CN106190774A - For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method - Google Patents
For capturing the micro-fluidic chip of circulating tumor cell and capture thereof and authentication method Download PDFInfo
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- CN106190774A CN106190774A CN201610590052.8A CN201610590052A CN106190774A CN 106190774 A CN106190774 A CN 106190774A CN 201610590052 A CN201610590052 A CN 201610590052A CN 106190774 A CN106190774 A CN 106190774A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- 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
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- 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
- B01L3/502707—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 the manufacture of the container or its components
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Abstract
The invention discloses a kind of micro-fluidic chip for capturing circulating tumor cell and capture thereof and authentication method.This micro-fluidic chip includes sample cell solution inlet port, the flow dividing structure being connected with sample cell solution inlet port, sample cell solution being shunted step by step, the cell focusing structure being connected with the outlet of flow dividing structure, the cell in sample cell solution being focused, outlet with cell focusing structure is connected, the circulating tumor cell in sample cell solution carries out the cell fence structure that captures, and the waste liquid outlet being connected with the outlet of cell fence structure.Sample cell solution can be carried out multi-stage diffluence by flow dividing structure by micro-fluidic chip of the present invention, to reduce the blocking of sample cell solution, the preparation of abundance is carried out for realizing quickly sorting, simultaneously under the effect of cell focusing structure, circulating tumor cell is central in runner by linear array, can directly and quickly flow in cell fence structure, to realize the fast Acquisition to circulating tumor cell.
Description
Technical field
The present invention relates to micro-fluidic chip, be specifically related to a kind of micro-fluidic chip for capturing circulating tumor cell and
Capture and authentication method.
Background technology
Circulating tumor cell (circulating tumor cells, CTCs) is an off primary lesion, attacks and enters and follows
The cancerous cell of loop systems, can be as diagnosis patient's prognosis or tumor recurrence, clinical of announcement neoplasm metastasis behavior, rationally guidance
The index of bodyization treatment, is the study hotspot of current oncology.But in tumour patient blood, the number of CTCs is the most rare, often
Milliliter blood contains only 1～100 CTCs, and in every milliliter of blood have up to a million leukocyte and number more red carefully
Born of the same parents, therefore detect the challenge that CTCs is this field face from peripheral blood fast and efficiently.
Microfluidic chip technology has that sample reagent consumption is few, analyze detection sensitivity high, high flux and high efficiency etc.
Advantage, has had research and utilization microfluidic chip technology to detect circulating tumor cell, has used more in micro flow chip
Catching method is to utilize for some tumor cell epithelial cell adhesion molecule (epithelial cell adhesion
Molecule, EpCAM) carry out specificity sorting.But EpCAM is as a kind of surface epithelial cell label, dissimilar
Tumor cell in expression degree different, be easily caused false positive and false-negative testing result.Furthermore according to tumor cell
With hemocyte difference in terms of particle diameter and deformability, with different physical methods, CTCs can be sorted.?
Conventional physical size screening technique is, different from CTCs size according to hemocyte, and the method using similar membrane filtration will
Less hemocyte filters out, and retains the CTCs that particle diameter is bigger, thus reaches the purpose of CTCs sorting enrichment.The method excellent
Point is that the CTCs response rate can reach 80%～95%；Shortcoming is that separation velocity is too slow, and efficiency is on the low side, and chip is easily hindered by hemocyte
Plug, causes sorting operation cannot be carried out.
Patent documentation CN102360010A discloses " a kind of integrated microfluidic chip for capture of cancer cells in whole blood ", but this technology
The mixing runner of scheme is S-shaped, there is also separation velocity too slow, and efficiency is on the low side, and chip is easily blocked by hemocyte, causes point
The defects such as selection operation cannot be carried out.
Patent documentation CN103409371A varies in size according to cell size, by design discoid structure, utilize every
Stopper top and cover plate spacing, make small size cell pass through, and make large scale tumor cell stay inside depressed part, reach
Purpose to capture cell.The shortcoming of this patent is that this chip actual acquisition area only has a coil structures around runner, works as periphery
One circle is paved with after target cell under fluid pressure effect, and target cell is the most easily extruded, and causes capture rate relatively low.
It addition, existing utilize micro flow chip capture CTCs method, major part capture tumor cell after by transfer to from
Heart pipe carries out the analysis of CTCs, and this transfer process can cause a large amount of loss cell, additionally completes follow-up qualification in centrifuge tube also
Comprise excessive risk operation (such as, be centrifuged, rinse and hatch) of loss target cell, therefore there is the defect being difficult to overcome.
