440, control module 472 can substantially stop in channel 434 Measure the stream of material 1910.

The step 108 executed in microfluidic device 400 may include one detected close to isolation rail 436,438,440 Or more proximal openings 452 local reaction 2002, show be loaded into channel 434 measurement material 1910 point Analyse the reaction of object component 1904.If the local reaction 2002 of any proximal openings 452 close to isolation rail 436,438,440 It is detected, can determine whether those any local reactions 2002 detected show one in isolation rail 436,438,440 The positive performance of a or more micro- target 1202,1204,1206 of biology.In some embodiments, human user can be observed The join domain 442 of channel 434 or fence 436,438,440 is to be monitored local reaction 2002 and determine that the part is anti- Answer the 2002 positive performances for whether showing the micro- target 1202,1204,1206 of biology.In other embodiments, control module 472 can To be configured as executing the function.The process 2500 of Figure 25 is to be monitored to local reaction 2002 for executing and determine the office Whether portion's reaction 2002 shows showing for the operation of the control module 472 of the positive performance of the micro- target 1202,1204,1206 of biology Example.

At step 2502, the control module 472 of implementation procedure 2500 can pass through camera or other image capture apparatus (be not shown, but can be the element of control/monitoring device 480 of Fig. 4 A) come capture channel 434 or isolation rail 436,438, At least one image of 440 join domain 442.The example of time for exposure for capturing each image includes 10 milliseconds to 2 Second, 10 milliseconds to 1.5 seconds, 10 milliseconds to 1 second, 50 to 500 milliseconds, 50 to 400 milliseconds, 50 to 300 milliseconds, 100 to 500 millis Second, 100 to 400 milliseconds, 100 to 300 milliseconds, 150 to 500 milliseconds, 150 to 400 milliseconds, 150 to 300 milliseconds, 200 to 500 Millisecond, 200 to 400 milliseconds or 200 to 300 milliseconds.Control module 472 can capture such image or multiple images. If control module 472 captures an image, which can be following so-called last image.If control module 472 capture multiple images, then the image that two or more are captured can be combined image to the end by control module 472 In.For example, control module 472 can average the image that two or more are captured.In some embodiments, it controls Molding block 472 can capture and equalize at least 10,20,30,40,50,60,70,80,90,100,110,120,130,140, 150,160,170,180,190,200 or more the images captured are to generate last image.

At step 2504, control module 472 can identify any sign of local reaction 2002 in last image. As discussed above, the example of local reaction 2002 includes shining (for example, fluorescence), and therefore control module 472 can be directed to Last image is analyzed in the luminous of any proximal openings 452 close to isolation rail 436,438,440.Control module 472 can With the local reaction 2002 for being programmed to identify using any image processing techniques in last image.In this example, as schemed Shown in 20, control module 472 can detecte the local reaction 2002 of the proximal openings 452 close to isolation rail 436,438.

At step 2506, control module 472 can by each local reaction 2002 detected at step 2504 with Corresponding isolation rail 436,438,440 is associated.For example, control module 472 can be proceed as follows the step: will be in step The each local reaction 2002 detected at 2504 with have to react 1002 nearest proximal openings 452 isolation rail 436, 438,440 is associated.In the example of fig. 20, control module 472 can be related to isolation rail 436,438 by reaction 2002 Connection.

Control module 472 can be for each isolation rail for detecting reaction associated there at step 2506 436, the step 2508 and 2510 of 438,440 execution Figure 25.Relative to the example of Figure 20, control module 472 can therefore for every Step 2508 and 2510 is executed from fence 436, then repeats step 2508 and 2510 for isolation rail 438.

At step 2508, it is associated with current isolation rail 436 anti-can to determine that needle detects for control module 472 Answer 1002 positive findings for whether showing the micro- target 1202 of (multiple) biologies in current fence 436.For example, control module 472 The data about the reaction 1002 detected can be extracted from the last image obtained at step 2502, and are determined and extracted To data whether show positive findings.Any amount of various criterion can be used.For example, the reaction 2002 detected can be with It is luminous, and it is more than that threshold value, light emission luminance are more than threshold value, shine that the standard for determining positive findings, which may include luminous intensity, Color is fallen within the scope of predetermined color etc..If the reaction that control module 472 confirmly detects is the positive at step 2508, Then control module 472 can continue to step 2510, and wherein current isolation rail 436 can be identified as wrapping by control module 472 Containing the micro- target 1202 of positive organisms.If being determined as feminine gender at step 2508, control module 472 can be in step The reaction that will test at 2506 next isolation rail 438 associated with it repeats step 2508.

