CN101743303B

CN101743303B - cell sorting system and method

Info

Publication number: CN101743303B
Application number: CN200880019323.8A
Authority: CN
Inventors: W·F·巴特勒; 张海川; P·玛常德; K·阿恩; 张毅; J·弗朗西斯; B·雷; E·涂
Original assignee: North & Co Ltd
Current assignee: North & Co Ltd
Priority date: 2007-04-20
Filing date: 2008-04-11
Publication date: 2016-12-14
Anticipated expiration: 2028-04-11

Abstract

The invention provides equipment and the method for micro-manufacture fluorescence-activated cell sorter of switch based on quick, the positive control being carried out cell sorting by microfluidic channel network.This sorter can carry out the sorting of the cell mass of low stress, high efficiency a small amount of cell (i.e. 1,000 100,000 cells).Present invention resides in the microfluidic channel network packaging in self-contained type's plastic box body, it can make, with aseptic disposable form, the interconnection that microfluidic channel network carries out macro-scale instrument.Alone or in combination optics and/or fluid switching power is used to realize switching.

Description

Cell sorting system and method

Technical field

The present invention relates to make in microfluidic channel network firmly to provide switch, this switch can pass through by separative selection The target cell of network is with the method and apparatus separated with non-target cell by described target cell and collect them.It is particularly interesting that Optics switching power or fluid switching power.

Related application

This application claims the U. S. application No.11/781 submitted on July 23rd, 2007, the priority of 848, this application requirement The priority of the U.S. Provisional Application No.60/925.563 that on April 20th, 2007 submits to.

Background technology

Traditional Fluorescence Activated Cell sorter (FACS) is widely used 1 in studies and clinical application.These instruments Very quick, the analysis of multiparameter and sorting can be carried out, but typically require bigger sample size, it is desirable to be trained Operator is for Operation and maintenance, and sterilizing difficulty.FACS instrument can be analyzed and lack to 10,000 and up to count with ten million The cell of meter.But, less than 100, during 000 cell, sorting executive capability dies down¹.Other separation method such as magnetic bead is not Cell as much is required as FACS, but they puzzlements of being assembled by non-specific binding, cell and globule, and be subject to The puzzlement of the probability of subsequent process steps is may interfere with to globule self.Therefore, the valuable of primary tissue is derived from order to sort Small sample or cell, it is possible to operation is containing the small sample amount of low cell number and allows efficient recovery to be sorted the cell of cell mass Sorter solves the problem in science of uniqueness.

Micro-manufacture cell counter can sort few to 1, and 000 cell disappears in wieldy closed system simultaneously Consume less reagent.The latter is important, because unlike traditional FACS instrument, not producing aerosol, decreasing and divided Select the pollution risk of cell, and decrease the risk that use bio-hazardous materials carries out operating.Have been described for several micro-manufacture Cell sorter, but major part is all " concept confirms (proof of concept) ".Fu, et al.²Report with 17 thin The flux of born of the same parents/second carries out 30 times of enrichments of bacillus coli (E.coli).After sorting, antibacterial only has 20% can survive, and And the separating purity in target bank is 30%.In follow-up research³In, flux increases to 44 cell/seconds, but target Purity drops below 10%, and response rate reported values is 39%.Wolff, et al.⁴Can be with 12,000 things from chicken red blood cell Flux separation globule (beads) of part/second, reaches 100 times of enrichments.But, the purity in target hole is about 1%.At these In research, enrichment is defined as the increase compared with initial concentration of the target group concentration in collecting hole.Purity represents sorting Accuracy and be to be sorted to enter to collect to be sorted the target cell percent relative to all cells in hole.The response rate is determined The quantity ratio that justice is the cell number counted by fluorescence detector and the cell being recovered from collection hole.Latter two research is at miniflow Using pressure switch in body device, it switches whole stream, and therefore switches in the interior any particle comprised of fluid plug.At this Mechanical compliance in a little switches causes fluid switch speed to be the rate-limiting step of flux³.There was reported electric flow control, example Such as electric osmose^2,5,6Or dielectrophoresis^7,8,9, but high electric-force gradient and physical chemistry to the ionic strength buffered limit for carefully It it is non-ideal condition for born of the same parents.

Buican et al.⁹First proposed use optical force for making particle be deflected through fluid passage.By light beam quilt The power being applied on particle is determined by the opposing optical character of luminous power and particle and surrounding fluid medium thereof.For a diameter of For the biological cell of about 10 μm, it is possible to obtain the power of about 1pN/mW.Although optical force is less, but cell deflection is made to enter Power necessary to adjacent flow logistics is the least, and such as, 900pN moves the cell of 10 μ m diameter, and 20-40 μm cell is at several millis Laterally across logistics in second.This power is to hinder by the viscous overcoming the cell being under the speed that this transverse movement is comprised to be subject to Power necessary to power.

United States Patent (USP) 6 is can be found in about the principle of optical force and general background technology, 744,038, it is merged in this in full Literary composition is as reference.

The multiple air pressure modulating system for carrying out particle sorting in microfluidic devices well known in the art.Pass through Alternately changing the air pressure of contact microfluidic device, the celliferous particle of bag flowed in microfluidic channel can be directed into required One or more branch roads, thus the sorting function of low cost can be realized.

Summary of the invention

As described below, these power are used for realizing switching in microfluidic channel network, can enter as cell sorting system Row operation.By detect upstream, the comfortable position of the switch microfluidic channel network in the fluorescence signal of target cell of flowing touch Send out switching, although can similarly use the such as light scattering of other detection mode to activate switch.Use switching guide cell or Particle enters one of multiple output flow logistics, and does not change basic flowing, thus the cell needed for collecting should for other With.Wish the laminar flow that the flowing in microfluidic channel is typically under extremely low Reynolds number.Therefore, with specific laminar flow layer Or any cell of flow stream flowing, when lacking under any power horizontal with this laminar flow layer, will stay on this flow stream In.Described switch applies power to cell and realizes this purpose exactly, and cell laterally shifts with laminar flow layer, makes cell from process The flow stream that bifurcated abutment is left in one outlet moves to leave the flow stream at bifurcated abutment through the second outlet.

In one embodiment, cell sorter includes: be suitable for accepting the one or more cells in fluid media (medium) Cell import；It is fluidly connected to the first and second buffer agent imports of cell import, to provide buffer agent solution to sorter； It is fluidly connected to cell import and the fluid passage of the first and second buffer agent imports；It is fluidly connected to the first horizontal stroke of fluid passage Circulation road；Being fluidly connected to the first and second outlets of fluid passage, these outlets are positioned at cross-flow passes downstream, and applicable detection is given The cell determining state the cell responding given state produce the detector of signal, and this detector is arranged in the first crossing current and leads to Road upstream position detection cell, be connected to detector and respond this signal and can be actuated in order to fluid in cross-flow passes The cross force switch of movement；Thus when the cell of given state being detected, cross force switch is activated to provide horizontal stroke to cell Xiang Li, so that signaling is so that its selectivity exits into the first or second outlet.

The present invention describes in detail in the following paragraphs for producing switching pass and for making switching method for optimizing Methodology, the design of microfluidic channel network and cell stream or the character of particle flux in microfluidic networks, in order to realize strengthening Separation performance.In the case of optics switches, optics switching is led to generally by illumination optical field is projected into microfluid The cell track establishing flow in road network, in microfluidic channel and work.Cell is right with the interaction of light field Cell produces power, and this power makes cell be horizontally transferred to the flow set up, thus cell from a flow stream to establish stream Amount moves to another flow stream, cell retention does not occur or does not significantly change the cell motion with former flow.

Hereinafter, term cell and particle both of which are understood to be and refer to any biological cell, biomone, sky So organic or inorganic particle and artificial organic or inorganic particle.The granularity of the cell being sorted in microfluidic channel network Scope is typically the particle size range of biological cell, and diameter is about 1 micron to about 50 microns.In more general terms, have about 100 nanometers Cell to about 100 micron diameters is by mean of the material standed for that the valve of microfluidic channel network carries out sorting.

An embodiment, use optical switch.Generally, laser has been used to produce the light being used in optical switch Bundle.The laser being currently used for optical switch is near-infrared continuous wave laser, it is known that it is close at the energy switched for display optics The viability of biological cell is not injured under degree and open-assembly time.If the infringement to particle is not problem, alternative laser Source can be considered for different application, including visible wavelength lasers or near ultraviolet wavelength laser, or wherein can use big Luminous flux carrys out very fast improved pulse laser.But, light beam source is not limited to laser, even if other of the present invention is discussed Employ laser to produce optics switching.

In still another embodiment, fluid switch can be used.Preferably, available based on pneumatic fluid switch.? In another embodiment, the microfluid wafer designed for cell sorter uses air pressure modulation.For flowing switching or cell The unilateral passage of sorting can be used in microfluidic device.Described channel geometries is effective for carrying out in microfluidic device Aerodynamic flow switching is prepared.In yet another aspect, a pair double block valve on unilateral passage makes the pneumatic handoff response time Minimize.In yet another aspect, it is provided that box body bearing is for the pneumatic control of microfluid wafer.

About the concrete application of fluid switching system, flow sorter switched system includes: be suitable for accepting fluid media (medium) Import；It is fluidly connected to the fluid passage of import；It is fluidly connected to the first and second cross-flow passes of fluid passage；It is connected to One and second first and second pneumatic operated valve of cross-flow passes；It is fluidly connected to the first and second outlets of fluid passage；These go out Mouth is positioned at the downstream of cross-flow passes；And control system, this control system is connected to the first and second valves, this control system offers Timing controling signal, to activate the first and second pneumatic operated valves, is characterised by the front opening that the first valve is opened at the second valve.Another Individual aspect, switching be further characterized in that the first valve after the second valve is opened and second valve close before close.? Another aspect, the second cross-flow passes and the second pneumatic operated valve can be cancelled to realize using the one side with 1 or 2 pneumatic operated valve to lead to Road sorts.

In still another embodiment, Microfluidic cell sorter uses optics switching and air pressure to modulate.Additionally provide light Learn design and opto-mechanical design.Double excitation lighting module and poorly lit module are preferably used.Efficient high NA (numerical aperture) thing Mirror is preferably used to collect fluorescence.

In the other side of the present invention, have employed fluorescence signal detection and processing system and method.ADC be optimized for by Analogue signal is converted into digital signal.In one embodiment, digital signal processing algorithm is implemented in FPGA.

