CN203525731U - Multistage sorting microfluidic device for rare cells - Google Patents

Multistage sorting microfluidic device for rare cells Download PDF

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CN203525731U
CN203525731U CN201320561583.6U CN201320561583U CN203525731U CN 203525731 U CN203525731 U CN 203525731U CN 201320561583 U CN201320561583 U CN 201320561583U CN 203525731 U CN203525731 U CN 203525731U
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runner
electrode
rare cell
fluidic device
rare
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易红
倪中华
黄笛
项楠
陈科
唐文来
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Southeast University
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Southeast University
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502776—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/088—Channel loops
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

Abstract

The utility model discloses a multistage sorting microfluidic device for rare cells. The multistage sorting microfluidic device comprises a runner structure and an electrode substrate which are encapsulated in a bonding manner, wherein the runner structure comprises a spiral runner, a secondary rubber, a lower branch runner, an upper bifurcated runner and a lower bifurcated runner, wherein a sample inlet is formed in one end of the spiral runner; the other end of the spiral runner is respectively connected with the secondary runner and the lower branch runner by a shrinking-expanding structure; the secondary runner is respectively connected with the upper bifurcated runner and the lower bifurcated runner; the upper bifurcated runner and the lower bifurcated runner are connected with a blood cell outlet; the lower bifurcated runner is connected with a rare cell outlet; the electrode substrate comprises a substrate body, and two electrode groups which are arranged on the bottom of the secondary runner. The multistage sorting microfluidic device disclosed by the utility model is low in cost, simple to operate, and easy for micromation integration, and the high-flux high-purity separation of the rare cancer cells can be realized.

Description

A kind of rare cell multipass sort micro-fluidic device
Technical field
The utility model belongs to analyzing and testing apparatus field, relate to a kind of rare cell particle multipass sort micro-fluidic device, particularly the high flux of a kind of integrated micro flow body inertia effect and dielectrophoresis technology, high-purity rare cell particle multipass sort micro-fluidic device.
Background technology
The accurate detection of cell is of great significance at tool aspect the biomedical applications such as prevention, diagnosis, treatment and RESEARCH ON CELL-BIOLOGY of disease.But conventionally clinically, research to as if a heterogeneous populations, wherein comprised a large amount of cell information, the rare cell that makes to detect wherein will become very difficult; For example, detect and enter the rare cancer cell in blood circulation, its number ratio to human normal haemocyte is about 1:10 9, in 1mL blood, only there are several rare cancer cells, therefore detect very difficult.
The new method that microflow control technique is controlled as a kind of high efficiency fluid or particle, can realize the functions such as sample preparation, reaction, focusing, sorting and detection, is the important research content in the multidisciplinary fields such as biomedicine, analytical chemistry.At present, adopting micro-fluidic cell sorting generally based on monotechnics, can brief overview be following a few class by its mechanism: the first kind is the technology of controlling based on complex micro structure; Equations of The Second Kind is the passive method based on fluid, as adopted inertia flow, extrusion flow, waterpower sorting, field flow separation etc. to control technology; The 3rd class is the technology of controlling that adopts gravity and sedimentation method to carry out sorting; The 4th class is the technology of controlling that adopts imitative biological method; The 5th class is the sorting technology according to water liquid two phasic properties; The 6th class is the sorting technology by magnetic, sound, the outfield such as optical, electrical.Though above-mentioned each method has all obtained certain research effect, but be applied to rare cell sorting and still have deficiency separately: as film sorting, extrusion flow sorting, waterpower sorting, field flow sorting, gravity and sedimentation sorting, the two phasic property sortings of water liquid, magnetic separation, photometric sorting all exist the too low problem of operation flux, thereby limit its further business application; And there is the problem that separating purity is not high in the methods such as columnar arrays sorting, inertia flow sorting, waterpower sorting, the two phasic property sortings of water liquid.In addition, also there is some other defect in above-mentioned method for separating, as film sorting, sound sorting easily cause damage to cytoactive; Extrusion flow sorting only differs in larger and just can obtain good separating effect at two kinds of particle sizes; Magnetic, sound, optical, electrical sorting cause apparatus expensive, complicated operation owing to having introduced power consumption outfield.