In the example shown in Figure 20, it is assumed that determine local reaction associated with isolation rail 436 at step 2508 2002 be the positive, and local reaction 2002 associated with isolation rail 438 is negative (is detected for example, shining, but its is low In for determining that isolation rail 438 is positive threshold value).As previously mentioned, the proximal openings 452 of neighbouring isolation rail 440 are not examined Measure reaction.Therefore, isolation rail 436 is only identified as with the micro- target of positive organisms by control module 472.Although in Figure 25 not It shows, but a part as process 2500, isolation rail 438,440 can be identified as feminine gender by control module 472.

Return to Fig. 1, at step 110, process 100 can by step 108 the micro- target of biology of positive test with The micro- target of biology being negative is tested to separate.It is in yin for subsequent characteristics test at step 108 that Figure 26 and Figure 27, which is shown wherein, Property the micro- target 1002 of biology be moved in the channel 252 of microfluidic device 200 and then flush out the logical of microfluidic device 200 The example in road 252.Figure 29 is shown the micro- target 1204,1206 of negative organisms and the positive organisms in microfluidic device 400 wherein The example that micro- target 1202 separates.

As shown in figure 26, the test micro- target 1002 of biology that each of is negative can keep fence 256 at step 110 In selected and be captured by ligh trap 2602.Negative micro- target is marked as 1802 in Figure 26.Then, ligh trap 2602 can be with It is moved in channel 252 from holding fence 256.As shown in figure 27, trap 2602 can be closed in channel 252, and medium The micro- target 1802 of negative organisms can be flushed out channel 252 and (also, optionally, flushed out by 244 stream 804 (for example, convection current) Flow region 240).Measurement material 1702 can diffuse out fence 256, and stream 804 can also rinse measurement material 1702 Channel 252 out.

Ligh trap 2602 can be generated and be operated as described above.For example, as shown, the micro- target of each negative organisms 2602 can individually be trapped and are moved in channel 252 from holding fence 256.Alternatively, more than one negative organisms Micro- target 2602 can be captured by single trap 2602.For example, can have the more than one micro- mesh of biology in single fence 256 Mark 2602.Anyway, the micro- target 2602 of two or more negative organisms can be selected and parallel in fence 256 Ground is moved in channel 252.

Detector 224 can capture the image of all or part of flow regions 240 (including the micro- mesh of biology in fence 256 The image of mark 1002), and these images can help to the micro- identification of target 2602 of individual negative organisms, trap and move out Specific fence 256 simultaneously enters in channel 252.Detector 224 and/or selector 222 are (for example, be configured as Fig. 3 A and Fig. 3 B DEP device) can be therefore micro- mesh for that will be directed to micro- target that characteristic test is positive be negative for characteristic test Mark one or more examples of the separator separated.

As shown in figure 27, for the micro- target 1802 of negative organisms in channel 252, the stream 804 of medium 244 can will be biological Micro- target 1802 flushes out channel 252, also, in some instances, flushes out microfluidic device 200 (for example, passing through outlet 210).For example, stream 804 can be resumed or increase if stream 804 is previously stopped or slows down.

Alternatively, the micro- target 1002 of biology of positive test can be moved to channel from fence 256 at step 108 It is washed away in 252 and by stream 804 at step 110 from channel 252.In such an example, it is tested at step 104 and 108 The micro- target 1002 of biology being positive can be collected to be stored in microfluidic device 200, further elsewhere Handle, be transmitted to another device (not shown) etc..The micro- target 1802 of biology that test is negative at step 108 then can be with It removes and abandons from holding fence 256.

As shown in Figure 28 and Figure 29, measurement material 1910 can be rushed out (2802) channel 434 (Figure 28).Then, as schemed Shown in 29, the micro- target 1204,1206 of biology tested in the microfluidic device 400 being negative at step 108 can be from isolation Fence 438,440 is moved in channel 434, and the micro- target 1204,1206 of negative organisms can be removed from channel 434 (for example, passing through The stream (being not shown, but can be similar to the 2802 of Figure 28) of medium in channel 434).It can be as discussed above for that will give birth to The micro- target 1202,1204,1206 of object from channel 434 be moved to any mode in isolation rail 436,438,440 (for example, DEP, Gravity etc.) the micro- target 1204,1206 of biology is moved in channel 434 from isolation rail 438,440.