Optionally, it is provided that the autoregistration of box body.The present invention solves the autoregistration problem loading box body in systems.

In other side, the present invention relates to the application software designed for microfluid sorter instrument.With recordable, lossless Become estranged at a high speed (10 mbit or higher) form repeatedly can perform to apply with process between from external equipment to original number According to shunting.

In other embodiments, it is provided that microfluid box body adds dress station.The present invention describes for adding dress microfluid The dress that adds of equipment is stood.

Aspect at other, it is provided that for growing the dilution cloning of the selection of monitoring and cell such as fetus cells.This Invention describes the dilution cloning of the selection for growing monitoring and described cell.

Accompanying drawing explanation

Fig. 1 is the plane graph at the Y-shaped sorting abutment in microfluidic channel network.

Fig. 2 is to be incorporated to sheath stream hold under the arm in reducer coupling chalaza and the Y-shaped sorting abutment connected by main thoroughfare and stream Cell has the plane graph of the microfluidic channel network of 50/50 shunting, and it is collectively known as 50/50 optic network.

Fig. 3 a and 3b is to be incorporated to sheath stream hold reducer coupling chalaza and the Y-shaped sorting abutment connected by main thoroughfare under the arm and flow In cell by means of differential sheath flow velocity the plane graph of microfluidic channel network of deflection shunting, its together with optical switch by It is referred to as the optic network of sheath stream deflection.

Fig. 4 a and 4b is to be incorporated to sheath stream hold reducer coupling chalaza and the Y-shaped sorting abutment connected by main thoroughfare under the arm and flow In cell by diversity exit width the plane graph of microfluidic channel network of deflection shunting, it is together with optical switch It is referred to as the optical switch network of sheath stream deflection.

Fig. 5 is 50/50 optical switch network with bi-directional laser line optical switch.

Fig. 6 is 50/50 optical switch network with two-way laser spot optical switch.

Fig. 7 a, 7b and 7c are that the laser line optical in having the larger microfluidic channel network exported more than two is opened The plane graph closed.

Fig. 8 a, 8b and 8c show the optical design designed for regulation and/or interrupted optical switch.

Fig. 9 a and 9b is and the transfer of cell levelling row or the laser spot optical switch of transfer angled with cell stream The plane graph of sheath stream deflection optical switch network.

Figure 10 a, 10b, 10c and 10d show the single lasing light emitter and trigger decision method used for cell detection Detector arrange and the diagram of selection of time/trigger.

Figure 11 a, 11b, 11c and 11d show two lasing light emitters and trigger decision method used for cell detection Detector arrange and the diagram of selection of time/trigger.

Figure 12 is the representative of the mask for the microfluidic channel network in both bottom and top glass substrate Property design diagram, when these substrates combined formation single network its provide the cell stream in main thoroughfare 2 dimension Sheath stream holds contracting under the arm.

Figure 13 shows the 3-dimensional example of the design described in Figure 12.

Figure 14 is the illustration of the side view of microfluidic channel network, and it provides cell stream in vertical direction with then at water Square to sequentially sheath stream hold contracting under the arm, in main thoroughfare produce cell stream completely 2 dimension sheath streams hold contracting under the arm.

Figure 15 is the 3-dimensional example of the microfluidic channel network described in Figure 14.

Figure 16 is when bottom and top glass substrate are combined together to form the microfluidic channel net shown in Figure 14 and 15 During network, for the diagram of the representative photolithography mask design of both bottom and top glass substrate.

Figure 17 is the representative embodiment of the mask for complete microfluidic channel network, this microfluidic channel Network has T-and holds reducer coupling chalaza under the arm and be connected to the T-bifurcated abutment of outlet, for implementing the cell sorting of optically-based switch Method.

Figure 18 is the representative embodiment of the mask for complete microfluidic channel network, this microfluidic channel Network has triangle and holds reducer coupling chalaza under the arm and be connected to the Y-bifurcated abutment of outlet, for implementing the cell of optically-based switch Method for separating.

Figure 19 shows the preferred embodiment party of the microfluidic channel network in complete Microfluidic cell sort wafers Case.

Figure 20 shows self-contained type's disposable cassette of the microfluidic channel network cell sorter for optically-based switch The preferred embodiment of body.

Figure 21 shows the preferred of the optical system of the microfluidic channel network cell sorter for optically-based switch Embodiment.

Figure 22 shows that the microfluidic channel network cell sorter of optically-based switch is held for the multiple of optical switch The representative performance of row.

Figure 23 is the prior art in the microfluidic channel network of display racetrack based on pneumatic fluid switching Y-shaped sorting abutment plane graph.

Figure 24 a and 24b is the dual pathways fluid switch having and showing possible racetrack under different pneumatic dividing potential drops Microfluidic channel in Y-shaped sorting abutment plane graph.

Figure 25 a and 25b is the single channel fluid switch having and showing possible racetrack under different pneumatic dividing potential drops Microfluid channel in Y-shaped sorting abutment plane graph.

Figure 26 shows the preferred of the Y-shaped sorting abutment in the microfluidic channel with single channel fluid switch Embodiment.

Figure 27 shows and makes pneumatic handoff response minimal timeization in single channel fluid switch and control pneumatic mode A pair double block valve.

Figure 28 a and 28b shows that the traffic flow of a pair double block valve and crossing channel, as the chart of the function of time, makes Obtain pneumatic handoff response minimal time and control the pneumatic mode in dual pathways fluid switch.

Figure 29 is the box body bearing of the pneumatic control for microfluid wafer.

Figure 30 shows the measurement of the fluorescent calibration globule carried out on instrument as herein described.

Figure 31 a shows the fluorescence microscopy images of the Jurkat cell with DAP1-dyeing, and Figure 31 b shows tool There is the fluorescence microscopy images of the target Jurkat cell of CellTracker Green.

Figure 32 shows the station adding dress microfluid box body.The present invention describes and stands for the dress that adds adding dress microfluidic device.

Figure 33 shows for growing supervision and the dilution cloning of fetus cells selection.The present invention describes for growing prison The dilution cloning that control and fetus cells select.

Figure 34 is the shallow microfluidic channel in 1 dimension adfluxion.The present invention describes and overcomes for microfluid fluidic cell The method of the original speed dispersion in 1 dimension adfluxion of art or sorting.

Figure 35 shows the cell/particle detection in heterogeneous microfluid droplet.The present invention describes some for detecting The method of the cell/particle counting in leggy droplet of fluid.

Figure 36 is the microfluidic outlet port port configurations for carrying out effective sample collection.The present invention describes in use micro- If fluid device carries out the method for dry sample collection after cell or particle sorting.

Figure 37 is the microfluidic device for repeatedly sample analysis or sorting.The present invention describes several for repeatedly sample Product are analyzed or the microfluidic device of sorting.

Figure 38 is the microfluid analysis wafer for sample analysis and recovery.The present invention describes several for lower Dilution factor sample analysis and the microfluidic device of recovery.

Detailed Description Of The Invention

Fig. 1 shows (i.e. have a main input channel 11 and from bifurcated abutment at 1 × 2 microfluidic channel network The network of stretch out two outlets 12 and 13) in be used for the embodiment of optical switch 10 of sorting cells.Bifurcated engages " Y " geometry of point is as it is shown in figure 1, but it be also possible to use other shapes of bifurcated such as " T " geometry.Generally, these Microfluidic channel is formed in optical clear substrate such that it is able to optical switch and other cell detection optics device are projected In stand in channel.This substrate typically, but without limitation, for glass, quartz, plastics, such as polymethyl methacrylate Etc., and other can cast or spendable polymer (such as polydimethylsiloxane, PDMS or SU8) (PMMA).Miniflow The degree of depth of body passage typically, but without limitation, is that 10 μm are to 100 μm.The width of microfluidic channel is typically, but unrestricted Property, for 1 to 5 times of the degree of depth.The masking carrying out substrate of glass, then carry out passage isotropic etching micro-to manufacture In the case of fluid passage, cross section is typically rectangle, or the rectangle with quadrant angle.

Flox condition is provided so that when light beam (deriving from laser in this case) is switched off or blocks, thus light beam does not has When inciding on dot area, all cells will preferably flow into one of outlet, the such as outlet 13 on right side.When light beam quilt When being turned or switched on, light beam is incident on dot area, and is drawn by cell and the produced optical force of light beam interaction Outlet 12 on the left of guided cell entrance.In this embodiment, the optical figuring being selected for guiding cell is relative to fluid The direction of flowing elongated laser lighting line at an angle.Optical gradient forces (Optical gradient forces) makes Cell laterally leaves the main streams circuit of cell, goes out so that the cell being switched is then departed from main thoroughfare entrance one Mouth such as 12, and the cell not being switched exits into another outlet such as 13 from the main streams of cell.At microfluid In channel network flox condition be set and controlled can pass through direct-driven pump, air pressure pumping, electrokinetics, capillarity, Gravity or other means that can produce fluid flowing complete.

With regard to flux (time rate in the sorting region at cell entrance top, bifurcated abutment), (target cell goes out yield at target Mark in mouthfuls 12) and purity (exporting the ratio of the cell number of 12 intracellular targets numbers/always at target) for the performance of sorting mechanism, Being affected by various factors, each factor affects the execution of optical switch.Optical switch can be with several such as following ginsengs Number is characterized: be projected into the shape of optical figuring in the sorting dot area of microfluidic channel network, figure relative to The position at bifurcated abutment, optical figuring is relative to its home position and any motion of shape, and what optical switch activated continues Time, for producing wavelength and the power of the lasing light emitter of optical switch figure, etc..The particular value of these parameters of optical switch Selection especially determined by following factor: the topology of micro-fluidic channel system and geometry, in micro channel systems Flow velocity (cell speed), (cell is the central authorities in main thoroughfare to control the ability of the position of the cell of flowing in main thoroughfare Flowing is still offset to side flowing), it is achieved the amount of the necessary cell displacement of reliable switching, the degree of depth of passage, the shape of passage Shape, and cell power produced with optical switch interaction.