For overcoming the deficiency of single-stage sorting, there is in recent years the research of some multipass sorts, the thinking of these researchs is the pretreatment using one-level operation as secondary sorting, to optimize the effect of secondary sorting; Or adopt secondary refining-sorting, the result of one-level ragging is further purified.Yet existing multipass sort research is still the technology based on single, is only by the simple combination of single sorting technology, has increased operational sequence and cost, is difficult to meet the high flux of rare cell sorting, highly purified requirement simultaneously.
Utility model content
Utility model object: in order to overcome above-mentioned defect, the purpose of this utility model is to provide high flux, the high-purity rare cell particle multipass sort micro-fluidic device of a kind of integrated micro flow body inertia effect and dielectrophoresis technology, realizes high flux, the high-purity sorting of rare cell/particle.
Technical scheme: a kind of rare cell multipass sort micro-fluidic device that the utility model provides, comprises flow passage structure and the electrode basement of bonding packaging; Described flow passage structure comprises helical flow path, secondary runner, inferior division runner, upper fork runner, lower fork runner, described helical flow path one end is that sample inlet, the other end are connected with secondary runner, inferior division runner respectively by reducing and expansion structure, described secondary runner is connected with lower fork runner with upper fork runner respectively, described upper fork runner is connected with haemocyte outlet with inferior division runner, and described lower fork runner is connected with rare cell outlet; Described electrode basement comprises substrate body and two arrays of electrodes group; Described electrode group is positioned at secondary runner bottom.
As improvement, described electrode group is oblique cutting profile of tooth, is mirror-image arrangement; By the oblique cutting claw pole group of two groups of mirror-image arrangement is set, last electrode group applies the negative dielectrophoresis signal of low frequency, and all cells is deflected to the side near flow path wall; Latter one group applies high frequency negative dielectrophoresis signal, and by rare cell particle, along electrode direction deflection, and haemocyte still moves along main flow direction, thereby makes rare cell particle effectively separated with haemocyte.
As another kind, improve, the vertical cross-section of described helical flow path is rectangle or that outside is high, inner side is low is trapezoidal; When the vertical cross-section of helical flow path is rectangle, its depth-to-width ratio is below 1, preferably 0.1-1; Helical flow path cross section is low depth-to-width ratio rectangle, can make larger rare cell particle farther with less haemocyte particle equilbrium position apart, is beneficial to separation.
As another kind, improve, described reducing and expansion structure and helical flow path link vertical cross-section size are than little with secondary runner, inferior division runner link vertical cross-section size.Described reducing and expansion structure upstream polycondensation Chu Yu helical flow path UNICOM, downstream sudden expansion place forms a shorter straight channel, and is divided into two, and respectively rare cell particle flux being imported to top set's runner is dielectrophoresis runner, and haemocyte particle flux is directed into inferior division runner.
As another kind, improve, described secondary runner is connected with reducing and expansion structure by top set's runner, and the vertical cross-section size of top set's runner is less than inferior division runner vertical cross-section size; Inferior division runner be haemocyte particle flux branch width to be greater than top set's runner be rare cell particle flux branch width, thereby reduce the fluid volume of the required processing of top set's runner sorting, and then reach the object that reduces flow velocity.
As another kind, improve, the vertical cross-section of described secondary runner is rectangle, and its depth-to-width ratio is 0.05-0.01; Top set's runner is that dielectrophoresis cross section of fluid channel is ultralow depth-to-width ratio rectangle, further to reduce the flow velocity in runner, guarantees that dielectrophoretic force has enough response times on particle.