After step 108 and 110, process 100 is selected also according to the test executed at step 108 at step 104 The micro- target (for example, 1002,1202,1204,1206) selected is classified.In addition, at step 104 selection and also directed to Micro- target of the follow-up test positive test at step 108 place, which can stay in, keeps fence (for example, 256,436,438,440) In, while negative micro- target can be removed.

As discussed above, step 108 and 110 can be repeated and therefore be performed n times, and wherein n is integer 1 (at this In the case of kind, step 108 and 110 is executed once without repeating) or greater than 1.It is executed at the step 108 repeated every time Follow-up test can be different test.Alternatively, repeat step 108 at execute follow-up test can be with it is aforementioned The identical test of previously execution of the test or step 108 that are executed at step 104.The micro- mesh of biology loaded at step 102 Therefore mark (for example, the micro- target of biology) can be carried out a series of n+1 tests.In some embodiments, every in n+1 test It once can be different test, and in some embodiments, different features can be directed to each time in n+1 test Test.Process 100 can therefore can sub-elect from the original mixture of the micro- target of biology and test for n+1 time (its each time It is different) group of positive test, and in some embodiments, process 100 can be from the initial mixing of the micro- target of biology The group for n+1 different characteristic positive test is sub-elected in object.

Alternatively, process 100 can select the micro- target of biology at step 104, then according to wherein biological micro- target The test quantity of the step 108 of positive test (is performed simultaneously or repeats step 108) to carry out the micro- target of selected biology Classification.It is desirable that multiple features are assessed in this way for including numerous applications of Identification of the antibodies.For example, multiple comment Estimate can help to it is any in following: identification conformation specific antibody (for example, different features can be it is specific anti-for bonding The ability of the antibody analysis object of former tripe systems elephant)；Antibody analysis object epitope mapping (for example, different features can be it is viscous Tie the various genes of antigen or the ability of chemical modification form)；Assess antibody analysis object cross-species reactivity (for example, Different features, which can be, comes from different plant species (such as mankind, mouse, big rat and/or other animal (examples for being bonded to Such as, experimental animal)) isogeneic antibody analysis object ability；And the IgG homotype of antibody analysis object.For example, " Dhungana et al. (2009), Methods Mol.Biol.524:119-34 (honest Ghana et al. (2009), molecular biosciences Method 524:119-34) " in the generation of the antigen of the chemical modification of the epitope mapping for antibody is described.

Whole process 100 can be repeated one or more times.Therefore, after executing n times step 108 and 110, step 102 to 106 can also be performed k times again, then execute n times step 108 and 110 again.For each repetition of process 100, Quantity k needs not be identical quantity.Similarly, for each repetition of process 100, quantity n needs not be identical quantity. For example, stream 804 shown in Figure 27 can be by new life for the specific duplicate last repetition step 108 and 110 of process 100 The mixture of the micro- target of object is loaded into the channel 252 of microfluidic device 200 as shown in Figure 8, therefore it can be in miniflow A part of the step 102 of the process next time 100 executed on body device 200.

Similarly, process 100 can be repeated quickly and easily as many times as required on microfluidic device 400.For example, process 100 can be repeated, To retest or reanalyse the micro- mesh of positive organisms being maintained in its isolation rail 436,438,440 at step 110 Mark；Assuming that initial testing is performed to the multiple biological targets of each isolation rail, in low-density (for example, every One micro- target of biology of a isolation rail) under retest and reanalyse the micro- target of positive organisms；Step 108 next time The new biology micro- target being loaded into microfluidic device 400 is tested or analyzed when repeating；For different points at step 110 Analysis object testing of materials or analysis be maintained in its isolation rail 436,438,440 the micro- target of positive organisms (for example, by using The measurement material 1910 of detection second or other interested analyte is designed as to repeat step 108) etc..

Figure 30 shows another example.As shown, after step 110 has been carried out, can permit be maintained at every From one or more micro- targets of biology (for example, 1202) in fence (for example, 436) in its isolation rail (for example, 436) Generate the clonal population 3002 of the micro- target of biology.It is then possible to using all or part of processes 100 (for example, step 108 and 110) test or analyze group 3002.Alternatively, as discussed above, the micro- target of biology can be surveyed by separation and again Examination.In another alternative scheme, (for example, in any of step 106 or 108 before process 100 has been completed Later, but prior to step 110), it can permit the micro- target of biology and grow into group.