Summary of the invention
For the deficiencies in the prior art, the purpose of the present invention aims to provide a kind of miniflow for capturing circulating tumor cell
Controlling chip and capture thereof and authentication method, this chip operation is convenient, rapidly and efficiently rate can capture circulating tumor cell, and can be direct
The subsequent analysis being directly circulated tumor cell in chip is identified.
For achieving the above object, the present invention adopts the following technical scheme that
For capturing the micro-fluidic chip of circulating tumor cell, including sample cell solution inlet port, with sample cell solution
The flow dividing structure that import is connected, is shunted step by step by sample cell solution, is connected with the outlet of flow dividing structure, by sample
The cell focusing structure that cell in cell solution is focused, the outlet with cell focusing structure is connected, by sample cell
Circulating tumor cell in solution carries out the cell fence structure captured, and be connected with the outlet of cell fence structure useless
Liquid exports.
It is connected by the bus structure in branching shape between outlet and the waste liquid outlet of described cell fence structure, confluxes
The sample cell waste liquid that cell fence structure flows out is collected in waste liquid outlet by structure step by step.
Described cell fence structure is arranged by the microtrabeculae of positive six prisms or regular triangular prism and forms.
Described cell fence structure includes inner ring cell fence structure and outer ring cell fence structure；Described outer ring cell grid
Hurdle structure includes the interconnecting part being connected with the outlet of cell focusing structure and and the channel part that is connected mutually of interconnecting part, described company
Logical portion is horn-like progressively to expand along the flow direction of sample cell solution, and channel part is arranged at outside inner ring cell fence structure
Enclosing, the import of inner ring cell fence structure is positioned at interconnecting part and relative with the outlet of cell focusing structure.
Described inner ring cell fence structure is made up of the microtrabeculae of 1-3 row's parallel interval arrangement, and the gap between microtrabeculae is 6-
8um。
The microtrabeculae that described outer ring cell fence structure is made up of 4-6 microtrabeculae arranges arranged in parallel forming, microtrabeculae row and level
Direction is 20 ° of-40 ° of angles, and the gap between microtrabeculae is 6-8 μm.
Described flow dividing structure is made up of multi-stage diffluence passage and is distributed in branching shape, and two shuntings in every level shunt passage are logical
Angle between road is 30 °-45 °.
The described serpentine-like corrugated of cell focusing structure.
A kind of method capturing circulating tumor cell, comprises the steps:
(1) use the micro-fluidic chip described in wash buffer claim 1, remove micro-fluidic chip air entrapment；
(2) aqueous solution of Kolliphor P188 is passed through the micro-fluidic chip described in claim 1, is coated 2-10min,
Reduce non-specific adsorption between cell and micro-fluidic chip；—
(3) test fluid being passed through the micro-fluidic chip described in claim 1, be circulated tumor cell capture, circulation is swollen
Oncocyte and part leukocyte capture to cell fence structure, and other blood cells flow out from waste liquid outlet.
The immunofluorescence dyeing authentication method of a kind of circulating tumor cell, comprises the steps:
(1) fixative is passed through the micro-fluidic chip described in claim 1, after sample introduction, fixes cell 10-30min；
(2) buffer is passed through the micro-fluidic chip described in claim 1, replaces the fixative in micro-fluidic chip；
(3) Triton X-100 is passed through the micro-fluidic chip 10-30min described in claim 1；
(4) buffer is passed through the micro-fluidic chip described in claim 1, replaces the Triton X-in micro-fluidic chip
100 solution；
(5) CK18-FITC, CD45-PE and DAPI are taken, the anti-diluent that adds, obtain diluent, then diluent is passed through power
Profit requires the micro-fluidic chip described in 1, hatches 2-12 hour under conditions of 4-37 DEG C；
(6) buffer is passed through the micro-fluidic chip described in claim 1, replaces in micro-fluidic chip and be not bonded to cell
Antibody staining liquid, dye complete, identify under microscope.
The beneficial effects of the present invention is:
Sample cell solution can be entered by flow dividing structure by the present invention for the micro-fluidic chip of circulating tumor cell capture
Row multi-stage diffluence, to reduce the blocking of sample cell solution, carries out the preparation of abundance, gathers at cell simultaneously for realizing quickly sorting
Under the effect of close-burning structure, linear array in runner central authorities, can directly and quickly be flowed into cell fence structure by circulating tumor cell
In, to realize the fast Acquisition to circulating tumor cell.