As another kind, improve, the material of described flow passage structure is selected from a kind of in dimethyl silicone polymer, epoxy resin, polymethyl methacrylate, Merlon, glass, silicon and quartz.
As another kind, improve, described electrode basement is formed by ito glass etching, or makes by the method that sputter figure on glass or silicon materials dissolve metal electrode.
Beneficial effect: low, simple to operate, the easy of integration microminiaturization of rare cell multipass sort micro-fluidic device cost that the utility model provides, by integrated inertia sorting and dielectrophoresis sorting two-stage runner, make full use of the high flux advantage of inertia sorting and the high accuracy advantage of dielectrophoresis sorting, overcome existing sorting chip only based on certain monotechnics, be difficult to realize the deficiency of high flux and high-purity sorting simultaneously, two kinds of modes combine works in coordination collaboratively, realizes the high flux of rare cancer cell, high-purity sorting.
Particularly, the one-level runner of this device is the high flux advantage that helical flow path makes full use of inertial technology, by Dean stream and inertia in curved runner, move, rare cancer cell particle and haemocyte particle are focused to different equilbrium positions, and by reducing and expansion structure, import respectively secondary runner and haemocyte exports, and after sorting the sample volume of rare cancer cell particle flux much smaller than the sample volume of haemocyte particle flux.By the ragging of one-level runner, can under high-throughout condition, get rid of most of haemocyte, only make the sample that fraction contains rare cancer cell and residual blood cell enter secondary runner, thereby alleviate the flux pressure of dielectrophoresis sorting.Secondary runner is by being laid on two groups of oblique cutting tooth electrodes of runner bottom, the rare cancer cell and the residual blood cell that enter are wherein applied to negative dielectrophoretic force, wherein last group of electrode applies low frequency signal, make rare cancer cell and residual blood cell all along electrode direction deflection, at flow path wall, one row of side place is into a line, to optimize separating effect; Rear one group of electrode applies high-frequency signal, only makes rare cancer cell along electrode direction deflection and residual blood cell still flows along former direction, thereby realizes both accurate separation.Dielectrophoresis sorting, as a kind of active sorting technology, has the advantage that precision is high.
This rare cell multipass sort micro-fluidic device can be widely used in the fields such as clinical diagnosis, biological study, biochemical analysis, is particularly useful for the earlier detection of rare cell in body fluid, the aspects such as chemotherapy drug susceptibility test in cytology level.
Accompanying drawing explanation
Fig. 1 is the structural representation of the utility model rare cell multipass sort micro-fluidic device.
Fig. 2 is the explosive view of the utility model rare cell multipass sort micro-fluidic device.
Fig. 3 is the structural representation of flow passage structure.
Fig. 4 is the structural representation of electrode basement.
Fig. 5 is the schematic diagram of helical flow path inertia sorting.
Fig. 6 is the schematic diagram of sudden expansion structure place cell particle sorting.
Fig. 7 is the schematic diagram of electrode group dielectrophoresis sorting in secondary runner.
The specific embodiment
Below the embodiment of the utility model patent is elaborated; the present embodiment is implemented take the utility model patented technology scheme under prerequisite; provided detailed embodiment and concrete operating process, but the protection domain of the utility model patent is not limited to following embodiment.
In the present embodiment, the material of flow passage structure 1 is dimethyl silicone polymer (PDMS), also can select the good materials of optical property such as glass, epoxy resin, polymethyl methacrylate (PMMA), Merlon (PC), silicon and quartz to make.Chip manufacture technique, bonding techniques that different materials is corresponding may there are differences, and can select respective material according to processing conditions and application demand.Flow passage structure 1 in the present embodiment is made by soft lithography, and the formpiston used of casting adopts the processing of SU-8 maskless lithography, and this technology has flexible high, the advantage such as cost of manufacture is low and the process-cycle is short.Also can make formpiston by the technology such as lithography of wet method/deep reaction ion etching, ultraprecise machined, metal plating and the photosensitive circuit plate of the photoetching technique based on chrome mask/printing film mask, silicon.