Although describing specific embodiments of the present invention and application in the present specification, these embodiments and application It is merely exemplary, and can have many modifications.For example, the process 100 of Fig. 1 and the process 2500 of Figure 25 are only examples, and And modification is expected.Thus, for example, at least some steps of process 100 and/or process 2500 can be shown to be different from Sequence execute, and some steps may be performed simultaneously or can be with other step Overlapped Executions.As other examples, mistake Journey 100,2500 may include unshowned other step or some steps shown in lacking.

Example

Example 1- screening secretion can bond the mouse boosting cell of the IgG antibody of mankind CD45.

Screening is executed to identify that secretion is bonded to the mouse boosting cell of the IgG type antibody of mankind CD45.The experimental design packet Include following steps:

1. generating the microballon for being coated with CD45 antigen；

2. obtaining mouse boosting cell；

3. by loading cells into microfluidic device；And

4. measuring antigentic specificity.

Experiment reagent used includes reagent as shown in Table 1

Table 1- reagent

Generate the microballon for being coated with CD45 antigen

The microballon for being coated with CD45 antigen is generated in the following manner:

By 50 μ g carrier-free CD45 in 500 μ L PBS (pH 7.2) settling flux.

Slide-A-Lyzer is rinsed with 500 μ L PBS^TMMini cup, is then added microcentrifugal tube.

The CD45 solution that 50 μ L concentration are 0.1 μ g/ μ L is added to the slide-A-Lyzer after rinsing^TMIn mini cup.

170 μ L PBS are added in 2mg NHS-PEG4- biotin, 4.1 μ LNHS-PEG4- biotins are added later To the slide-A-Lyzer comprising CD45 antigen^TMIn mini cup.

CD45 antigen culture EZ-Link is used at room temperature^TMNHS-PEG4- biotin 1 hour.

After culturing, by slide-A-Lyzer^TMMini cup from microcentrifugal tube remove, and be placed into second it is micro from In 1.3ml PBS (pH 7.2) in heart pipe, and first 1 hour period is cultivated at 4 DEG C in the case where rocking. slide-A-Lyzer^TMMini cup is then transferred in the third microcentrifugal tube comprising the fresh PBS of 1.3ml (pH 7.2), and And second 1 hour period is cultivated at 4 DEG C in the case where rocking.The final step is repeated a further three times, in total into Row 5 are cultivated for 1 hour.

100 μ L biotinylation CD45 solution (about 50ng/ μ L) are moved into the pipe of label.

The microballon coated with Streptavidin of 500 μ L Spherotech companies is moved into microcentrifugal tube, 3 times (1000 μ L/ flushing) is rinsed in PBS (pH 7.4), then with 3000RCF centrifugation 5 minutes.

Microballon is resuspended in 500 μ l PBS (pH 7.4), to generate the microballon concentration of 5mg/ml.

The microballon coated with Streptavidin of the Spherotech company of 50 μ L biotinylated protein matter and settling flux is mixed It closes.At 4 DEG C in the case where rocking culture mix 2 hours, be then centrifuged 5 minutes at 4 DEG C with 3000RCF.Supernatant It is dropped and the microballon coated with CD45 rinses 3 times in 1mL PBS (pH7.4).Then, microballon is with 3000RCF at 4 DEG C It is centrifuged again 5 minutes.Finally, the CD45 for being coated with microballon is generally resuspended in the PBS of 500 μ L pH 7.4 and is stored in 4 DEG C Under.

Obtain mouse boosting cell

It obtains the spleen by the CD45 mouse being immunized and places it in DMEM medium+10%FBS.It is cut using scissors Broken spleen.

Broken spleen is placed in 40 μm of cell filters.It is rinsed individual cells by cell mistake Filter.Glass bar for rupture spleen further and force it is unicellular by cell filter, later again with 10ml pipette come It is rinsed individual cells and passes through cell filter.

Red blood cell is cleaved by commercial external member.

Cell makes former splenocyte with 2e with 200xG rapid centrifugation by 10ml pipette⁸The concentration of cells/ml It is resuspended in DMEM medium+10%FBS.

By loading cells into microfluidic device

Splenocyte is directed in micro-fluid chip and is loaded onto fence, and each fence includes 20 to 30 cells. 100 μ L media were flowed with 1 μ L/ seconds by device to remove undesired cell.Temperature is set to 36 DEG C, and with 0.1 μ L/ Velocity perfusion culture medium 30 minutes of second.

