CN105062866A - Disposable separating chip module for peripheral blood circulating tumor cell and application method of disposable separating chip module - Google Patents
Disposable separating chip module for peripheral blood circulating tumor cell and application method of disposable separating chip module Download PDFInfo
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Classifications
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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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- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention provides a disposable separating chip module for a peripheral blood circulating tumor cell and an application method of the disposable separating chip module. The disposable separating chip module comprises a micro-fluidic chip, a blood sample storage tank, a PBS sample storage tank, a waste liquid storage tank, an enrichment recycling liquid outlet, a connecting guide pipe (1) of the micro-fluidic chip and the blood sample storage tank, a connecting guide pipe (2) of the micro-fluidic chip and the PBS sample storage tank, a connecting guide pipe (3) of the micro-fluidic chip and the waste liquid storage tank, and a connecting guide pipe (4) of the micro-fluidic chip and the enrichment recycling liquid outlet. The invention further provides the application method of the disposable separating chip module. The micro-fluidic chip adopts a dual-inlet and dual-outlet structure and has an erythrocyte removing efficiency of 100%. A clinical blood sample requires no pretreatment process of attenuation or erythrocyte lysis, so that plenty of time for separating operation can be saved, and the activity of the circulating tumor cell after separation and enrichment can be effectively guaranteed.
Description
Technical field
The invention belongs to lesion detection technical field, particularly relate to the disposable separating chips module for Peripheral Circulation tumour cell and using method thereof.
Background technology
Circulating tumor cell (CTCs) is the tumour cell entering peripheral blood from tumour primary lesion or metastatic lesion, detect circulating tumor cell significant to prevention and control of cancer, the rapid evaluation, individualized treatment etc. of external early diagnosis, chemotherapeutics can be effectively applied to.For the conventional sense means of tumour, such as iconography, tumour, when being less than one centimeter, is difficult to confirm as exception.Can be seen by research both at home and abroad, a lot of tumour has had tumour cell to enter blood circulation when 2-4 millimeter, and from this angle, it has immeasurable meaning for early diagnosis.The advantage of blood serum tumor markers detection method can be imaging diagnosis provide reference data, low cost and be applicable to popular examination, and shortcoming is the mark not having high degree of specificity, often occurs false positive and false negative result; The advantage of histopathology is beneficial to make a definite diagnosis, and shortcoming has the inspection of wound property, can not be used for detecting and for the INFORMATION OF INCOMPLETE of transfer.Compare above traditional method, the transfer index that advantage is non-dipped type, susceptibility is high, good, the rapid evaluation of chemotherapy and radiation effect, prognosis evaluation, the detection recurrence of circulating tumor cell detection method, expense is low and be applicable to popular examination, and therefore circulating tumor cell detection method has broad application prospects in clinical tumor prevention and control field.Easy not Nikon (Immunicon) company through U.S. FDA certification passes through scientific research for many years, a large amount of scientific researches has been done at a hundreds of patient, finally reach a conclusion, after the treatment by detecting the number of circulating tumor cell, its prognosis situation of determination patient that can be clear and definite.
At present, the CellSearch that American I mmunicon company researches and develops is only had
tMsystem becomes a standard protocols, in 2004 by the clinical application of FDA (Food and Drug Adminstration) (FDA) access.This is a sorting detection technique summarizing immune magnetic sorting technology and immunocytochemical method, identifying specifically, in conjunction with target cell, completing the screening of tumour cell under the effect of externally-applied magnetic field by fixing EpCAM antibody on magnetic bead.But the shortcoming of the method is complicated operation, need multistep to complete, be in the circulating tumor cell of leaf phase for some, the expression of its EpCAM surface antigen is very low even not to be expressed, and therefore the method is difficult to the existence this class circulating tumor cell being detected.