Accompanying drawing explanation
Fig. 1 is the structural representation of the micro-fluidic chip that the present invention captures for circulating tumor cell；
Fig. 2 is the structure enlarged diagram of cell focusing structure；
Fig. 3 is the structure enlarged diagram of cell fence structure；
Fig. 4 is the design sketch of the micro-fluidic chip capture H1299-GFP cell that the present invention captures for circulating tumor cell；
In figure: 1, sample cell solution inlet port；2, flow dividing structure；3, cell focusing structure；4, cell fence structure；5, useless
Liquid exports；6, bus structure；21, split channel；41, inner ring cell fence structure；42, outer ring cell fence structure；420, even
Logical portion；421, channel part.
Detailed description of the invention
Below, in conjunction with accompanying drawing and detailed description of the invention, the present invention is described further:
Embodiment 1
As it is shown in figure 1, the present invention includes sample cell solution inlet port for the micro-fluidic chip of circulating tumor cell capture
1, the flow dividing structure 2 being connected with sample cell solution inlet port 1, sample cell solution being shunted step by step, say, that logical
Cross flow dividing structure 2 and sample cell solution can be split into a plurality of fluids step by step, to reduce the blocking of sample cell solution；With point
The cell focusing structure 3 that the outlet of flow structure 2 is connected, is focused by the cell in sample cell solution, say, that warp
Sample cell solution after shunting can enter directly in cell focusing structure 3, and cell focusing structure 3 is to larger-size cell
(leukocyte that such as circulating tumor cell and portion size are bigger) focusing effect is preferable, and the cell after focusing focuses on knot at cell
Structure 3 exit position is arranged in a linear in runner central authorities；Outlet with cell focusing structure 3 is connected, by sample cell solution
Circulating tumor cell carry out the cell fence structure 4 that captures, cell fence structure 4 forms trapping region, say, that through meticulous
Circulating tumor cell after born of the same parents' focusing structure 3 focuses on can enter directly in cell fence structure 4, cell fence structure 4
Directly capturing circulating tumor cell, the sample waste after capture can be expelled directly out from waste liquid outlet 5.
From the foregoing, the present invention can be by sample by flow dividing structure 2 for the micro-fluidic chip that circulating tumor cell captures
Product cell solution carries out multi-stage diffluence, to reduce the blocking of sample cell solution, carries out the preparation of abundance for realizing quickly sorting,
Simultaneously under the effect of cell focusing structure 3, linear array in runner central authorities, can directly and quickly be flowed into by circulating tumor cell
In cell fence structure 4, to realize the fast Acquisition to circulating tumor cell.
Additionally, in order to improve this micro-fluidic chip acquisition speed to circulating tumor cell further, above-mentioned cell grid
Being connected by the bus structure 6 in branching shape between outlet and the waste liquid outlet 5 of hurdle structure 4, bus structure 6 is by cell fence
The sample cell waste liquid that structure 4 flows out is collected in waste liquid outlet 5 step by step, say, that can be thin by sample by bus structure 6
Born of the same parents' waste liquid converges step by step, it is to avoid sample cell waste liquid converges suddenly and causes blocking, to realize the quick stream of sample cell solution
Logical, thus improve the acquisition speed to circulating tumor cell further.
Wherein, chance in the once experiment of inventor, when above-mentioned cell fence structure 4 by positive six prisms or just
When the microtrabeculae arrangement of triangular prism forms, in identical pillar gap, under the conditions of identical flow velocity, the narrow gap flow pressure of both pillars
Less, cell is less easily extruded.
It addition, in order to improve the micro-fluidic chip of the present invention capture rate to circulating tumor cell further, as it is shown on figure 3,
Above-mentioned cell fence structure 4 includes inner ring cell fence structure 41 and outer ring cell fence structure 42, wherein, inner ring cell fence
Structure 41 forms core trapping region, and outer ring cell fence structure 42 forms aided capture district；This outer ring cell fence structure 42 wraps
Include interconnecting part 420 that the outlet with cell focusing structure is connected and and the channel part 421 of interconnecting part 420 phase linking, wherein,
This interconnecting part 420 is horn-like progressively to expand along the flow direction of sample cell solution, so, can be easy to improve sample cell molten
The flow velocity of liquid.Channel part 421 is then arranged at the periphery of inner ring cell fence structure 41, so, owing to outer ring cell fence is tied
The circulating tumor cell that the existence of structure 42 is possible to prevent in sample cell solution is not extruded, and plays the effect of duplicate protection, from
And can realize to circulating tumor cell efficient, accurately capture.The import of inner ring cell fence structure 41 is then positioned at interconnecting part 420
Interior and relative with the outlet of cell focusing structure 3, so, in the sample cell solution of the overwhelming majority can be made to flow into rapidly
Circle cell fence structure 41 in, with realize to circulating tumor cell efficient, accurately capture.