Antigentic specificity measurement

Preparation includes 1:2500 sheep anti-Mouse F (ab ') 2-Alexa568 cell medium.

100 μ L CD45 microballons include diluted sheep anti-Mouse F (ab ')-Alexa of 1:2500 in 22 μ L568 it is thin Settling flux in born of the same parents' medium.

The CD45 microballon of settling flux is then flowed into the main channel of micro-fluid chip with 1 L/ seconds rates of μ, until its neighbour Close includes the fence of splenocyte, but only on the outside of the fence.Then, fluid flowing is stopped.

Then, the position that micro-fluid chip is imaged to determine microballon in bright field.

Then, the image of cell and microballon is captured using Texas red optical filter.It shoots within every 5 minutes in 1 hour Image, 1000 milliseconds of each classification duration and gain are 5.

As a result

It observes development of the positive signal on microballon, reflects that IgG Isotype antibody diffuses out specific fence and spreads Into main channel, they can bond the microballon coated with CD45 in main channel.The bonding of anti-CD45 antibody and microballon allows Mountain sheep anti mouse IgG-568 is associated with microballon and generates detectable signal.Referring to Figure 31 A to Figure 31 C and white arrow.

Using method of the invention, every group of splenocyte associated with positive signal can be separated and by it with individual cells It is moved in new fence and is redeterminated.In this way, the individual cells for expressing anti-CD45IgG antibody can be detected It surveys.

In addition to any modification previously shown, without departing from the spirit and scope of the present invention, this field skill Art personnel are contemplated that many other modifications and alternative arrangement.Therefore, although most real above in association with being presently considered to Border and most preferred scheme are with special case and datail description information, but it will be apparent that without departing substantially from sheet described herein In the case where the principle and concept of invention, those of ordinary skill in the art can carry out many modifications, including but not limited to, shape Formula, function, mode of operation and use.As it is used herein, in all fields, example and embodiment are illustrative and do not answer It is construed as limiting.Although should also be noted that term " step " is used in this, which can be used for letter Singly cause the attention to the different piece of described method and is not meant to any portion of starting point of demarcation method or stops Point, or limit in any other manner.

Claims

1. a kind of microfluidic device including enclosing boundary, comprising:

Base portion is arranged microfluidic circuit structure on the base portion and limits the lid of microfluidic circuit jointly, wherein described Microfluidic circuit includes:

Flow region is configured to contain the stream of first fluid medium；

One or more entrances, the first fluid medium can be input into the flow region by the entrance；

One or more outlets, the first fluid medium can be removed by the outlet；

Microfluidic channel, at least part including the flow region；And

Microfluid isolation rail, comprising:

Isolation structure, the area of isolation including being configured to contain second fluid medium, the area of isolation have single opening； And

The area of isolation is connected to the flow region on fluid by join domain, wherein the join domain includes entering To the microfluidic channel proximal openings and enter the distal openings of the area of isolation, wherein the proximal openings Width W_conIn the range of 20 microns to 100 microns, the join domain from the proximal openings to the distal openings Length L_conFor the width W of the proximal openings of the join domain_conAt least one times,

Wherein the area of isolation of the microfluid isolation rail is the bypassed area domain of the microfluidic device.

2. the apparatus according to claim 1, wherein the channel at the proximal openings of the join domain Width is between 50 microns and 500 microns.

3. the apparatus according to claim 1, the length of the join domain of the proximal openings to the distal openings L_conFor the width W of the proximal openings of the join domain_conAt least 2.0 times.

4. the apparatus according to claim 1, wherein the join domain of the proximal openings to the distal openings Length L_conWith the width W of the proximal openings of the join domain_conSize is adjusted to so that with no more than 5.0 μ L/ seconds Flow rate be less than by the first fluid medium that the microfluidic channel flows to the penetration depth in the isolation rail The length L_con。

5. the apparatus according to claim 1, wherein the proximal openings of the join domain have at 20 microns and 60 Width W between micron_con。

6. the apparatus according to claim 1, wherein from the proximal openings to the join domain of the distal openings Length L_conBetween 60 microns and 300 microns.