Electrode basement 2 in the present embodiment adopts mask-free photolithography process technology, ito glass is carried out to graphical also chemical etching and form, and also can make by deposit metal electrodes photolithography patterning in glass or silicon materials substrate.In above-mentioned various processing method, the characteristics such as the machining accuracy that different technologies can reach and shape of cross section are not identical yet, should select according to sorting object and physical device condition.
Device described in the present embodiment is for high flux, the high-purity sorting of the rare cancer cell of blood, also can be used for sorting or the purification of rare cell in other body fluid (as urine, saliva, hydrothorax, ascites, phlegm etc.), also can expand be applied to two kinds vary in size, electric conductivity difference or other different particle sortings of dielectric properties.
Rare cell multipass sort micro-fluidic device, is shown in Fig. 1 and 2, comprises flow passage structure 1 and electrode basement 2, by flow passage structure 1 and electrode basement 2 bonding packagings are formed.
Flow passage structure 1, is shown in Fig. 2, comprises helical flow path 11, secondary runner 12, inferior division runner 13, upper fork runner 14, lower fork runner 15; Helical flow path 11 near-ends are connected with sample inlet 16, helical flow path 11 far-ends extend into straight runner and are connected with reducing and expansion structure 17 near-ends, reducing and expansion structure 17 far-ends are divided into two, are divided into secondary runner 12 and inferior division runner 13, and inferior division runner 13 is directly connected with haemocyte outlet 18; Secondary runner 12 far-ends are divided into 15 two of fork runner 14 and lower fork runners equally, and wherein upper fork runner 14 is connected with haemocyte outlet 18, and lower fork runner 15 exports 19 with rare cell and is connected.
For cell particle can be focused in the interior realization of helical flow path 11, the height h of helical flow path 11 should meet 0.07<ap/h<0.3, and wherein ap is particle diameter; Simultaneously, the vertical cross-section shape of helical flow path 11 should be designed to low depth-to-width ratio rectangle (depth-to-width ratio AR=h/w<1), to the particle through helical flow path 11 is carried out to sorting along width of flow path direction, reduces the impact of particle interphase interaction as far as possible; In other embodiments, also can be designed to that outside is high, inner side is low trapezoidal for cross section of fluid channel.In addition, between the runner of helical flow path 11, spacing should be greater than width of flow path, and to avoid flow path wall to cross thin, generation is out of shape.Reducing and expansion structure 17 near-end both sides flow path walls are mutually the angle of 120 °, then form a wider straight runner, that is to say, reducing and expansion structure 17 and helical flow path 11 link vertical cross-section sizes are than little with secondary runner 12, inferior division runner 13 link vertical cross-section sizes.Secondary runner 12 is connected with reducing and expansion structure 17 by top set's runner 10, and the vertical cross-section size of top set's runner 10 is less than inferior division runner 13 vertical cross-section sizes, so that only have a small amount of rare cell sample to flow to into secondary runner 12.Secondary runner 12 vertical cross-section be ultralow depth-to-width ratio rectangle (depth-to-width ratio AR=h/w is 0.01-0.05), guaranteeing should to make runner as far as possible wide under the condition that PDMS micro-structural is not caved in, fully to reduce the sample flow rate in secondary runner 12, meet the requirement of dielectrophoresis sorting, optimize secondary separating effect; Secondary runner 12 near-ends and top set's runner 10 connecting place both sides flow path walls are also symmetrical hexagonal angle, to relax gradually runner to required width.