Owing to the diameter of circulating tumor cell is between 12～25 μm, the diameter of leukocyte is between 8～14 μm, red blood cell diameter
Between 6～8 μm, and contrasting with circulating tumor cell, erythrocyte and leukocyte are easier to deform upon, and the most above-mentioned inner ring is thin
Born of the same parents' fence structure 41 is made up of the microtrabeculae of 1-3 row's parallel interval arrangement, and the gap between microtrabeculae is 6-8um, above-mentioned outer ring cell
The microtrabeculae that fence structure 42 is made up of 4-6 microtrabeculae arranges arranged in parallel forming, and microtrabeculae row are 30 ° of angle (microtrabeculaes with horizontal direction
Row can reduce fluid to a certain extent and flow out the speed of outer ring cell fence structure in 30 ° of angles, thus decrease cell
It is extruded), the gap between microtrabeculae is 6-8 μm, so, by above-mentioned design, can be by the erythrocyte in sample cell solution
Trapping region is overflowed so that the cell that trapping region is captured number the most greatly is circulating tumor cell, and the accuracy rate of capture is high with leukocyte
Reach more than 95%.
Wherein, above-mentioned flow dividing structure 2 is made up of multi-stage diffluence passage and is distributed in branching shape, in every level shunt passage
Angle between two split channels 21 is 30-45 °, when the angle between two split channels 21 is 30-45 °, it is possible to further
Promote the flow velocity of sample cell solution.Certainly, above-mentioned flow dividing structure 2 be not limited only to described in the present embodiment in crotch
The mode of shape, it would however also be possible to employ the mode of other multi-stage diffluences, as long as can shunt sample cell solution, it is to avoid occurs
The phenomenon of blocking.
As in figure 2 it is shown, the above-mentioned serpentine-like corrugated of cell focusing structure 3, so, under the effect of fluid inertia force,
After snakelike corrugated channel, the cell in fluid can be gathered in the centre position of fluid automatically.Certainly cell focusing structure 3 is also
Can be in the way of using other shapes, as long as being capable of the cell in fluid being gathered in the centre position of fluid automatically i.e.
Can.
Meanwhile, in the present embodiment, in order to improve micro-fluidic chip capture rate of the present invention and capture accuracy further,
Above-mentioned split channel 21 is provided with 16, say, that cell focusing structure 3 and cell fence structure 4 are also provided with
Article 16,.
Embodiment 2
A kind of method capturing circulating tumor cell, comprises the steps:
(1) PBS solution being passed through the micro-fluidic chip in embodiment 1, waste liquid outlet 12 connects 15ml centrifuge tube, opens gas
Press pump, rinses micro-fluidic chip 3min, removes micro-fluidic chip air entrapment；
(2) aqueous solution of 1%P188 is passed through the micro-fluidic chip in embodiment 1, opens sampling pump sample introduction and be coated 3min,
So can reduce the non-specific adsorption on micro-fluidic chip surface in cell and embodiment 1；
(3) blood is diluted after 5 times from, the relatively DABAI cell capture of circulating tumor cell and part cell dia through PBS
The trapping region of micro-fluidic chip to embodiment 1, other blood cells can flow out from waste liquid outlet；
As shown in Figure 4: be 10 by 1ml cell concentration4The H1299 cell suspension of cell/ml is passed through the miniflow in embodiment 1
After control chip, the capture region at micro-fluidic chip is captured, and testing result shows: the H1299-GFP cell quilt of 95%
Trapping region retains.
Embodiment 3
The immunofluorescence dyeing authentication method of a kind of circulating tumor cell, comprises the steps:
(1) by 4% paraformaldehyde to be passed through the micro-fluidic chip in embodiment 1 under 500mbar pressure, sample introduction fixes cell
10min；
(2) by PBS to be passed through the micro-fluidic chip in embodiment 1 under 500mbar pressure, micro-fluidic chip is replaced
In paraformaldehyde solution；
(3) by Triton X-100 that volume fraction is 0.2% to be passed through above-mentioned micro-fluidic core under 500mbar pressure
Sheet, is passed through micro-fluidic chip 10min, to increase the circulating tumor cell film permeability to staining reagent；
(4) by PBS to be passed through the micro-fluidic chip in embodiment 1 under 500mbar pressure, micro-fluidic chip is replaced
In Triton X-100 solution；
(5) take 10ul CK18-FITC, 10ul CD45-PE and 0.5ul concentration is the DAPI of 1mg/ml, the anti-dilution that adds
Liquid is mended to 100ul, obtains diluent, diluent is then passed through above-mentioned micro-fluidic chip, hatches 12 little under conditions of 4 DEG C
Time；
(6) by PBS to be passed through the micro-fluidic chip in embodiment 1 under 500mbar pressure, micro-fluidic chip is replaced
In be not bonded to the antibody staining liquid of cell；
(7) micro-fluidic chip of antibody mediated immunity reaction will be completed in fluorescence microscopy Microscopic observation, record observed result.