Determinacy lateral shift (DLD) refers to that the bulkrage collision rift generation lateral shift exceeding critical radius in micro-pillar array is converged to side, and small volume material flows through the phenomenon of array according to its former flow direction.Based on the physical property of CTCs cell size (12 ~ 30 μm) with hemocyte size (5 ~ 15 μm) difference, the DLD array micro-fluidic chip of sorting enrichment critical radius between CTCs and hemocyte can be designed and produced.The people such as Du Jinghui (Du Jinghui, Liu Zongbin, peep. the micro-fluidic chip of sorting enrichment cycles tumour cell fast. Chinese Journal of Health Laboratory Technology, 06 phase in 2013 .) set up trilateral microtrabeculae determinacy lateral shift array micro-fluidic chip, chip by a tunnel inlets, central enrichment runner exit, both sides non-lipid mass flow pathway export and specular trilateral microtrabeculae display form, trilateral microtrabeculae DLD array micro-fluidic chip can realize 2ml/min, 95% high-recovery human peripheral blood tumour cell sorting.Because this research adopts single entrance chip, be difficult to realize high-recovery, and blood needs to carry out decaploid and amasss dilution, disengaging time can extend greatly; After the blood diluted is passed into chip, red corpuscle and white corpuscle are evenly distributed in whole chip, so it is the same that red corpuscle collects mouth ratio out with the Dilution ratio of blood from pregnant solution, inside the collection liquid finally obtained, erythrocytic concentration is identical with the concentration of hemodilution.Very large impact can be caused on observation containing red corpuscle in pregnant solution, affect the purity of circulating tumor cell, and then analysis and research are carried out to circulating tumor cell cause disadvantageous effect.
Summary of the invention
The present invention is directed to the above-mentioned defect of prior art, be provided for disposable separating chips module and the using method thereof of Peripheral Circulation tumour cell, the circulating tumor cell that can realize in human peripheral blood carries out quick, accurate, highly active separation and concentration.
The present invention adopts following technical scheme: for the disposable separating chips module of Peripheral Circulation tumour cell, comprise micro-fluidic chip, blood sample liquid storage tank, PBS(phosphate buffered saline buffer) linking conduit that exports of sample liquid storage tank, waste liquid liquid storage tank, the outlet of enriching and recovering liquid, the linking conduit of micro-fluidic chip and blood sample liquid storage tank, the linking conduit of micro-fluidic chip and PBS sample liquid storage tank, the linking conduit of micro-fluidic chip and waste liquid liquid storage tank and micro-fluidic chip and enriching and recovering liquid;
Described micro-fluidic chip is based on the design of determinacy lateral shift array principle.
Preferably, the width of described micro-fluidic chip is 3-5mm, and length is 5-7cm; The critical radius of micro-fluidic chip is 6.5-8.5 μm.
Preferably, the width of described micro-fluidic chip is 4mm, and length is 6cm.
Preferably, described micro-fluidic chip comprises one and supplies the pipeline of blood circulation and multiple structural unit, the center line of described pipeline is parallel with flow direction, and the microtrabeculae of described structural unit to be cross-sectional shape be equilateral triangle, described microtrabeculae is axle angularly symmetric offset spread centered by the center line of described pipeline.
Preferably, the trilateral of described trilateral microtrabeculae has a limit vertical with the direction of fluid.
Preferably, micro-fluidic chip runner height is 30-100 μm; The length of side of equilateral triangle is 20-40 μm.
Preferably, micro-fluidic chip runner height is 75 μm; The length of side of equilateral triangle is 30 μm.
Preferably, described microtrabeculae is divided into multirow, is projected as 30-50 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, and the spacing of adjacent rows microtrabeculae is projected as 45-70 μm, the wherein middle line parallel of x-axis and described pipeline in y-axis.
Preferably, be projected as 40 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, the spacing of adjacent rows microtrabeculae is projected as 58 μm in y-axis.
Preferably, the pitch angle of described micro-pillar array is 1-5 degree.
Preferably, the pitch angle of described micro-pillar array is 3.1 degree.
Preferably, described micro-fluidic chip adopts two entrance structure, is blood sample entrance and PBS buffer inlet respectively.
Blood and PBS damping fluid enter micro-fluidic chip from blood sample entrance and PBS buffer inlet respectively, flow into after meeting in micro-fluidic chip region and can form three fluid streams regions, both sides are blood flow region, centre is PBS buffer fluid region, the reason forming this phenomenon is because fluid can not mix between fluid under the state of laminar flow, so the cell being only greater than micro-fluidic chip critical size just can flow into PBS buffer fluid region from blood flow region, and red corpuscle is a kind of cell of offset flat shape, no matter be that diameter or width are all little than critical size, so only region can be flowed through at red corpuscle, therefore, disposable separating chips module of the present invention is removed erythrocytic efficiency and can be reached 100%.
Preferably, described micro-fluidic chip adopts two outlet liquid collecting structure, be the outlet after intermediate flow road junction is connected with chip both sides respectively, be separated the pregnant solution obtained and flow out from the enriching and recovering liquid outlet of intermediate flow channel, the waste liquid port after waste liquid then connects from chip both sides flows out.
The material that disposable separating chips module uses is COC plastics, wherein substrate is made up of two-layer COC thin plate, the working method of injection moulding is adopted to obtain one piece with unicom pipeline and the COC thin plate with extraneous connection outlet, then shaping through the sealing-in of thermal bonding mode with the COC thin plate of another block surfacing, each liquid storage tank of substrate inner Shi You pipeline UNICOM is connected with micro-fluidic chip interface.