Electrode basement 2, is shown in Fig. 4, comprises the two arrays of electrodes group 22 that substrate body 21 and etching form; Two arrays of electrodes group 22 is all positioned at secondary runner 12 bottoms.Wherein, the first electrode group 23 and the second electrode group 24 are mirror, and wherein the first electrode group 23 comprises the first electrode 25 and the second electrode 26, the second electrode groups 24 comprise third electrode 27 and the 4th electrode 28.The wide parallel pole that the first electrode 25 is connected by three upper ends forms, the wide parallel pole that the second electrode 26 is connected by three lower ends forms, and the first electrode 25 and the second electrode 26 are the cross arrangement of oblique cutting dentation, six parallel poles forming the first electrode 25 and the second electrode 26 after arranging are wide, etc. gap evenly distributed, the sample main flow direction in electrode direction and secondary runner 12 is 15 ° of angles.Equally, the wide parallel pole that third electrode 27 is connected by three upper ends forms, the wide parallel pole that the 4th electrode 28 is connected by three lower ends forms, third electrode 27 and the 4th electrode 28 are the cross arrangement of oblique cutting dentation, six parallel poles forming third electrode 27 and the 4th electrode 28 after arranging are wide, etc. gap evenly distributed, the sample main flow direction in electrode direction and secondary runner 12 is 165 ° of angles.In other embodiments, the parallel pole number that the first electrode 25, the second electrode 26, third electrode 27 and the 4th electrode 28 comprise and the angle of parallel pole and main flow direction all can redesign by demand.Width, the integral width that the whole height of electrode group 22 is greater than secondary runner 12 is less than the length of secondary runner 12.In addition, the first electrode 25, the second electrode 26, third electrode 27 are connected from different extraction electrodes respectively with the 4th electrode 28, adopt signal generator, can to the first electrode group 23, apply compared with the negative dielectrophoresis signal of low frequency, the second electrode group 24 is applied to the negative dielectrophoresis signal of higher-frequency by extraction electrode.
The encapsulation process of this device is as follows: after flow passage structure 1 and electrode basement 2 micro-structure surfaces are cleaned up, the present embodiment utilizes UV/ozone to carry out surface modification treatment to flow passage structure 1 and electrode basement 2 surfaces, realizes the irreversible bonding of flow passage structure 1 and electrode basement 2.In other embodiment, also can carry out the irreversible bonding that surface modification realizes flow passage structure 1 and electrode basement 2 by means such as oxygen plasma treatment.After bonding, the first electrode group 23 and the second electrode group 24 will be laid on the bottom of secondary runner 12, form secondary dielectrophoresis sorting chip.In addition, in other embodiments, also can be at the edge of flow passage structure 1 and electrode basement 2 more symmetrical alignment marks, alignment mark can be processed together with the micro-structural of soft lithography/metal thin-film pattern metallization processes and flow passage structure 1 and electrode basement 2, by stereomicroscope and alignment mark, realize flow passage structure 1 and electrode basement 2 accurate bonding in position.
After treating integral device encapsulation, in sample inlet 16, insert microtubule and connect the external fluid driving arrangements such as syringe pump, for the introducing of specific flow velocity sample, haemocyte outlet 18 and rare cell export 19 places insert microtubules for sorting after collection and the derivation of sample liquid.
Below to elect job step and the principle that example is set forth this multipass sort device dividing of rare cancer cell in blood as.