It will be apparent to those skilled in the art that can technical scheme as described above and design, make other various
Corresponding change and deformation, and all these change and deformation all should belong to the protection domain of the claims in the present invention
Within.
Claims (10)
1. the micro-fluidic chip being used for capturing circulating tumor cell, it is characterised in that include sample cell solution inlet port, with
The flow dividing structure that sample cell solution inlet port is connected, is shunted step by step by sample cell solution, with the outlet of flow dividing structure
The cell focusing structure being connected, being focused by the cell in sample cell solution, the outlet with cell focusing structure is connected
Logical, the circulating tumor cell in sample cell solution is carried out the cell fence structure that captures, and with cell fence structure
The waste liquid outlet that outlet is connected.
2. micro-fluidic chip as claimed in claim 1, it is characterised in that the outlet of described cell fence structure and waste liquid outlet
Between be connected by the bus structure in branching shape, the sample cell waste liquid that cell fence structure is flowed out by bus structure is step by step
It is collected in waste liquid outlet.
3. micro-fluidic chip as claimed in claim 1, it is characterised in that described cell fence structure is by positive six prisms or positive three
The microtrabeculae arrangement of prism forms.
4. the micro-fluidic chip as described in claim 1 or 2 or 3, it is characterised in that described cell fence structure includes that inner ring is thin
Born of the same parents' fence structure and outer ring cell fence structure；Described outer ring cell fence structure includes being connected with the outlet of cell focusing structure
Logical interconnecting part and the channel part being connected mutually with interconnecting part, described interconnecting part is horn-like along the flow direction of sample cell solution
Progressively expanding, channel part is arranged at the periphery of inner ring cell fence structure, and the import of inner ring cell fence structure is positioned at interconnecting part
Interior and relative with the outlet of cell focusing structure.
5. micro-fluidic chip as claimed in claim 4, it is characterised in that described inner ring cell fence structure is arranged parallel by 1-3
Spaced microtrabeculae is constituted, and the gap between microtrabeculae is 6-8 μm.
6. micro-fluidic chip as claimed in claim 4, it is characterised in that described outer ring cell fence structure is by 4-6 microtrabeculae
The microtrabeculae row of composition are arranged in parallel to be formed, and microtrabeculae row are 20 ° of-40 ° of angles with horizontal direction, and the gap between microtrabeculae is 6-8 μm.
7. micro-fluidic chip as claimed in claim 1, it is characterised in that described flow dividing structure is made up of also multi-stage diffluence passage
Being distributed in branching shape, the angle between two split channels in every level shunt passage is 30-45 °.
8. micro-fluidic chip as claimed in claim 1, it is characterised in that the described serpentine-like corrugated of cell focusing structure.
9. the method capturing circulating tumor cell, it is characterised in that comprise the steps:
(1) use the micro-fluidic chip described in wash buffer claim 1, remove micro-fluidic chip air entrapment；
(2) aqueous solution of Kolliphor P188 is passed through the micro-fluidic chip described in claim 1, is coated 2-10min；
(3) test fluid being passed through the micro-fluidic chip described in claim 1, be circulated tumor cell capture, circulating tumor is thin
Born of the same parents and part leukocyte capture to cell fence structure, and other blood cells flow out from waste liquid outlet.
10. the immunofluorescence dyeing authentication method of a circulating tumor cell, it is characterised in that comprise the steps:
(1) fixative is passed through the micro-fluidic chip described in claim 1, after sample introduction, fixes cell 10-30min；
(2) buffer is passed through the micro-fluidic chip described in claim 1, replaces the fixative in micro-fluidic chip；
(3) Triton X-100 is passed through the micro-fluidic chip 10-30min described in claim 1；
(4) buffer is passed through the micro-fluidic chip described in claim 1, replaces the TritonX-100 in micro-fluidic chip molten
Liquid；
(5) CK18-FITC, CD45-PE and DAPI are taken, the anti-diluent that adds, obtain diluent, then diluent is passed through right and wants
Seek the micro-fluidic chip described in 1, hatch under conditions of 4-37 DEG C 2-12 hour；
(6) buffer is passed through the micro-fluidic chip described in claim 1, replaces and micro-fluidic chip is not bonded to the anti-of cell
Body dyeing liquor, dyes complete, identifies under microscope.
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