Generally, when cell is introduced in the stream being positioned at main thoroughfare, they can be in any lateral position in stream Move down along passage.Therefore, according to the difference of cell lateral attitude, known to the pressure driven flow in microfluidic channel Parabola (for cylindrical microfluidic body passage) or quasi-parabola (for more common cross section) VELOCITY DISTRIBUTION, and Cell is caused to move at different rates.This will make it difficult to all cells bias current flow to outlet i.e. 13, such as Fig. 1 institute Show.Any optics switching using this flow geometry to carry out will necessarily cause small throughput and can obtain for producing optics switching The service efficiency of laser power low.Use any fluid switching that this flow geometry carries out, due in detection region and The variable cell flight time between Zone switched, low flux, purity and yield will be caused.Use suitable flox condition Can help to alleviate these restrictions that the performance to optics switching or fluid switching causes.

Suitable flox condition can be established in many aspects.In one embodiment, sheath stream as shown in Figure 2 is used Scheme, by holding shrinking born of the same parents under the arm input logical from the left side 21 of cell input channel stream 20 and both sides, right side 22 all additional cushion agent stream Road stream 20, it is achieved that the cell central 1-dimension concentration in main thoroughfare becomes single file (at plane graph for flatly).By from often Side has equal stream it is achieved thereby that keep a cell in the central authorities of main thoroughfare.This stream produces divided fluid stream face effectively 23, as in figure 2 it is shown, and its produce fluid and cell the most at last and shunt at bifurcated junction point 50/50.Use this microfluid Passage design and flox condition carry out optics switching and require optical switch with sorting target cell from mixed cellularity group, and this optics is opened Close energetically target cell is switched to one outlet the most as shown in Figure 1 12 and non-target cell is switched to another outlet the most such as Shown in Fig. 1 13.This design of micro fluidic channels and flox condition is used to carry out fluid switching to sort from mixed cellularity group Target cell requires fluid switching, and target cell is switched to an outlet i.e. 12 shown in Fig. 1 and is cut by non-target cell by energetically Change to another outlet i.e. 13 shown in Fig. 1.One embodiment of this embodiment be such as Figure 24 in dual pathways fluid open Close.

As an alternative, as shown in Fig. 3 a-b, by applying unequal stream to sheath circulation road both sides, the thin of concentration can be made The central authorities of born of the same parents' place on line deviation main thoroughfare.This causes from input channel 30 effectively to grouping teaching 33 1 in main thoroughfare Lateral deviation from cell oblique flow.The side that cell flows to main thoroughfare inclination is contrary with the side that its mesotheca stream has higher flow rate. It is to say, when right side sheath buffer agent 32 flows faster than left side sheath buffer agent 31, cell circuit in main thoroughfare towards The left side deflection of stream, as shown in Fig. 3 a-b.But, left side sheath stream also can have a higher flow velocity, thus promotes cell circuit court The right side deflection of main thoroughfare.Fig. 3 a-b also show fluorescence detector 34 and optical switch 35.Fluorescence detector is used as Determine the mechanism which kind of cell is to be sorted, and will hereinafter be discussed in detail.From Fig. 3 b it is clear that effective Sorting relates to making signaling cross over grouping teaching, arrives fluorescence feminine gender non-target cell microfluidic channel 36 from leaving bifurcated abutment Flow stream enter leave bifurcated abutment arrive fluorescent positive target cell microfluidic channel 37 flow stream.Sheath buffer agent The manipulation of flow velocity can realize by the following means: uses direct-driven pump, air pressure pumping, electrokinetics, capillarity, weight Power or other means producing fluid stream are individually controlled the flow velocity in respective wing passage, or by designing miniflow especially Body sheath network, by balance carefully pressure drop in each microfluidic channel with ensure to occur central authorities' stream (50/50 shunting) or Bias current.

One alternative, and to realize all cells inlet flow 40 before fluoroscopic examination 44 in main thoroughfare preferred Flowing into output microfluidic channel i.e. means for fluorescence feminine gender passage 46 is: use equal sheath buffer flow rate 41 and 42 to obtain Central must hold contracting under the arm, then pass through relative to fluorescent positive outlet 47, there is bigger volumetric fluid from bifurcated abutment and flow into Fluorescence feminine gender outlet 46, makes the preferred bias current of cell enter fluorescence feminine gender passage.These means are as shown in Fig. 4 a-b, and wherein left side goes out Mouth 46 is wider than right-side outlet 47.Grouping teaching 43 is arranged into the right side of centrally located cell stream by this configuration effectively.Therefore, When cell is in desired location, then uses optical switch or fluid switch 45 that target cell is transferred across grouping teaching and enter the right side The target cell fluorescent positive outlet of side.These means are wider than left side outlet but equally effective by right-side outlet, target whereby Cell is transferred leap grouping teaching by optical switch or fluid switch, and described grouping teaching is located in the cell stream of central authorities now Left side, and be therefore sorted entrance left side outlet.Therefore, by designing microfluidic channel egress network especially or logical Cross the outlet back-pressure controlled energetically in respective outlet, the outlet needed for cell flows into can be controlled.

Either using central authorities' stream or use deviation stream, the cell stream of concentration and the respective distance in divided fluid stream face are Domination is the displacement size realizing the necessary cell of switching reliably eventually.This arranges further as realizing the switching of reliable optics The length of required laser rays and laser power or for realize the amplitude of the air pulsing required by the switching of reliable fluid with Persistent period.Cell stream and grouping teaching the closer to, then required displacement is the shortest, and assorting room becomes more effective.In order to be divided The purity of monoid strengthens and for high flux, and the single switch of unidirectional layout, it is as optical switch or fluid switch, it is desirable to sample Product stream deviation grouping teaching.By this way, the incidence rate of mistake sorting minimizes.For the sample that size is uneven, such as its The diameter of middle cell and fragment can be from the single cell suspension of 1 micron to 50 micron of primary tissue not etc., with flux as generation Valency facilitates bigger deviation to be favourable.For more uniform sample such as cell line or polystyrene beads, optional Select less deviation to allow bigger flux.

A kind of alternative plan as described design is to use bi-directional optical switch, and it uses two laser rays. When using these means, required cell to the outlet of laser rays sorting, another laser rays sorts other thin all Born of the same parents enter another outlet.These means can be divided with 50/50 shunting configuration shown in Fig. 2 or the skew shown in Fig. 3 and Fig. 4 Use from configuration.In the later case, when cell not Zone switched interior time, two location status at laser can be selected Lower any one open this laser, or also can interrupted this laser during this period.Optical switch also can be made into bi-directional optical as follows Switch: have two that be located just at above bifurcated abutment, be incident on Zone switched mirror image laser rays, it is opened independently With any one in guiding cell to export to two sent from bifurcated abutment.

The diagram of bi-directional optical switch of laser rays is used as shown in Figure 5 in 1 × 2 microfluidic networks.Similar is two-way Optical switch it be also possible to use and is directed into the laser spot of passage either side and is achieved, as shown in Figure 6.With mono-directional optical switch Identical, single lasing light emitter can be used in bi-directional optical switch, or, bi-directional optical switch can use two independent lasing light emitters. Bi-directional design can be provided that certain performance benefit relative to unidirectional design.First benefit is that purity may maximize, because Each cell is guided by laser.Second benefit is that fluid stream is simplified, because equal stream can be brought out from two Each of output port, instead of using certain predetermined flow ratio rate.

Although only considered the most in this manual flow through one input main thoroughfare enter bifurcation arrive two go out 1 × 2 design of micro fluidic channels of mouth, but it is used as the microfluidic networks with 1 × N or M × N number of output ".At these more In big network, the laser rays modulated by the independence with arbitrarily large number or independent cross-flow passes can be realized optics and switch Or fluid switching.Some embodiments are as shown in figs. 7 a-c.It addition, cell also by identical sorter by feed back repeatedly with Increase the purity being sorted cell, or, passage can be also used for the cascade of multipass sort and is arranged.

When operating the optical switch of unidirectional layout or bidirectional arrangements, it may be considered that two kinds of different active modes: passive Mode or active mode.Passive mode is so that the state of optical switch is on state or closed mode, can with what cell Flow through this passage unrelated.In this case, it is not required that know when cell enters Zone switched or have how many cell to enter Zone switched, therefore, according to the state of laser, all cells in Zone switched is switched.As an alternative, at active mode Under, cell is first detected when it enters detection/selection region, is then switched according to certain decision process.Fig. 3 a-b With the example that Fig. 4 a-b shows this mode, which use just be disposed in Zone switched before fluorescence detector. In this case, all of fluorecyte is directed into an outlet, and all of non-fluorescence cell is directed into another Outlet.Other non-fluorescence detection/selection technique for decision process includes the flight time (Time-Of-Flight), dissipates Penetrate, imaging, electric capacity or any detection mode identifying required cell.Unrelated with detection/system of selection, can use use actively The switching of mode, to open a cell mass with the sorting of another cell mass according to certain decision process.

In order to utilize active mode, light beam must be modulated to respond decision process but be turned on and off.With used Laser number unrelated, or no matter optical switch is unidirectional or two-way, and laser can be modulated in many ways, described Mode includes using electrooptic modulator, modulates laser power, interrupted laser, uses liquid crystal modulator, uses galvanometer and use Acousto-optic modulator.For using the bi-directional optical switch of two laser, single laser can be opened or closed independently； But, when using single lasing light emitter, two different orientations of optical switch circuit can be by using polarization rotator (such as liquid Brilliant manipulator) and two different circuit diagram shapes in each be that each in two independent polarizations is achieved.Similar Ground, it is possible to use acousto-optic modulator or galvanometer mirror regulate the position of the single luminous point being used as optical switch, or Two-axis acousto-optic manipulator or twin shaft galvanometer mirror can be used to draw two and to be used as the different of bi-directional optical switch Circuit shape.

Fig. 8 shows the regulation for carrying out optical switch and/or the discontinuously three kinds of different possible optical designs made. In Fig. 8 a, from towards and manufacture bi-directional optical switch by the directed single light beam (laser) of liquid crystal modulator (LCM). LCM is polarization rotator, and therefore, if light beam is polarized in a direction, it will pass straight through polarization beam apparatus (PBS), wear Cross cylindrical lens and produce linear, through another PBS, then pass through some focusing optics, described focusing optics On Zone switched for line focus to microfluid.This produces the bi-directional optical switch of single line effectively, and it is for being switched to cell In one of passage output of bifurcated.In order to cell being switched in another outlet, it is necessary to produce image line.This is changed by rotation The LCM becoming light beam polarization is achieved.Therefore, when light beam is incident on a PBS, it is directed into another path: it leads to Cross different cylindrical lens (producing linear), through another PBS, it guides light beam to pass back through focusing optics, institute State focusing optics image line is focused on microfluid Zone switched on.It is noted that use cylindrical lens is to produce use Linear in bi-directional optical switch；As an alternative, cylindrical lens can be removed, produce the luminous point for optical switch.At Fig. 8 b In, do not use the combination of LCM and PBS, it is possible to use acousto-optic modulator (AOM), under with and without cylindrical lens, produce Line in bi-directional optical switch or luminous point.By building, AOM is linear needed for required by acquisition to be achieved for this.Separately Outward, AOM can be used to carry out interrupted light beam in the way of opening/closing, direct the light beam into the light closing condition for optical switch Door screen.Fig. 8 c shows the combination of the system described in Fig. 8 a and Fig. 8 b.Can use any configuration to replace AOM, described What configuration uses AOM to change the direction of light beam, uses galvanometer reflection uniaxially or biaxially according to required beam motion Mirror.