Helical flow path 11 is the grading principle of one-level inertia runner, see Fig. 5, with specific flow velocity, inject particle suspension in from sample inlet 16 toward runner, helical flow path 11 intake section random distribution have red blood cell, leucocyte and rare cancer cell, the about 6-9 μ of red blood cell diameter m wherein, leucocyte diameter 6-15 μ m, and rare cancer cell kind is different, vary in size, but generally all much larger than haemocyte size, in the present embodiment, take human breast cancer cell MCF-7 as example, getting its diameter is 16-24 μ m.The in the situation that of straight channel, due to the parabola shaped velocity profile of Poiseuille flow (Poiseuille flow), particle wherein will be subject to pointing to the shear-induced inertia lift F of wall lSeffect towards wall, move, and when particle is during near wall, because of particle from then symmetrical tail that rotation produces by wall, affected and produce a wall induction inertia lift F who points to runner center line lW, F lSwith F lWjointly formed inertia lift F l, cause particle that specific inertia migration occurs.And in curved runner except inertia migration, because near fluid runner center line has higher flow velocity compared with near fluid wall, cause the flowing through centrifugal force of fluid of runner and the imbalance of radial pressure gradient, cause the fluid of runner centerline outwards to flow.Closed flow is for meeting the conservation of mass, and the fluid at close outside wall surface 112 places will reflux along bottom surface on runner, so producing two whirlpools that direction of rotation is contrary in vertical main flow direction, be called Dean stream or Secondary Flow, because particle is subject to Dean, drags power F dDeffect, Dean stream size is proportional with the diameter of particle.Particle is at inertia lift F land Dean drags power F dDacting in conjunction under migrate to the equilbrium position near runner inner wall face 111, in Fig. 5, place, particle a place is stable equilbrium position; Though particle d is stress balance also, unstable, very little disturbance will be away from this position; All the other each position b, c, e, f, the stressed equal imbalance of g place particle.
The principle of sudden expansion structure 7 places cell particle sorting, is shown in Fig. 6, and in the exit of helical flow path 11, rare cancer cell 5, because of larger compared with red blood cell 3, leucocyte 4 sizes, is subject to larger Dean and drags power F dDeffect, thereby the more close internal face 111 in its equilbrium position, but due to the flexibility of cell particle and the dispersiveness of size, still have a small amount of participation haemocyte to be dispersed in rare cancer cell 5 particle fluxes.When cell particle moves in sudden expansion structure 17, because of wall induction inertia lift F lWvariation, all cells is to moving to new equilbrium position near the direction of wall, and now between equilbrium position and the haemocyte equilbrium position of rare cancer cell 5 spacing become greatly, be beneficial to sorting.At sudden expansion structure 17 far-ends, rare cancer cell 5 and a small amount of remaining red blood cell 3, leucocyte 4 enter fork runner, are directed in secondary runner 12; And red blood cell 3, the leucocyte 4 of the overwhelming majority enter inferior division runner 13, be directly directed into haemocyte outlet 18.In addition, the width of top set's runner 10, much smaller than the design of inferior division runner 13 width, makes to only have fraction sample to enter in secondary runner 12, thereby reduces the flux pressure of secondary runner 12.
In secondary runner 12, the principle of electrode group dielectrophoresis sorting, is shown in Fig. 7, and secondary runner 12 has formed secondary dielectrophoresis sorting chip jointly with the first electrode group 23 and the second electrode group 24 that are laid on its bottom.By the first electrode group 23 and the second electrode group 24 being applied to the ac signal of CF, to in secondary runner 12, form inhomogeneous field, and the cell of the secondary runner 12 of flowing through will be induced the asymmetric dipole moment of generation under inhomogeneous field effect, thereby produce dielectrophoretic force F dEP.Dielectrophoretic force F dEPsensing and the size of size and the dielectric constant of electric-field intensity, electric field frequency, electric-force gradient, particle and surrounding medium and electric conductivity, particle etc. relevant, in the present embodiment, cell particle by secondary runner 12 is applied to negative dielectrophoresis effect, make it a little less than electric-field intensity, away from the direction of electrode, move.In the present embodiment because secondary runner 12 cross sections are ultralow depth-to-width ratio rectangle, there is larger cross-sectional area, and the sample volume entering in secondary runner 12 is less, cause sample flow velocity in secondary runner 12 extremely slow, thereby cell particle have the driving of time enough