7. the apparatus according to claim 1, wherein the microfluidic device further includes dielectrophoresis construction, the dielectrophoresis Construction includes first electrode, electrode activation substrate and second electrode, wherein the first electrode is the microfluidic device A part of first wall, the electrode activation substrate and the second electrode are one of the second wall of the microfluidic device Point,

Wherein the electrode activation substrate provides dielectrophoresis region at the inner surface of electrode activation substrate.

8. device according to claim 7, wherein electrode activation substrate is chemically activated by light.

9. device according to claim 7, wherein electrode activation substrate includes:

A. light-guide material；Or

B. semiconductor material, multiple doped layers, electric insulation layer and conductive layer including forming semiconductor integrated circuit.

10. device according to claim 7, wherein first wall of the microfluidic device is the lid, it is described micro- Second wall of fluid means is the base portion.

11. device according to claim 10, wherein the lid and/or the base portion light transmission.

12. device described in any one of -11 according to claim 1, wherein limiting the barrier of the microfluid isolation rail From the surface of the base portion of the microfluidic device pass through entire flow region extend to it is opposite with the surface described micro- The inner surface of the lid of fluid means.

13. a kind of process of the biological cell of analysis in microfluidic devices, described device include enclosing boundary, the boundary that encloses includes:

Flow region is configured to contain the stream of first fluid medium；

One or more outlets, the first fluid medium can be removed by the outlet；

Microfluidic channel, at least part including the flow region；And

At least one microfluid isolation rail, comprising:

Wherein the area of isolation of at least one microfluid isolation rail is the bypassed area domain of the microfluidic device,

The process includes:

One or more biological cells are loaded at least one described isolation rail；

The biological cell for cultivating the loading for a period of time, is enough that the biological cell is made to generate interested analyte；

The neighbouring join domain from least one isolation rail in the channel is to the described close of the channel Setting captures micro- target at end opening, and the micro- target of capture includes that specific can bond the interested analyte extremely Few a type of affinity agent；And

It is monitored to the micro- target of capture is bonded to the interested analyte.

14. process according to claim 13, wherein loading includes being loaded into one or more biological cell In the area of isolation of at least one isolation rail.

15. process according to claim 13, wherein loading one or more biological cell and including:

It is flowed into one group of biological cell in the channel of the microfluidic device；And

Described group of one or more biological cells are moved in each of at least one described isolation rail.

16. process according to claim 15 further includes rinsing to remove after loading at least one described isolation rail Stay any biological cell in the channel.

17. process according to claim 15, in which:

Loading one or more biological cell further includes that selection from described group meets each biology of preassigned carefully Born of the same parents；And

When the biological cell is in the join domain or the isolation of the channel or at least one isolation rail When in region, selection is performed.

18. process according to claim 13, being provided with the micro- target of capture includes:

Flow the micro- target of capture in the channel, and

Substantially stop the flowing, so that described capture the neighbouring company from least one isolation rail of micro- target Connect the proximal openings in region.

19. process according to claim 13, wherein the micro- target of capture includes label.

20. process according to claim 13, wherein it includes that will capture micro- mesh that setting, which captures micro- target, in the channel The mixture of mark and label object is arranged into the channel.

21. process according to claim 20, wherein the label object includes fluorescence labels.

22. process according to claim 13, wherein the interested analyte is antibody.

23. process according to claim 22, wherein the affinity agent of at least one type is special by the antibody Property identification antigen.

24. process according to claim 23, wherein the antigen is protein, carbohydrate, lipid, nucleic acid, generation Thank object, antibody or combinations thereof.

25. process according to claim 22, wherein the affinity agent of at least one type is Fc molecule, antibody, egg White matter A or protein G.

26. process according to claim 13, in which:

The microfluidic device includes multiple isolation rails, and each isolation rail includes fluid isolation structure, the fluid Isolation structure includes the join domain that the area of isolation is connected to the channel on area of isolation and fluid；And

Be arranged it is described capture micro- target include with the mixture for capturing micro- target or the micro- target of capture and label object substantially The channel of the neighbouring opening from the join domain of the multiple isolation rail to the channel of filling.

27. process according to claim 13, in which:

The length L of the join domain of at least one isolation rail_conIn the range of 20 microns to 200 microns, and

The process further includes that any stream in the channel is kept to be less than maximum allowable flow rate V_max。

28. process according to claim 13, wherein the microfluidic device is according to claim 1 any one of -12 The microfluidic device.

29. process according to claim 13 further includes rinsing to remove the micro- target of the capture from the channel.