Using method of the present invention is as follows:
(1) blood sample to be measured and PBS damping fluid are added blood sample liquid storage tank and PBS sample liquid storage tank respectively, the outlet of enriching and recovering liquid is placed 1.5ml sterile centrifugation tube and is received enriching and recovering liquid;
(2) disposable separating chips module is put into the Module-embedding mouth of separometer, air pressure control interface respectively with blood sample liquid storage tank and PBS sample liquid storage tank Butt sealing;
(3) instrument is opened, micro-fluidic chip starts processing blood, realize the concentration and separation of circulating tumor cell according to the size of cell dia, concentration and separation liquid flows out from concentration and separation outlet and accesses 1.5ml sterile centrifugation tube, and the waste liquid after separation can be stored in waste liquid liquid storage tank.
Preferably, adopt air pressure sample introduction control blood sample and PBS damping fluid to pass into the mode of micro-fluidic chip, inlet pressure is 1000-2000mbars.
H1299 cell is joined the blood of simulating cancer patient in phosphate buffer soln or normal human blood, detected result shows: the H1299 cell of about 100% is by concentration and separation, improve 50 times before H1299 cell concn after separation is comparatively separated, detection speed reaches 2 milliliters of per minutes.
The invention has the beneficial effects as follows: the micro-fluidic chip adopting two inlet fluid design, compare the micro-fluidic chip only having blood entry port, there is separating effect more clearly, double-entry micro-fluidic chip reaches 100% in the erythrocytic efficiency of removal, and the micro-fluidic chip of single entrance can only reach 90% at most, and double-entry micro-fluidic chip can not produce any impact to the recovery of target cell.
Compared with the circular configuration adopting the array structure of equilateral triangle and general document to adopt, its critical size of separation is more accurate, and the cell on the critical size of design can realize the recovery of more than 95%; Micro-fluidic chip adopts trilateral micro-column structure, and trilateral microtrabeculae has a limit vertical with horizontal direction.The result calculated through fluid simulation draws, the triangular array of vertical direction can form a more symmetrical velocity field, and the array of equilateral triangle limit and horizontal direction parallel then can form an asymmetrical velocity field.So adopt the triangular array of vertical direction can be better than the triangular array of horizontal direction, the cell on the critical size of design can realize the recovery of more than 98%.
Adopt disposable separating chips module, the benefit brought is easy to use, quick, simple, as long as change disposable separating chips module when processing different clinical blood sample, accomplishes zero cross-contamination.
The speed of this cover instrument system process clinical blood sample can reach a milliliter per minute rank, and clinical blood sample is without the need to dilution or the pretreatment process of splitting erythrocyte, a large amount of lock out operation time can be saved, the activity of effective guarantee separation and concentration Posterior circle tumour cell.
The present invention adopts disposable separating chips modular operation, and convenience provides possibility for clinical medical application.The material that disposable separating chips module of the present invention uses is COC plastics, greatly reduces chip manufacturing cost, greatly can reduce the testing cost of Peripheral Circulation tumour cell like this, for the health check-up project being generalized to normal people establishes cost basis.
Accompanying drawing explanation
Fig. 1 is instrument one-piece construction figure of the present invention;
Fig. 2 is disposable separating chips module of the present invention;
Fig. 3 is micro-fluidic chip cut-away view of the present invention;
Fig. 4 is the real work situation of the two inlet microfluidic control chip of the present invention;
Fig. 5 is the direction of micro-fluidic chip inside triangle microtrabeculae;
Fig. 6 is that trilateral microtrabeculae direction is to the analog result of divided fluid stream;
Fig. 7 is the recovering state utilizing micro-fluidic chip of the present invention to export the H1299 cell enrichment Ethylene recov adding normal blood to;
Fig. 8 utilizes micro-fluidic chip of the present invention to the recovering state of H1299 cell waste liquid port adding normal blood to;
Fig. 9 is the projection of microtrabeculae spacing in x-axis and y-axis in micro-fluidic chip of the present invention.
Reference numeral:
The disposable separating chips module of 1-; 2-Pneumatic controller system;
11-micro-fluidic chip; 12-blood sample liquid storage tank;
13-PBS sample liquid storage tank; 14-waste liquid liquid storage tank;
15-enriching and recovering liquid exports; 111-blood sample entrance;
112-PBS buffer inlet; 113-waste liquid port;
Q-flow direction.