response inhomogeneous field.The ac signal that the first electrode group 23 and the second electrode group 24 is applied to different frequency, the cell in secondary runner 12 is subject to fluid and pulls power (F d) and negative sense dielectrophoretic force (nF dEP) acting in conjunction, and haemocyte and rare cancer cell 5 are because of dielectric properties and vary in size and be subject to different dielectrophoresis active forces, show different kinetic characteristics.At the first electrode group 23 places, apply compared with the low-frequency ac signal of telecommunication, red blood cell 3, leucocyte 4, rare cancer cell 5 will all be subject to negative dielectrophoretic force nF dEP1pull power F with fluid d1effect, and at both F that makes a concerted effort 1effect under along the direction of electrode, move, and finally migrate to the upside wall place of secondary runner 12, arrange approx into a line, to increase the sharpness of separation of the second electrode group 24.At the second electrode group 24 places, apply higher-frequency ac signal, only have rare cancer cell 5 to be subject to negative dielectrophoretic force nF dEP2effect, and at nF dEP2pull power F with fluid d2the F that makes a concerted effort 2under effect, along electrode direction, move, finally migrate to the lower wall surface place of secondary runner 12, and flow into lower fork runner 15, finally from rare cell outlet 19, derive; And red blood cell 3, leucocyte 4 will only pull power F at fluid d2effect under still along main flow direction, move, and flow into fork runner 14, finally from haemocyte outlet 18, derive, realize the accurate sorting of red blood cell 3, leucocyte 4 and rare cancer cell 5.
The rare cell multipass sort device proposing in the present embodiment, integrated inertial technology and dielectrophoresis technology, can meet rare cell sorting high flux, high-precision sorting requirement simultaneously, have that cost is low, simple operation and other advantages simultaneously, the fields such as clinical diagnosis, biological study, biochemical analysis be can be widely used in, the earlier detection of rare cell in body fluid, the aspects such as chemotherapy drug susceptibility test of cytology level are particularly useful for.

Claims (8)

1. a rare cell multipass sort micro-fluidic device, is characterized in that: the flow passage structure (1) and the electrode basement (2) that comprise bonding packaging, described flow passage structure (1) comprises helical flow path (11), secondary runner (12), inferior division runner (13), upper fork runner (14), lower fork runner (15), described helical flow path (11) one end is sample inlet (16), the other end by reducing and expansion structure (17) respectively with secondary runner (12), inferior division runner (13) connects, described secondary runner (12) is connected with lower fork runner (15) with upper fork runner (14) respectively, described upper fork runner (14) is connected with haemocyte outlet (18) with inferior division runner (15), described lower fork runner (15) is connected with rare cell outlet (19), described electrode basement (2) comprises substrate body (21) and two arrays of electrodes group (22), described electrode group (22) is positioned at secondary runner (12) bottom.
2. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: described electrode group (22) is oblique cutting profile of tooth, is mirror-image arrangement.
3. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: the vertical cross-section of described helical flow path (11) is rectangle or that outside is high, inner side is low is trapezoidal; When the vertical cross-section of helical flow path (11) is rectangle, its depth-to-width ratio is below 1.
4. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: described reducing and expansion structure (17) and helical flow path (11) link vertical cross-section size are than little with secondary runner (12), inferior division runner (13) link vertical cross-section size.
5. a kind of rare cell multipass sort micro-fluidic device according to claim 1, it is characterized in that: described secondary runner (12) is connected with reducing and expansion structure (17) by top set's runner (10), and the vertical cross-section size of top set's runner (10) is less than inferior division runner (13) vertical cross-section size.
6. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: the vertical cross-section of described secondary runner (12) is rectangle, and its depth-to-width ratio is 0.05-0.01.
7. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: the material of described flow passage structure (1) is selected from a kind of in dimethyl silicone polymer, epoxy resin, polymethyl methacrylate, Merlon, glass, silicon and quartz.
8. a kind of rare cell multipass sort micro-fluidic device according to claim 1, is characterized in that: described electrode basement (2) is formed by ito glass etching, or makes by the method that sputter figure on glass or silicon materials dissolve metal electrode.
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