30. process described in 6 or 29 according to claim 1, in which:

The length L of the join domain of at least one isolation rail_conIn the range of 20 microns to 200 microns, wherein The length L_conGreater than in the channel with maximum allowable flow rate V_maxThe penetration depth D of the medium of flowing_p, and

Described rinse includes making the scouring media in the channel to be less than the maximum allowable flow rate V_maxFlowing.

31. a kind of machine-readable storage device, make to control the nonvolatile of equipment implementation procedure in microfluidic devices for storing Property machine readable instructions, the microfluidic device include enclose boundary, the boundary that encloses includes:

Flow region is configured to contain the stream of first fluid medium；

One or more outlets, the first fluid medium can be removed by the outlet；

Microfluidic channel, at least part including the flow region；And

At least one isolation rail, comprising:

Wherein the area of isolation of at least one isolation rail is the bypassed area domain of the microfluidic device,

The process includes:

Micro- target will be captured to be arranged into the channel, so that the micro- target of capture is arranged at the neighbouring institute in the channel The proximal openings of join domain are stated, the micro- target of capture includes being capable of the specificity bonding interested analyte The affinity agent of at least one type；And

32. storage device according to claim 31, wherein the step of biological cell of the culture loading is held Row a period of time, being enough (1) makes the biological cell of the loading generate interested analyte, and (2) keep the sense emerging The analyte of interest diffuses out the isolation rail and is diffused into the channel.

33. storage device according to claim 31, wherein the step for loading one or more biological cell Suddenly include:

It is flowed into one group of one or more biological cell in the channel of the microfluidic device；And

One or more biological cells in described group are moved at least one described isolation rail.

34. storage device according to claim 33, wherein the step of one or more the biological cell of the movement It suddenly include the isolation that described group of one or more biological cell is moved to at least one isolation rail In region.

35. storage device according to claim 33, in which:

The microfluidic device includes the multiple isolation rails for being fluidly coupled to the channel, each isolation rail packet Fluid isolation structure is included, the fluid isolation structure includes that the area of isolation is connected to described lead on area of isolation and fluid The join domain in road；And

The moving step includes that individually one or more biological cell is moved in the multiple isolation rail In each.

36. storage device according to claim 33, in which:

The microfluidic device includes dielectrophoresis construction；And

By controlling the step of moving one or more biological cell described in the dielectrophoresis construction complete.

37. storage device according to claim 33, wherein the step of one or more the biological cell of the movement Suddenly include:

Capture multiple images in one group of biological cell in the microfluidic device；

Selection meets one or more biological cells of preassigned from described group；And

One or more selected biological cell is moved at least one described isolation rail.

38. the storage device according to claim 37, wherein for select the preassigned of biological cell for more than Threshold size.

39. the storage device according to claim 37, wherein for select the preassigned of biological cell for Cross section is with 5 microns to 20 microns, 20 microns to 40 microns or the circular shape of 100 microns to 500 microns of diameter.

40. storage device according to claim 33, wherein one or more the selected biology of the movement The step of cell includes the dynamic dielectric swimming power that capture and the mobile biological cell are generated in the microfluidic device.

41. storage device according to claim 31, wherein the step of setting capture micro- target includes:

Control the stream of the medium in the channel so that it is described capture micro- target and flow to neighbouring entered from the join domain Region in the channel of the opening in the channel；And

Substantially stop the stream.

42. storage device according to claim 41, wherein the step of stream for controlling the medium in the channel wraps Include driving on fluid with the channel attached inlet tube and outlet upper valve of the microfluidic device.

43. storage device according to claim 33, wherein the process is further comprising the steps of:

Any biological cell flushing that will be left in the channel removes out except the microfluidic device, wherein the flushing Step complete the step being loaded into one or more biological cell at least one described isolation rail it After execute.

44. storage device according to claim 31, wherein the step of setting capture micro- target includes setting Capture the micro- target of multiple capture in the join domain for being located at least one isolation rail in micro- target.

45. storage device according to claim 31, wherein the step of setting capture micro- target includes basic Described in the micro- target filling of the capture of the opening of the upper join domain with neighbouring at least one isolation rail Channel.

46. storage device according to claim 31, wherein the step of setting capture micro- target includes using Dielectrophoretic force.

47. storage device according to claim 46, wherein the dielectrophoretic force is constructed by photoelectricity tweezers and provided.

48. storage device according to claim 31, wherein the micro- target of capture includes label.