Embodiment
Embodiment 1
As Figure 1-4, the disposable separating chips system for Peripheral Circulation tumour cell of the present invention, comprises disposable separating chips module 1 and Pneumatic controller system 2.As Figure 2-3, described disposable separating chips module 1 comprises micro-fluidic chip 11, blood sample liquid storage tank 12, PBS sample liquid storage tank 13, waste liquid liquid storage tank 14, enriching and recovering liquid outlet 15, the linking conduit of micro-fluidic chip 11 and blood sample liquid storage tank 12, the linking conduit of micro-fluidic chip 11 and PBS sample liquid storage tank 13, the linking conduit of micro-fluidic chip 11 and waste liquid liquid storage tank 14 and micro-fluidic chip 11 and exports the linking conduit of 15 with enriching and recovering liquid.The material of disposable separating chips module is COC, wherein substrate is made up of two-layer COC thin plate, the working method of injection moulding is adopted to obtain one piece with unicom pipeline and the COC thin plate with extraneous connection outlet, then shaping through the sealing-in of thermal bonding mode with the COC thin plate of another block surfacing, each liquid storage tank of substrate inner Shi You pipeline UNICOM is connected with micro-fluidic chip interface.Wherein blood sample liquid storage tank 12 is connected with Pneumatic controller system sealing as shown in Figure 1 with PBS sample liquid storage tank 13, realizes stable sample introduction pressure.
Described micro-fluidic chip comprises one and supplies the pipeline of blood circulation and multiple structural unit, the center line of described pipeline is parallel with flow direction, the microtrabeculae of described structural unit to be cross-sectional shape be equilateral triangle, described microtrabeculae is axle angularly symmetric offset spread centered by the center line of described pipeline.
Described micro-fluidic chip is two entrance structures, is blood sample entrance 111 and PBS buffer inlet 112 respectively.Waste liquid after micro-fluidic chip 11 and enriching and recovering liquid flow out from waste liquid port 113 and enriching and recovering mouth 15 respectively, and wherein the outlet of enriching and recovering liquid adopts independent interface to be received in the container of user by enriching and recovering liquid.
As Fig. 4, blood and PBS damping fluid enter micro-fluidic chip from blood sample entrance 111 and PBS buffer inlet 112 respectively, flow into after meeting in micro-fluidic chip region and can form three fluid streams regions, both sides are blood flow region, centre is PBS buffer fluid region, the reason forming this phenomenon is because fluid can not mix between fluid under the state of laminar flow, so the cell being only greater than micro-fluidic chip critical size just can flow into PBS buffer fluid region from blood flow region, and red corpuscle is a kind of cell of flats, no matter be that diameter or width are all little than critical size, so only region can be flowed through at red corpuscle, therefore, disposable separating chips module of the present invention is removed erythrocytic efficiency and can be reached 100%.
The width of described micro-fluidic chip is 3mm, and length is 5cm; The critical radius of described micro-fluidic chip is 6.5-8.5 μm; Micro-fluidic chip runner height is 30 μm; The length of side of equilateral triangle is 20 μm; As shown in Figure 9, described microtrabeculae is divided into multirow, is projected as 30 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, and the spacing of adjacent rows microtrabeculae is projected as 45 μm, the wherein middle line parallel of x-axis and described pipeline in y-axis; The pitch angle of described micro-pillar array is 1 degree.
Embodiment 2
For the disposable separating chips module of Peripheral Circulation tumour cell, wherein, micro-fluidic chip 11 comprises one and supplies the pipeline of blood circulation and multiple structural unit, the center line of described pipeline is parallel with flow direction, the microtrabeculae of described structural unit to be cross-sectional shape be equilateral triangle, described microtrabeculae is axle angularly symmetric offset spread centered by the center line of described pipeline.
Micro-fluidic chip uses material to be COC, and the processing mode of employing injection moulding obtains the runner layer with array microtrabeculae, realizes irreversible fixation by the COC substrate of heat-sealing connection and another block flat surface.
The width of described micro-fluidic chip is 4mm, and length is 6cm; The critical radius of described micro-fluidic chip is 6.5-8.5 μm; Micro-fluidic chip runner height is 75 μm; The length of side of equilateral triangle is 30 μm; Described microtrabeculae is divided into multirow, is projected as 40 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, and the spacing of adjacent rows microtrabeculae is projected as 58 μm, the wherein middle line parallel of x-axis and described pipeline in y-axis; The pitch angle of described micro-pillar array is 3.1 degree.