When realizing the optimal switching efficiency of unidirectional or bi-directional optical switch, it may be considered that the multiple change of optical figuring. As it has been described above, laser rays has been used as optics switching figure.Laser rays can be generated by techniques below: cylindrical lens, Scanning galvanometer mirror or acousto-optic modulator, diffraction optics, customize refractive optics, or other technology any.Up to now, Through using cylindrical lens, by scanning galvanometer or by using acousto-optic modulator to produce laser rays.The length of laser rays It can be random length or can be short as single point.Laser rays can have higher intensity and intensity court in top online Line end to taper into.It addition, laser rays is probably curve arc, it makes the outbound course optimization of cell.It addition, laser The angle of line or shape can change (i.e. rotating to realize the optimization of output) in real time.For using the enforcement of multiple outlets Journey, it is possible to create the 2D space laser line of any arbitrary patterns is to realize the direction optimization of each output cell.As replacing In generation, laser rays can be produced by discrete spots array.

In order to improve sorting mechanism performance for flux, yield efficiency and purity aspect further, have been built up Optical switch is so that when selected cell flows down towards bifurcated abutment along main thoroughfare, laser spot is thin with selected Born of the same parents are scanned side by side, thus increase the total interaction time between cell and laser.The laser spot that optical switch uses With straight line along the length of main thoroughfare towards bifurcated abutment pan-down.Can be with the wall of main thoroughfare by the line of spot scan Parallel (Fig. 9 a) or can be relative to cell flow stream (Fig. 9 b) at an angle.Therefore, described angle can be 0-90 degree.Sweep The ability retouching luminous point uses AOM or scanning galvanometer mirror to be achieved.To use fluorescence or other can identify required thin The detection mode of born of the same parents such as flight time, scattering, imaging or capacitance technology carry out the judgement being detected as basis of required cell and touch Luminescence switch is scanned.Cell position can be deviation position or middle position in main thoroughfare, and it has been arranged by light The length of the line of spot scan and for realizing the laser power effectively switching/sorting.Therefore, when required cell being detected, light Switch is unlocked, and luminous point manifests side by side with required cell.Then luminous point and selected cell are gone along track side by side, and Outlet needed for using optical force to guide selected cell to enter.

Two kinds described below help effectively to trigger optical switches or the means of fluid switch.Both of which typically makes Analyze migratory cell by time signal (temporal signal), and use the information to produce switching or sentencing of not switching Fixed.This time signal is substantially the measured value of the signal as time function, and it can be with regard to peak intensity and peak width two aspect For obtain distinctive time fingerprint.This signal can be fluorescence, scatters (such as forward scatter), electric capacity, imaging or any Can be in the way of identifying required cell.One means is to use the single lasing light emitter being combined with two or more detectors with reality Existing cell detection and cellular identification two aspect.Figure 10 a-d shows this use and fluorescence detector and forward scatter detector The means of one lasing light emitter of combination.The time signal obtained by these detectors is used as the information being switched and determined.Cell Exist and tested and when this signal is combined with the fluorescence signal intensity being in preset range by forward scatter signal；Then Should " gate " information be used for triggering optical switch.It is noted that and only show single fluorescence detector, but can use many Individual fluorescence detector is for refining cellular identification further.In such a scenario, equal flow velocity sheath buffer agent is used to make carefully Born of the same parents flow centrally located, use the outlet with different in width to produce grouping teaching on the right side of cell stream.But, as it has been described above, can To use any configuration to come the position of manipulation cell stream and grouping teaching.It addition, all there is error checking detector in two kinds of configurations, its inspection Whether cell is switched or is not switched.In this case, detection can scatter (such as forward scatter), electric capacity based on fluorescence, Imaging or any can be in the way of identifying required cell.

Figure 10 a-b shows when sorting parameter is feminine gender and optical switch or fluid switch is not triggered, detection Device arranges and selection of time/trigger diagram.These cells are entered principal fhiidic passage and are buffered by the sheath from both sides Agent is concentrated into as single file.When laser in cell travels through detection/selection region, simultaneously or almost simultaneously detect glimmering Optical signal and forward scatter signal.Although the existence of cell successfully by forward scatter signal detection to (at time t₁), but Fluorescence signal is less than gate level, and optical switch is not triggered (at time t₂).Therefore, error checking signal is not obtained (at time t₃), Because not having cell to be switched.As an alternative, Figure 10 c-d shows when sorting parameter is positive and optical switch or fluid When switch is triggered, detector arranges and selection of time/trigger diagram.Here, detection/selection region is travelled through when cell In laser time, the most simultaneously or almost simultaneously detect that both fluorescence signal and forward scatter are (at time t₁), but fluorescence Signal is in gate level, and optical switch or fluid switch are triggered (at time t₂).Obtain error checking signal (in the time t₃), because cell is switched.In the method, the triggered time is (at time t₂) it is from initial detection time (t₁) play the pre-of measurement If value (Δ t), and this Δ t value is determined relative to the position in detection/selection region by the speed of cell and optical switch. The method achieves effectively sorting satisfactorily；But as improving the means of triggering precision further, employ second method.

Figure 11 a-d shows this second method, which uses two lasing light emitters rather than a lasing light emitter.It addition, such as The same with single laser means as above, the time signal deriving from these detectors is used as the letter for being switched and determined Breath.Use a laser to realize cell detection individually before identifying/select region within a detection region.In this situation Under, detection can scatter (such as forward scatter), electric capacity, imaging or any detection that can identify required cell based on fluorescence Mode.Second laser is combined with two or more detectors and is used for realizing cell detection and cellular identification.It addition, in these feelings Under condition, qualification can scatter (such as forward scatter), electric capacity, imaging or any inspection that can identify required cell based on fluorescence Survey mode.For two sequentially cell detection steps purpose for make cell flow rate can obtain comfortable first detection (in the time t₁) and second detection (at time t₂The time difference (Δ t) between).Spacing (d) between known detector window will obtain flow velocity (v= D/ Δ t), it is combined with the known distance (x) deriving from optical switch or the fluid switch identifying window then to use this value, calculates light Learn the triggered time (t of switch₃=x/v).It addition, again send out when obtaining for the specific gate level of cellular identification step Raw switching.Although illustrate only single fluorescence detector for identifying, but multiple fluorescence detector can be used.In described feelings In condition, use the sheath buffer agent of equal flow rates to make cell stream centrally located, use the outlet with different in width at cell stream Right side produces grouping teaching.But, as set forth above, it is possible to use any configuration to come the position of manipulation cell stream and grouping teaching.It addition, All there is error checking detector in two kinds of configurations, whether its inspection cell is switched or is not switched.In this case, detection can be with base In fluorescence, scatter (such as forward scatter), electric capacity, imaging or any detection mode that can identify required cell.

Figure 11 a-b shows when sorting parameter is feminine gender and optical switch or fluid switch is not triggered, detection Device arranges and selection of time/trigger diagram.These cells are entered principal fhiidic passage and are buffered by the sheath from both sides Agent is concentrated into as single file.The existence of cell is when it is through detecting window region by forward scatter signal (at time t₁) inspection. When cell is through identifying/select window, it is thus achieved that the second forward scatter signal is (at time t₂), but, this signal be not in door Fluorescence signal intensity in control level is (at time t₂) combine, and optical switch or fluid switch are not triggered (at time t₃)。 Do not obtain error checking signal (at time t₄), because not having cell to be switched.Even if not sorting cells, use (t₁)、(t₂) and inspection The known distance (d) surveyed and identify between window obtains the flow velocity (v) of cell stream.This uses relationship below obtained: Δ t= (t₂)-(t₁) and v=d/ Δ t.

As an alternative, Figure 11 c-d shows when sorting parameter is positive and optical switch or fluid switch are triggered Time, detector arranges and selection of time/trigger diagram.Here, the existence of cell when its through detection window region again by Forward scatter signal is (at time t₁) inspection.When cell through qualification/select window, it is thus achieved that the second forward scatter signal (time Between t₂), but, this signal and the fluorescence signal intensity being in gate level are (at time t₂) combine, and optical switch or stream Body switch is triggered (at time t₃).Obtain now error checking signal (at time t₄), because cell is switched.In the method, touch Send out time (t₃) it not preset value, but use cell flow rate rate (v) and the known distance between optical switch and qualification window D () calculates the triggered time.This uses relationship below obtained: Δ t=(t₂)-(t₁)；V=d/ Δ t；(t₃)=x/v.The party Method allows to be more effectively carried out sorting, because it can solve the fluctuation of cell flow rate, and triggers optical switch the most more accurately Or fluid switch.The additional benefits that the method switches for optics is, for each single cell, it is possible to regulation swashs The speed that light luminous point moves down along passage, so that its speeds match with the cell as above measured, so that cell and light Learn the interaction time maximization between the laser spot of switch.The transfer velocity of laser spot is by by changing driving of AOM Move device and be changed.

Another improves the efficiency of separation and to be simultaneously introduced the method for triggering method as above be to use passage design to make Cell concentrates in main thoroughfare, and the design of described passage produces authentic sample core, thus this core is wrapped completely by sheath buffer agent Enclose.Cell can cause the variability of cell detection and fluorescence intensity along the variability of the position of channel height.Guarantee at cell In main thoroughfare central authorities core flow in can improve the efficiency of separation because this make due to cell radial distribution caused any Variability minimizes, and it is mobile to realize the distance of effectively sorting to control cell needs.This core flow can use sheath to buffer Agent carries out 2 dimensions of inlet flow logistics and holds contracting under the arm and be achieved.