49. storage device according to claim 48, wherein the label is fluorescence labels.

50. storage device according to claim 48, wherein described pair captures described micro- target to be bonded to the sense emerging The step of analyte of interest is monitored includes the image for capturing the cold light from the label.

51. storage device according to claim 31, in which:

The step of setting includes by the label object including label of the neighbouring opening and the mixing of the micro- target of capture Object is arranged from the join domain into the channel.

52. storage device according to claim 48, wherein described pair captures described micro- target to be bonded to the sense emerging The analyte of interest is monitored the figure including capturing multiple cold light from the label or the label object including the label As and to the cold light that detects equalizes.

53. storage device according to claim 50, wherein described pair captures described micro- target to be bonded to the sense emerging The step of analyte of interest is monitored further includes determining described in the region of the opening from the neighbouring join domain Whether cold light is more than threshold intensity.

54. storage device according to claim 53, wherein the process is further comprising the steps of: when from described neighbouring Region the cold light be more than the threshold intensity when, will include the join domain described at least one isolation rail know One or more biological cells of the expression interested analyte Wei not included.

55. storage device according to claim 31, further includes:

After the micro- target of capture is bonded to the step of interested analyte is monitored by described pair of execution, institute It is further comprising the steps of to state process: rinsing and removes the micro- target of the capture from the channel.

56. storage device according to claim 31, in which:

The length L of the join domain of at least one isolation rail of the microfluidic device_conAt 20 microns to 200 In the range of micron, and

The process further includes that any stream in the channel is kept to be less than maximum allowable flow rate V_maxThe step of.

57. the process that the micro- target of biology in a kind of pair of microfluidic device is classified, the microfluidic device include enclosing boundary, institute Shu Wei circle includes:

Flow region is configured to contain the stream of first fluid medium；

One or more outlets, the first fluid medium can be removed by the outlet；

Microfluidic channel, at least part including the flow region；And

Microfluid isolation rail, comprising:

Wherein the area of isolation of the microfluid isolation rail is the bypassed area domain of the microfluidic device,

The process includes:

By the micro- target load of biology in the flow path into microfluidic device；

The first test for fisrt feature is executed to the micro- target of the biology in the flow path；

The micro- target of multiple positive organisms is moved in the microfluidic device and does not involve space, wherein in response to described first Test, the multiple micro- target of positive organisms are directed to the positive test of the fisrt feature；And

The second test for second feature is executed to the micro- target of the multiple positive organisms not involved in space.

58. process according to claim 57, wherein the fisrt feature is the particular organisms shape of the micro- target of biology State.

59. process according to claim 58, wherein the particular biological status corresponds to the spy of the micro- target of biology Determine appearance.

60. process according to claim 58, wherein the particular biological status corresponds to the micro- objective expression of biology Cell surface marker.

61. process according to claim 57, in which:

Execution first test includes the radiation that the micro- target of the multiple positive organisms is detected in the flow path, The radiation wherein detected shows the fisrt feature.

62. process according to claim 61, in which:

The multiple micro- target of positive organisms includes the measurement material for being bonded to interested analyte；And

The measurement material emits the radiation.

63. process according to claim 62, wherein described the step of executing the described first test further includes only will transmitting Be greater than intensity threshold intensity the radiation the micro- target of biology in it is multiple be identified as it is described first spy The positive test of sign.

64. process according to claim 62, wherein described the step of executing the described first test further includes only will transmitting Be less than intensity threshold intensity the radiation the micro- target of biology in it is multiple be identified as it is described first spy The positive test of sign.

65. process according to claim 57, wherein the fisrt feature or the second feature include to interested The expression of analyte.

66. process according to claim 65, wherein the interested analyte is protein.

67. process according to claim 57, wherein the step of the movement includes only will be from the flow path The multiple micro- target of positive organisms does not involve in space described in being moved to.

68. process according to claim 57, wherein described, not involve space include inside the microfluidic device The area of isolation of one or more isolation rails.

69. process according to claim 57, in which:

The described micro- target of multiple positive organisms is moved in the microfluidic device include: the step of not involving space

First light pattern is projected in the microfluidic device, to activate the electrode zone in the inner surface of electrode activation substrate Place in one or more dielectrophoresis electrodes, to surround and trap the micro- target of the positive organisms；And

Mobile first light pattern projected in the microfluidic device, thus by the captured micro- mesh of positive organisms Mark, which is guided from the flow path to described, not to be involved in space.