The method requires bottom substrate and bottom substrate；Each formed therein which microfluidic channel network.Figure 12 a-b and Figure 13 shows the method realizing this purpose, and passage design the most on a substrate is design on another substrate Mirror image.Therefore, when two basis set are fitted together, using passage relative to each other to design, channel network covers and is formed Complete fluid conduit systems.Figure 12 a-b shows a kind of kind of design using this method, and sample channel is shown as dotted line.Should The key feature of method is to ensure that sample channel is more shallow than sheath passage, so that the sample line when substrate is assembled together Seem as entrance abutment, hole.This wherein can be observed cell and enter abutment, then held under the arm from four sides as shown in figure 13 Contracting, produces core of sample, and it flows into the central authorities of main thoroughfare.It is noted that passage can be lost by wet chemical etching technique or laser Carved glass or quartz and formed, or by be shaped in plastics or polymer or embossing and formed.

Another kind of method relates to having a series of cross aisle, and it is arranged such that in first abutment/cross point, Cell is promoted by a vertical wall towards main thoroughfare, and next abutment/cross point forces this cell stream vertically into master Want the central authorities of passage, then flow in main thoroughfare from the contracting stream of finally holding under the arm of both sides the 3rd abutment/intersection Sample central authorities surrounding produce complete sheath buffer agent covering.This as shown in Figure 14 and Figure 15, a possible passage figure Solution is as shown in figure 16.In this embodiment, at abutment (A) place, sample flows into this abutment from bottom substrate and flows downwardly into Being positioned at the passage of bottom substrate, wherein sheath buffer agent in side is from flowing sideways into abutment.Sample connects towards the next one when its continuation Somewhat collected to neutralize during chalaza (B) flowing and shifted onto the upper wall of foot passage.At abutment (B) place, sample is along foot passage Top flows to abutment (B) from abutment A.Now second sheath buffer agent flows into abutment (B) and sample from bottom substrate It is pushed down to be positioned at the middle part of the passage of bottom substrate.Sample continues on the middle part of foot passage and engages towards the next one Point (C) flows.Now the 3rd sheath buffer agent is from two side inflow abutments (C), and sample is held under the arm and shortened into as single file.Sample is existing Surrounded by sheath buffer agent as core of sample when it continues flowing, both horizontally and vertically position in main input channel In central authorities.

Another method uses the shallow passage that its axle is vertical with 1 dimension sheath buffer agent, so that the impact of parabolic velocity dispersion Minimize.Period in 1 dimension adfluxion in microfluidic channel, particle in the logistics of entrance central authorities or cell are only along a direction Held under the arm contracting.Contract and flowing to vertical direction with holding under the arm, still retain parabolic velocity pattern.Particle flow near passage central authorities Hurry up, and slow near the particle flow of conduit wall.Result is, particle or cell are so that the synchronization of detection event and handover event becomes Complicated VELOCITY DISTRIBUTION moves down along circulation road.Figure 34 shows and overcomes the solid problematic side of period in 1 dimension microfluid adfluxion Method, using in vertical direction can channel depth compared with the granularity of particle or cell.Such as, particle or cell is a diameter of 10 microns, then channel depth can be 15 microns.In this case, particle or cell occupy the major part in channel depth direction, So that they experience make VELOCITY DISTRIBUTION be able to the speed that mean deviation makes the velocity dispersion in passage narrow.

All of microfluidic channel network design described in Fig. 1-16 has utilized traditional mask and has been covered The isotropic etching of mould substrate of glass is generated in substrate of glass.Isotropic etching typically produces such microfluid and leads to Road, i.e. this microfluidic channel are entreated in the channel has degree of depth d_eWith the top at passage, there is width w=w_p+2×d_e, wherein w_p It it is the width of the litho pattern limiting passage.The bottom profile of passage has radius at each edge due to isotropic etching d_eQuadrant profile and to be etched the top of passage be unlimited.Substrate of glass, typically glass cover-slip, quilt It is thermally bonded to have in the substrate of etched microfluidic channel, to seal the top of passage and to complete microfluidic channel network. Before thermal, generally in bottom substrate, turn hole, enter and microfluidic channel network of going out for fluid stream to provide Through hole, but, as an alternative, it is possible in etched bottom substrate rather than in bottom substrate internal drilling.The degree of depth of passage d_eSpeed and the difference of the persistent period of etching step according to chemically etching process and different.The degree of depth of microfluidic channel is typically But it is 10 microns to 100 microns without limitation.The width of microfluidic channel typically but is 2 to the 5 of the degree of depth without limitation Times.This is by using typically on mask but be that the line of 5 microns to 400 microns is achieved without limitation.As front Described, other substrate, such as plastics or the plastic polymer maybe can cast can be used.In these cases, miniflow Body passage typically has a rectangular cross section, but the passage being but similar in substrate of glass in further aspect.Wherein produce The size of the substrate of glass of microfluidic channel network typically but is 5 millimeters × 5 millimeters to 25 millimeters × 50 millis without limitation Rice, gross thickness typically but is 500 microns to 2 millimeters without limitation.Bottom substrate typically has identical size, thickness Typically but be 300 microns to 1 millimeter without limitation.The diameter of through hole typically but be without limitation 200 microns to 600 Micron.The substrate completed, has microfluidic channel network and combined covering plate, and this covering plate has through hole for fluid stream The fluid bore entered and go out, is referred to as microfluid sort wafers or for simplicity being referred to as wafer.

Microfluidic channel network shown in Fig. 1-16 generally only describes the local geometric shape of inlet microfluidic body passage, Sheath buffer agent holds reducer coupling chalaza passage, cellular identification and optical switch main thoroughfare under the arm, and main thoroughfare is to the bifurcation of outlet. This description needs to be extended providing region to produce bank in macro-scale fluid means or box body in each passage Connection, it provides and the interface of through hole as above, thus provides the entrance from network of the fluid stream and go out.These The cross section of each in microfluidic channel and length need to be adjusted to ensure according to being selected to achieve in passage The difference of the technology of flowing controls the flowing in whole microfluidic channel network.The cross section of these passages and length thereof All determined by the figure being used for generating mask.

Figure 17 shows an embodiment of the mask for complete microfluidic channel network, described microfluidic channel Network has intake channel, two the sheath passages holding reducer coupling chalaza under the arm to T-and two outlets separated from T-bifurcated abutment.Should Mask is designed to provide the volume of 7: 1 and holds contracting ratio seven times of cell inlet velocity (sheath flow velocity be) under the arm.The length quilt of passage It is designed to provide for sufficient pressure drop, enabling use lazy flow syringe pump or the low-pressure pneumatic controller of standard To establish flowing.This design also reflects as can only use two pumps to need balance pressure, a pump to lead to for cell import Road and a pump keep at atmosheric pressure for two sheath passages, outlet.Sheath channel entrance is positioned at the end at design top, Cell intake channel beginning is less than this, in two sheath passage central authorities, and long enough, thus offer is arranged to 7: 1 and holds contracting under the arm The suitable pressure drop of ratio, and two outlets are positioned at the end on bottom left and right side.

Figure 18 shows that the another kind at the triangle abutment being incorporated with for holding reducer coupling chalaza and Y-bifurcated abutment under the arm is real Executing scheme, its design provides the volume of 10: 1 and holds contracting ratio under the arm.This design is geometry in the design shown in other side and Figure 17 Similar.Other designs many are substantially possible, but they total common traits be need to provide fluid to enter and Go out and provide suitable pressure drop and pressure balance for establishing the method selected by fluid stream.Use similar design condition Produce for manufacturing the mask that foregoing 2 dimensions hold the microfluidic channel network of contracting flow network under the arm.

Figure 19 shows the preferred embodiment of the microfluidic channel network in complete microfluid sort wafers.Really Determine, for cell sample stream with for two inlet ports of sheath buffer agent stream, to identify for fluorescent positive target cell and use Two outlet port in the fluorescence i.e. refuse of feminine gender non-target cell.Wafer is 24 millimeters × 40 millimeters.It is etched the thickness of substrate It it is 1.1 millimeters.The combined thickness covering plate is 550 microns.Deep 50 microns of microfluidic channel.Cell import microfluidic channel Wide 110 microns.Sheath stream and outlet microfluidic body channel width 150 microns, key microfluidic body passage is also such.Sheath stream holds reducer coupling chalaza under the arm It is reverse equilateral triangle, every length of side 300 microns, pass through the base of triangle by cell intake channel at the top at abutment Two the sheath streams being connected to every side from main thoroughfare by vertex of a triangle with the bottom at abutment hold contracting passage under the arm.This is micro- Network of fluid passages designs through optimization all to use the pneumatic control of liquid stream to set up network flow in all four port.

Microfluid is connected to wafer can use various ways.One mode is the flexible miniflow using and being directly connected to port Body pipeline, by gluing or use the various pipeline jointer that can be attached to wafer surface in port.This pipeline can directly connect Receiving syringe pump or similar system, it provides volume for disposing cell sample and sheath buffer agent, and for making these volumes Flow through wafer and pressure is provided.Use and require that each sample is cleaned and be reloading with to pump for the syringe pump disposing sample volume And produce carry a kind of sample or a kind of sample probability to lower a kind of sample contamination.

The method of the improvement being connected to wafer for microfluid uses box body, and this box body uses ultraviolet-curing bonding Agent, PSA (contact adhesive) bonding sheet or other traditional associated methods such as thermal are directly attached on wafer.This box Body has four embedded banks, and it provides respectively to cell intake channel, two sheath passages (deriving from a bank) and two The interface of each in outlet connects.This box body provides sterile working's cell sample and is sorted target cell and refuse Probability, because they can be fully restricted in the volume of this box body before and after cell sorting.For this The liquid stream of box body and lamellar system can be provided by using two gas pressure regulators, and described gas pressure regulator pressurizes individually Cell import and sheath buffer agent bank, the microfluidic channel network arrival flowing through wafer with induction is in the outlet under atmospheric pressure Bank.

By using four pneumatic controllers to provide the flow control method of improvement, described four pneumatic controllers are independent Ground pressure cell import, sheath buffer agent, target cell collect each in bank and waste collection bank.This flowing controls system System provides the volume that regulation individually foregoing holds contracting junction point under the arm at sheath and holds contracting ratio under the arm, regulates key microfluidic body channel Interior cell flow rate is for fluorescence analysis and optical switch and regulates the split ratio at switching bifurcation to be capable of bias current Ability.

Figure 20 shows the preferred embodiment of the disposable box body of self-contained type, this box body be cell sample volume, sheath delay Electuary volume and two the outlet collected volume being respectively used to target cell and refuse provide fluid reservoir.Box body is by acrylic compounds Plastics are made or can be manufactured by machine or cast and form.If suitably, other plastics available or suitable material replace propylene Acids plastics.The usual tapered shape of cell sample volume, the port towards import microfluidic channel is tapered.Preferably In embodiment, import bank comprises polypropylene dummy slider so that cell adhesion minimizes and therefore makes cell yield maximize.Will Wafer UV binding agent is adhered to optical window region, and connects from the outlet port of wafer and the ir volume of each of which Mouthful.With stinging, lid being sealed ir volume, this is stung has boring to lid, the connection between pneumatic controller and single bank. This lid includes that silicone rubber gasket is to help to seal against cartridge body.Also be incorporated to the polypropylene filter of 0.1 micron with Gas transmissive and liquid-tight interface is produced between box body volume and external environment.This maintains the nothing on box body Bacterium condition and make any biohazard that user or instrument are caused pollute minimize.

Prepare box body to carry out cell sorting: first pass through and use the ordinary syringe with luer cooperation, buffer with sheath Agent solution adds dress microfluidic channel network by sheath port.By this way, to fill 800 micro-through adding dress and sheath bank for passage Rise and each outlet bank fills 200 microlitres.Cell sample bank is sucked out the buffer fluid of excess, then uses and moves liquid The cell sample of 5-25 microlitre is placed in sample input bank by pipe.Then plus tray cover and press and be pressed onto position, it is provided that wherein Carry out the self-contained systems of cell sorting operation.

Box body is designed to be placed on bearing, and the main thoroughfare of wafer is arranged so that projection optics is opened by this bearing The optical imaging system closed in light beam entrance passage is properly aimed and focuses on entrance passage.Box body bearing also includes pressure discrimination Tube sheet, this plate has 4-6 port, and they are connected to four pneumatic controllers by external pipe.Each manifold port uses O Shape circle is sealed on its respective tray cover port, and these seal and use cam-locking mechanism by facing to tray cover Press manifold and be made leak-free.Figure 29 shows the preferred embodiment of the box body bearing for fluid switching, its Two pneumatic operated valves are integrated on manifold, in order to shorten volume and the path-length of air pulsing.Without intervenient pipeline, Manifold can carry out the joint of air pressure and the air pressure port deriving from source.This makes to realize faster switch speed for higher Performance having augmented derive from the performance benefits of the switching configuration shown in Figure 27-28.

For optics switching optical system preferred embodiment as shown in figure 21.To have and be connected to sting covering The main body of pneumatic manifolds be arranged so that the Zone switched lens combination seen above box body and from below of being positioned at of optics In the focus of both the lens combinations seen.The output beam deriving from 488nm laser is projected and enters just position through imaging system In the main thoroughfare as shown in Fig. 3-7 and Fig. 9-11 of sorting region upstream, derive from fluorescent positive target to provide for detection Exciting of the fluorescence of cell.Fluorescent emission is by collected by same lens and by dichroic mirror and suitable fluorescence emission filter It is imaged onto on photomultiplier tube.Derive from the signal of photomultiplier tube to carry out processing by electronic equipment and derive from cell to measure Fluorescence level also determines the existence of fluorescent positive target cell in the flow stream in main thoroughfare.Fluorescence exciting wavelength is not limited to 488 nano wave lengths, and can also is that any wavelength being applicable to identify the fluorogen of target cell.If using different exciting lights According to, then the wavelength of fluorescence emission filter must correspondingly change.When identifying fluorescencepositive cell, electronic equipment triggers AOM With guide derive from IR-laser, be typically the light beam of 1070nm laser operations between comfortable 5W to 20W output at light Learn switching position and enter main thoroughfare.In preferred embodiments, AOM cuts to produce the optics as described in Fig. 9 b through control Change figure, although foregoing arbitrary optics changing method can be implemented.488 nanometer exciting lights are shone by the lens below box body It is imaged onto on photodiode.The signal detected by this photodiode is used to help to distinguish fluorescence labeled cell and portability Fluorescently-labeled relatively fractionlet, and identify the cell block being likely to be formed.These events are as being used for being sorted into target outlet Material standed for be eliminated.

Another preferred embodiment is introduced into suitable imaging and light filtering, with according to for exciting fluorescence The illumination that cell is carried out by 488nm laser provides forward scatter signal.These Optical devices will provide a range of angular sensitivity, Such as, but not limited to for the scope of 0.8 ° to 10 ° of forward scatter acquisition of signal.This signal can help to characterize except fluorescence Cell outside signal, and help to distinguish cell and fragment.Forward scatter illumination is not limited to fluorescence excitation laser, and can also be Other the wavelength any provided by the additional source of light being imaged properly in main thoroughfare.

Another preferred embodiment will be incorporated to other fluorescence detection channel, and the fluorescence being in different wave length is sent out by it Penetrate sensitivity, typically sensitive such as, but not limited to the fluorescent emission of 488nm to using single excitation wavelength.Each sense channel is also Enter to have the PMT of suitable dichroic mirror and the transmitting filter of the fluorescence emission wavelengths for other fluorogen.With this Mode can easily accommodate two to four fluorescence detection channel.The fluorogen using more than one by this way provides multiple The ability of examination criteria, thus identify and use optical switch to carry out the target cell sorted.

Another preferred embodiment will be incorporated to error checking ability, and it provides illumination optical, typically as crossing over net The narrow line of one of the passage in network, and be typically under longer wavelength, but it is not limited to derive from the 785nm of solid-state laser Wavelength, it is in beyond the wave-length coverage that fluoroscopic examination and forward scatter detect, but ratio is typically at 1070nm Optical switch wavelength short.This light source can be imaged properly in microfluidic channel network and can be used for detection through net to provide The line of the range of particle of any vertical plane in network.It provides the ability of the other performance checking optical switch performance and carries Supply the ability of the selection of time of the other trigger for optical switch as described in Figure 11.

Another preferred embodiment of optical system other be in by being incorporated to but be not limited to the optics of 750nm and shine Bright, such as, by the light deriving from LED carries out the illumination optical that bandpass filtering is generated, and use this optical illumination microfluid to lead to Region, road.This region will be imaged onto on ccd video camera by the band filter of 750nm, to provide at microfluidic channel net In bifurcated junction point and/or the visualization of the performance at the cell holding the flowing of contracting junction point under the arm in network.Before video camera Wave filter by enough block with fluorescence excite or detect relevant and with forward scatter/lateral scattering optics device and error Any shorter wavelength radiation that detection Optical devices are relevant.Wave filter also block derive from optical switch there is longer wavelength The light of 1070nm.

It is horizontal configuration that the preferred embodiment of the box body shown in Figure 20 is designed to keep microfluidic channel network, So that all of passage and outlet/inlet port are in identical vertical-horizontal.This makes gravity lead to by microfluid The impact of the pressure drop in road minimizes, and causes the more stable He more controlled flowing in network.But, gravity will convection current Cells in vivo has impact, particularly when cell from cell sample bank by enter cell import microfluidic channel time.In order to Help to control gravity on the impact of cell sedimentation in this bank and gravity to cell in the cell flow rate holding contracting junction point under the arm The side of being preferable to carry out of the sorter of the impact of the sedimentation in the slowest fluid in import microfluidic channel before increase Case is to increase the buoyancy of cell, so that the sedimentation of cell minimizes.Increasing buoyancy can be by making in sample buffer Complete with additive.The example of these rheology control additives, particularly has pseudoplastic behavior or shear thinning or the two that A bit, for xanthan gum, antler glue, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxyl Propyl cellulose, hydroxypropyl guar gum, Radix Acaciae senegalis, gum tragacanth, alginate, polyacrylate, carbomer.Other adds Agent includes Histopaque^TM, it is the mixture of ficoll and sodium amidotrizoate, and Optiprep^TM, it is that iodixanol is at water In the solution of 60%w/v.The concentration of these additives is according to being sorted the difference of density of cell and different.Such as, exist Optiprep^TMIn the case of, concentration can be 5% to 40%.Finally, the salinity of sample buffer and the interpolation of sucrose can be additionally used in The buoyancy of regulation cell.

For cell sample volume and for the buffer agent of sheath stream can be be sorted cell be bio-compatible and with For fluoroscopic examination pattern with for the compatible any buffer agent of the illumination optical of optical switch, say, that this buffer agent exists At fluorescence exciting wavelength/detection wavelength and optical switch wavelength, there is of a sufficiently low absorbance.The preferred enforcement of sheath buffer agent Scheme uses PBS/BSA, and one has the phosphate buffered saline (PBS) (PBS) of 1% bovine serum albumin (BSA) fraction 5, pH 7.2.The preferred embodiment of cell buffer agent use the PBS/BSA with 14.5%Optiprep for living cells sample and There is the cell sample that the PBS/BSA of 27%Optiprep fixes for various formalin.

The cell that be have rated in microfluidic channel network by flux, purity and the response rate of foregoing sorting is divided Select the performance of optics changing method.Box body described in Figure 20 measures described performance through optimization with permission, because it is by propylene Acids plastics are made, and the bottom of bank and waste collection bank collected by target is transparent, and can use inversion fluorescence microscopy Mirror carries out quantificational expression to being sorted the cell entering these banks with regard to number and fluorescent labeling two aspect.Have rated such as Fig. 3-11 Described several switch configuration.These mixture evaluating use HeLa: HeLaGFP cell of living 50: 50 are carried out, this mixture 1 side or 2 side fixed laser luminous points or 0 ° or 8 ° of 1 side laser scanning is used to sort.Laser is scanned under 240Hz.Swash The light opening time be 4 milliseconds and for all of switch mode laser power be 20W.For method scanning spot Speech, the IR laser spot of focusing moves about 70 microns along main thoroughfare.

As shown in figure 22, the bi-directional optical switch using laser spot as shown in Figure 6, for target cell-non-target cell 50: 50 mixture for, under the flux of up to 50 cell/seconds, it is provided that good purity and the result of the response rate.So And, under lower subgroup concentration (non-video data), laser power can not be effective to switch non-target cell, and overlaps by mistake Difference increases under higher cell through-rate.It addition, the small particles not being switched will pollute target bank.

Figure 22 also show the performance of 1 side changing method shown in Fig. 9, and it has fixed laser luminous point or at flow direction Hot spot that is upper parallel with liquid stream or that move in slight angle with stream.Core of sample flow stream is offset to Waste outlet, so that Inflow Waste outlet must be given tacit consent to by all cells under lacking optical switch.These methods both provide the performance of improvement, such as figure Shown in.The fact that the performance crossover of the two method imply that the triggering of optical switch is not optimal, and imply that such as figure The active of the optical switch shown in 10 and Figure 11 triggers will improve performance.

Figure 23 shows (i.e. have a primary input passage and two nets exported stretched out from bifurcated abutment 1 × 2 Network) in be used for the embodiment of pneumatic fluid switch of sorting cells or particle." Y " geometry at bifurcated abutment is such as Shown in Figure 23, but it be also possible to use other bifurcated such as " T " geometry.Generally, these microfluidic channel are at optical clear base The end, is formed such that it is able to by cell detection optics device projection stand in channel.This substrate typically, but without limitation, For glass, quartz, plastics, such as polymethyl methacrylate (PMMA) etc., and other can cast or spendable polymerization Thing (such as polydimethylsiloxane, PDMS or SU8).The degree of depth of microfluidic channel typically, but without limitation, is 10 microns To 100 microns.The width of microfluidic channel typically, but without limitation, for 1 to 5 times of the degree of depth.By substrate of glass Masking, then carrying out the isotropic etching of passage to manufacture in the case of microfluidic channel, cross section is typically rectangle, Or the rectangle with quadrant angle.

Flox condition be provided so that air pressure P0 ＞ P1 ＞ P2 and cell in a fluid preferentially from maximum pressure to Minimum pressure flows.When pressure P1 increases so that during P0 ＞ P2 ＞ P1, then fluid stream is disturbed and preferentially flows down " Y " when fluid Balance is re-established during the contrary branch road at abutment.This system can recover its initial shape by repairing the relation of P0 ＞ P1 ＞ P2 State.

The property of the sorting mechanism for flux (entering the time rate of the cell in the sorting region at top, bifurcated abutment) Can by fluid as shown in figure 23 backward-spread and limited.When fluid switch is activated, saw before setting up new flow path Observe direction reversing and the flow upstream of Particles Moving.The time delay induced by this motion limit switch speed with And realize acceptable yield efficiency (target outlet intracellular targets mark) and purity (target outlet intracellular targets number/ The ratio of total cell number) the frequency of event.

Figure 24 shows (i.e. have a primary input passage at 1 × 2 microfluidic channel network and stretch out from bifurcated abutment The networks of two outlets) in be used for the embodiment of dual pathways fluid switch of sorting cells." Y " at bifurcated abutment Geometry as shown in figure 24, but it be also possible to use other bifurcated such as " T " geometry.At analyzed area and bifurcated abutment Between arrange a pair of cross-flow passes.Cross-flow passes can be symmetrical dimensionally, or side exists with support more than opposite side Faster switching on one direction.Generally, these microfluidic channel are formed in optical clear substrate such that it is able to by cell In detection Optical devices projection stand in channel.This substrate typically, but without limitation, for glass, quartz, plastics, the most poly-first Base acrylic acid methyl ester. (PMMA) etc., and other that can cast or spendable polymer (such as polydimethylsiloxane, PDMS Or SU8).The degree of depth of microfluidic channel typically, but without limitation, is 10 microns to 100 microns.The width of microfluidic channel Typically, but without limitation, for 1 to 5 times of the degree of depth.Carry out substrate of glass masking, then carry out passage etc. Isotropic etch manufactures in the case of microfluidic channel, and cross section is typically rectangle, or the rectangle with quadrant angle.

Flox condition is provided so that when fluid switch (deriving from air pressure in this case) is switched off, thus at abutment During pressure P3=P4 in region, all cells will preferentially flow into one of outlet, the such as outlet on right side.When fluid switch exists Change pressure when making to be opened under P3 ＞ P4, fluid plug displacement flow stream, so that near the cell quilt of fluid plug Lead into left side outlet.In microfluidic channel network, being set and controlled of flox condition can pass through direct-driven pump, gas Press pump gives, electrokinetics, capillarity, gravity or other can produce fluid flowing means complete.

With regard to flux (time rate in the sorting region at cell entrance top, bifurcated abutment), (target cell is at target for yield efficiency Mark the mark in mouth 12) and purity (exporting the ratio of 12 intracellular targets numbers/total cell number at target) for sorting mechanism Performance, is affected by various factors, and each factor affects the execution of optics switching.Fluid switching is available such as following some Parameter characterizes: be projected into the pressure differential (P4 is to P3) in the sorting dot area of microfluidic channel network, and switching is logical Road is relative to the position at bifurcated abutment, and the persistent period that fluid switching activates, for producing the maximum pressure etc. of displacement of fluid Deng.The selection of the particular value of these parameters of fluid switch is especially by make decision: the topology of micro-fluidic channel system And geometry, the flow velocity (cell speed) in micro channel systems, the ability controlling flow cell position in main thoroughfare is (thin Born of the same parents are the central authorities' flowings in main thoroughfare or are offset to side flowing), it is achieved the amount of the necessary cell displacement of reliable switching, The degree of depth of passage, the shape of passage, and cell power produced with optical switch interaction.

Figure 25 shows (i.e. have a primary input passage at 1 × 2 microfluidic channel network and stretch out from bifurcated abutment The networks of two outlets) in be used for the embodiment of single channel fluid switch of sorting cells." Y " at bifurcated abutment Geometry as shown in figure 25, but it be also possible to use other bifurcated such as " T " geometry.Single cross-flow passes can be located to be analyzed On the either side of the main thoroughfare between region and bifurcation.Generally, these microfluidic channel in optical clear substrate by shape Become such that it is able to by cell detection optics device projection stand in channel.This substrate typically, but without limitation, for glass, stone English, plastics, such as polymethyl methacrylate (PMMA) etc., and other can cast or spendable polymer (the most poly- Dimethyl siloxane, PDMS or SU8).The degree of depth of microfluidic channel typically, but without limitation, be 10 microns micro-to 100 Rice.The width of microfluidic channel typically, but without limitation, for 1 to 5 times of the degree of depth.Cover in the photoetching carrying out substrate of glass Covering, then carry out the isotropic etching of passage to manufacture in the case of microfluidic channel, cross section is typically rectangle, or with The rectangle at quadrant angle.

Flox condition is provided so that when fluid switch (deriving from air pressure in this case) is switched off, thus at abutment When pressure in region is neutral, all cells is using preferentially flowing into the outlet in same side as wing passage, in these feelings It it is the outlet on such as right side under condition.When fluid switch in change pressure make to be unlocked under P3 ＞ P2 time, fluid plug displacement flow Logistics, so that the cell near fluid plug is directed into left side outlet.Flox condition in microfluidic channel network Be set and controlled can pass through direct-driven pump, air pressure pumping, electrokinetics, capillarity, gravity or other can produce fluid The means of flowing complete.

The preferred embodiment of single channel fluid switch is as shown in figure 26.Bias current is wider than left channel by right channel It is achieved.Switching channel is also wide than main thoroughfare along its major part path-length, and gradually becomes at fluid insertion point Carefully.It reduce the peak amplitude for being realized the air pressure that sorting must apply by concentration power in more small cross-sectional area.This Allow the flux operating faster and improving.

With regard to flux (cell enters the time rate in the sorting region at top, bifurcated abutment), yield efficiency (goes out at target The mark of the target cell in Kou) and purity (exporting the ratio of intracellular targets number/total cell number at target) for sorting mechanism Performance, is affected by various factors, and each factor affects the execution of optical switch.Fluid switch can be with such as following some Parameter characterizes: being projected into the maximum pressure in the sorting dot area of microfluidic channel network, switching channel is relative In the position at bifurcated abutment, the persistent period etc. that fluid switch activates.The selection of the particular value of these parameters of fluid switch Flow velocity (cell speed especially by make decision: the topology of micro-fluidic channel system and geometry, in micro channel systems Degree), (cell is the central authorities' flowing in main thoroughfare or deviation to control in main thoroughfare the ability of the position of the cell of flowing Flow to side), it is achieved the amount of the necessary cell displacement of reliable switching, the degree of depth of passage, and the shape of passage.For suckling Zooblast, has about 50 microns × 150 microns cross sections and has 0.5 to 1.0psi in the path-length of 20-50 millimeter The passage of pressure drop will realize the flow velocity of 1-5 microlitre per minute and to use the switching pressure of 1-3psi enough to realize per second up to a hundred The switching rate of individual event.In order to be generally applicable to the particle that 100 nanometers are to 100 microns, typical for sample and sheath stream Pressure drop is 0.1 to 10psi in the path-length of 10 to 100 millimeters.Sheath is selected: target pressurizing force in current chip design Ratio is with guarantee sample stream suitably to hold contracting at 2: 1.Channel cross-section can be made into 5 to 150 microns deep × 10 to 1,000 micro- Meter Kuan, according to the difference of the type of the cell or particle flowing through wafer and different.

Figure 27 shows bivalve switch configuration, its maximum pressure allowing to control pneumatic pressure pulses, the rise time, fall time With total time to activate fluid switch.Three ports can be connected to pneumatic supply, surrounding air or vacuum.In order to be switched fast, P1 is maintained at more than under the high pressure of surrounding air, and P2 and P3 keeps under ambient air.Under middle position, two valves all by Close or turn off.When needs handover event, first valve is opened, and it applies air pressure to switching channel.In short delay (example Such as less than 5-10 millisecond) rear second valve be opened, and excess pressure power is discharged into beyond port P3 rapidly by it from P1.First valve Be turned off in switching channel discharge residual compression to ambient level, then second valve is closed such that system returns it Middle position.This makes air pulsing more shorter than the on/off of single valve-pass circulation time (e.g., from about 5 to 10 milliseconds).Select ratio required The pressure P1 of maximum pressure high several times (such as there is the pulse of required 2psi peak value be 8psi) and with at port P2 and Surrounding air or vacuum at P3 combine, and pneumatic pressure pulses can have the shape of the maximum performance for fluid switch.

Figure 28 shows the bivalve configuration for the dual pathways fluid switch as shown in 24.In first passage, single Valve regulation high-voltage power supply, and second channel is in surrounding air.In this configuration, by the on/off of valve-pass cycle period (example Such as 5-10 millisecond) determine the shortest possible pulse.In the second configuration, valve is positioned on each wing passage of fluid switch.At this In the case of, two valves all can be used for regulating external pressure source.In the variable time more shorter than the on/off of valve-pass circulation time After delay, second valve can be opened after first valve.By this way, pneumatic pressure pulses can be shortened to increase fluid switch Speed and performance.

Have been built up cell sort instrument based on complete fluid switch.Use the measurement result that this instrument obtains Example is as shown in Figure 30-31.In Figure 31, measure fluorescent calibration globule (such as Spherotech SPHERO globule or BD Quantibrite globule) and show being clearly separated of two intensity.Mean intensity, standard deviation and CV statistics and manufacture Business's recommendation is consistent.Figure 31 shows the embodiment of sorting cells group.In this embodiment, CellTracker is used^TM The target group of the 1%Jurkat cell that Green (Invitrogen Corp.) dyes mixes the Jurkat cell group not being colored. It is the pressure of 0.3psi to sample that cell uses, and is 0 to sheath buffer agent, the pressure of 6psi and be the pressure of 1.5psi to switching valve Sort.About 35000 cells by analysis and 336 target cell is identified and sorting enters in target hole.To target The inspection of bank is found that 331 target cells in totally 369 cells.This results in be calculated as 99% the response rate and The purity of 90%.

In order to operate microfluid wafer, all microfluidic channel are carried out initial fluid and adds reload request with not voids Or the mode introducing grit is carried out, described bubble or grit convection current cause retardance.This needs under stable air pressure from cleaning High pressurized air source applies the air of reproducible volume.Typically, dustless system such as clean bench or laminar flow cleaning cupboard are to add dress Necessary to microfluidic device.Device as shown in figure 32, i.e. adds dress station, meets these and want in the way of simple and economical Ask.Add the clamp on dress station physically keep box body and provide the sealing entered for high pressure.Small-sized sub-micron porosity mistake Filter is attached on clamp supply pressure-air in dustless mode to the bank being filled with fluid.Detachable syringe can be used The air providing controlled volume maybe can use the automatic valve being connected to high-voltage power supply.This is uniformly distributed in whole microfluidic channel The fluid of the not bubbles of predetermined.

Figure 33 shows the cell dilution cloning for monitoring growth and separating clone group.There is several nanoliter volumes Micropore can use multiple material manufacture, described material to include, but without limitation, glass, quartz, plastics, the most poly-methyl-prop E pioic acid methyl ester (PMMA) etc., and other can cast or spendable polymer (such as polydimethylsiloxane, PDMS or SU8).The degree of depth of microfluidic channel typically, but without limitation, is 10 microns to 100 microns.The width allusion quotation of microfluidic channel Type ground, but without limitation, for 1 to 5 times of the degree of depth.Carry out substrate of glass masking, then carry out passage etc. to Property etching manufacture in the case of microfluidic channel, cross section is typically rectangle, or the rectangle with quadrant angle.Receive The example of metre hole array is as shown in figure 33.In this case, hole is 100 microns × 100 microns squares, and the degree of depth is 70 microns, And be to use PDMS flexible lithography method manufacture.This hole carries out sterilizing in the plasma and is inserted in 24 hole tissue culturing plates. By suspending, loading cells in media as well and passes through gravitational settling in nano-pore in each hole, and wherein they can periodically be carried out Monitor to observe growth kinetics or preferably clone colony in single time point results.This some holes be alternatively arranged as retaining element or It is merged in the output bank of sorting unit as removable dummy slider.

Figure 35 shows that carrying out cell/particle counting in heterogeneous microfluid droplet (is such as encapsulated in microfluidic channel In droplet in cell/particle) optical detecting method.Apply suitable signal processing, can be based on deriving from cell/particle Signal peak detection droplet in cell/population.In a method, whole passage is illuminated and single detection Device measures signal such as forward scatter signal, and it indicates presence or absence cell in droplet.More complicated detection is adopted With crossing channel and with flow to vertically arranged forward scatter detector array, in order to for counting and localization droplet in thin Born of the same parents/particle provides more preferable spatial resolution.In another method, single detector scatterometry additional dark field can be illuminated And imaging.When the forward position of forward scatter signal detection droplet, ccd video camera can be with synchronization to obtain the dark field image of droplet. Cell/particle will be bright spot in the picture, and it can be used for counting through processing and qualification.

In the case of many sorts, the target group of cell or particle may be less, but the surface area collecting bank is bigger. Particle may cling bank wall, it reduces collection efficiency.Figure 36 shows and makes the interaction on cell or particle and surface The means of littleization.In one approach, will be sorted target cell and be collected in closed area, this closed area is than displacement of fluid Less volume so that minimize at cell and the contact collected between bank wall.Due to surface tension, the liquid being collected exists Wetted droplet is formed on closed area.In another approach, the surface collecting bank can be by coating based on hydrocarbon or fluorine The silanization of hydrocarbon and become hydrophobic.Because hydrophobic coating, the liquid containing cell or particle being collected will be closed in close In the droplet of outlet opening.In another approach, can use and be independently matched with but detachable container is collected and is sorted cell. This single container can be used for processing further cell, and it will make loss cell minimize, and will be sorted cell from collection simultaneously Bank is transferred in another bank (such as porous culture plate).Collect container to have in bottom and lead in microfluidic device The hole of outlet opening.The diameter in hole can be less than a millimeter, thus surface tension prevents from, by this hole, seepage occurs.By flexible material The liner that (such as based on siloxanes rubber, PDMS) makes is inserted between collection container and microfluidic device for sealing And combination.

It is generally desirable to recovery sample after cell counting is analyzed.Figure 38 shows microfluidic device, and it is held shrinking born of the same parents under the arm and flows use Cell is reclaimed with lower dilution factor in analyzing.Sample uses the sheath buffer agent as shown in Fig. 2 and Figure 38 to carry out concentrating stream Dynamic.After sample by analysis region, microfluidic channel is split into three passages.Centre gangway is sample recovery approach, left Wing passage and right channel collect the sheath buffer agent of excess to reduce diluted sample.By suitable selector channel width and applying Pressure, diluted sample degree can be adjusted to 1: 1 dilution factor dilution factor (typically 1: 20) to sheath stream.At certain condition Under, under conditions of the dilutest sample, the sample being collected can instead concentrate.

Figure 37 shows the microfluidic device designed for repeatedly sample analysis or sorting.This design does not require can at loading Change box body after causing the different samples that fluorescence background changes or rearrange.The present invention includes three sample banks, two Sample bank is used for sample, and a sample bank is for buffer agent, so that two samples are away from cross-contamination.Pressed by regulation Power, each sample of injectable also carries out holding contracting under the arm for Flow Cytometry Analysis.Such as, two sample A and B are used, for A's Analyzing, the bank of sample A is applied maximum pressure, buffer agent bank is in middle pressure, and the bank of sample B is maintained at Under low-pressure.By alternately having the passage of maximum pressure, required passage will flow to analyzed area (maximum pressure) or its flowing It is prevented from (minimum pressure).Pure buffer agent can be introduced to allow to be clearly separated letter in analyzed area between each sample Number.The sample number can being loaded and sequentially operate can for each independent separation buffer channel being affixed to microfluid wafer It is added to 2.

Figure 38 also show the microfluidic device that can carry out having the multiplexing of dilution two samples of scalable Embodiment.This device have employed two sample channel A and B and separation buffer agent.In order to analyze sample A, use pressure below Set: P_sw_A ＜ P_sw_B ＜ P_B ＜ P_sb ＜ P_A ＜ P_sth.In order to analyze sample B, employ pressure below and set Fixed: P_sw_B ＜ P_sw_A ＜ P_A ＜ P_sb ＜ P_B ＜ P_sth.In order to prevent the cross-contamination between sample, separation buffer Agent can flow downward along passage between sample A and B measures.Using additional pressure port, this design can be amplified scale and be used In three or more samples.Figure 38 shows the embodiment of this device of switching between two samples.

Although being illustrated with embodiment the present invention above is had been described in considerable detail for being clearly understood that Purpose, but those of ordinary skill in the art obviously can carry out certain change and change according to the teachings of the present invention to it and not take off Spirit and scope from present specification.

Claims

1. Microfluidic cell sorter, comprising:

It is suitable for accepting the cell import of the one or more cells in fluid media (medium)；

It is fluidly connected to the first and second buffer agent imports of cell import, to provide buffer agent solution to sorter；

It is fluidly connected to cell import and the fluid passage of the first and second buffer agent imports,

Being fluidly connected to the first cross-flow passes of fluid passage, described first cross-flow passes is positioned at the first and second fluid cushion agent The downstream of import；

Being fluidly connected to the second cross-flow passes of fluid passage, described second cross-flow passes is positioned at the first and second fluid cushion agent The downstream of import, and the second cross-flow passes be positioned at fluid passage with on the first cross-flow passes opposite side；

Being fluidly connected to the first and second outlets of fluid passage, these outlets are positioned at the downstream of the first and second cross-flow passes,

The cell being suitable for detection given state the cell responding given state produce the detector of signal, and this detector is arranged Cell is detected for the upstream position at the first cross-flow passes,

Be connected to detector and the first and second cross-flow passes pneumatic cross force switch, its response signal and actuatable with produce Pneumatic pressure pulses, thus cause fluid to move in cross-flow passes, described pneumatic cross force switch includes:

It is connected to the first pneumatic operated valve of the first cross-flow passes；

It is connected to the second pneumatic operated valve of the second cross-flow passes；

Connect detector and the control system of pneumatic cross force switch, it is provided that timing controling signal actuates the first He in a sequential manner Second pneumatic operated valve, it is characterised in that the front opening that the first pneumatic operated valve is opened at the second pneumatic operated valve；

Thus when the cell of given state being detected, pneumatic cross force switch is activated to provide cross force to cell, thus Make signaling so that its selectivity exits into the first or second outlet.

2. the Microfluidic cell sorter of claim 1, the fluid stream wherein flowing through fluid passage is laminar flow.

3. the Microfluidic cell sorter of claim 1, wherein the first and second buffer agent imports are of different sizes.

4. the Microfluidic cell sorter of claim 1, wherein the first and second outlets are of different sizes.

5. the Microfluidic cell sorter of claim 4, wherein the first outlet has the fluid stream of bigger volume than the second outlet.

6. the Microfluidic cell sorter of claim 1, it is characterised in that the first pneumatic operated valve after the second pneumatic operated valve is opened and Closed before the second pneumatic operated valve cuts out.