Embodiment 3
For the disposable separating chips module of Peripheral Circulation tumour cell, wherein, micro-fluidic chip 11 comprises one and supplies the pipeline of blood circulation and multiple structural unit, the center line of described pipeline is parallel with flow direction, the microtrabeculae of described structural unit to be cross-sectional shape be equilateral triangle, described microtrabeculae is axle angularly symmetric offset spread centered by the center line of described pipeline.
The width of described micro-fluidic chip is 5mm, and length is 7cm; The critical radius of described micro-fluidic chip is 6.5-8.5 μm; Micro-fluidic chip runner height is 100 μm; The length of side of equilateral triangle is 40 μm; Described microtrabeculae is divided into multirow, is projected as 50 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, and the spacing of adjacent rows microtrabeculae is projected as 70 μm, the wherein middle line parallel of x-axis and described pipeline in y-axis; The pitch angle of described micro-pillar array is 5 degree.
Embodiment 4
As shown in Figure 5, Q is flow direction, and the trilateral of trilateral microtrabeculae of the present invention has a limit vertical with the direction of fluid (as Fig. 5 b), and the trilateral that comparative example is taked have limit parallel with flow direction (as Fig. 5 a).
As shown in Figure 6, the result calculated through fluid simulation draws, the a leg-of-mutton limit of the present invention triangular array vertical with flow direction can form a more symmetrical velocity field (Fig. 6 a), the leg-of-mutton limit triangular array parallel with flow direction then can form an asymmetrical velocity field (Fig. 6 b), therefore, the leg-of-mutton limit triangular array vertical with fluid flow direction is adopted can be better than the triangular array of horizontal direction.
Embodiment 5
Using method of the present invention is as follows:
(1) blood sample to be measured and PBS damping fluid are added blood sample liquid storage tank 12 and PBS sample liquid storage tank 13 respectively, place 1.5ml sterile centrifugation tube in enriching and recovering liquid outlet 15 and receive enriching and recovering liquid;
(2) disposable separating chips module 1 is put into the Module-embedding mouth of separometer, air pressure control interface respectively with blood sample liquid storage tank 12 and PBS sample liquid storage tank 13 Butt sealing;
(3) instrument is opened, micro-fluidic chip 11 starts processing blood, the concentration and separation of circulating tumor cell just can be realized according to the size of cell dia, concentration and separation liquid flows out from concentration and separation outlet 15 and accesses 1.5ml sterile centrifugation tube, and the waste liquid after separation can be stored in waste liquid liquid storage tank 15.
Wherein, adopt air pressure sample introduction control blood sample and PBS damping fluid to pass into the mode of micro-fluidic chip, inlet pressure is 1000-2000mbars.
As shown in Figure 7; In figure, white represents the H1299 cell after fluorescent dye, and by experiment, H1299 cell is joined the blood of simulating cancer patient in phosphate buffer soln or normal human blood, Fig. 8 is the recovering state of waste liquid port, does not substantially observe H1299 cell; Detected result shows: the H1299 cell of 98%, by concentration and separation, improves more than 50 times before the H1299 cell concn after separation is comparatively separated, and detection speed is 2 milliliters of per minutes.
The disposable separating chips module for Peripheral Circulation the tumour cell above embodiment of the present invention provided and using method thereof, be described in detail, apply specific case herein to set forth principle of the present invention and embodiment, the explanation of above embodiment just understands method of the present invention and core concept thereof for helping; Meanwhile, for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.
Claims (10)
1. for the disposable separating chips module of Peripheral Circulation tumour cell, it is characterized in that, comprise the linking conduit that micro-fluidic chip, blood sample liquid storage tank, PBS sample liquid storage tank, waste liquid liquid storage tank, the outlet of enriching and recovering liquid, the linking conduit of micro-fluidic chip and blood sample liquid storage tank, the linking conduit of micro-fluidic chip and PBS sample liquid storage tank, the linking conduit of micro-fluidic chip and waste liquid liquid storage tank and micro-fluidic chip and enriching and recovering liquid export.
2. disposable separating chips module according to claim 1, is characterized in that, the width of described micro-fluidic chip is 3-5mm, and length is 5-7cm; The critical radius of micro-fluidic chip is 6.5-8.5 μm.
3. disposable separating chips module according to claim 1, it is characterized in that, described micro-fluidic chip comprises one and supplies the pipeline of blood circulation and multiple structural unit, the center line of described pipeline is parallel with flow direction, the microtrabeculae of described structural unit to be cross-sectional shape be equilateral triangle, described microtrabeculae is axle angularly symmetric offset spread centered by the center line of described pipeline.
4. disposable separating chips module according to claim 3, is characterized in that, the trilateral of described trilateral microtrabeculae has a limit vertical with the direction of fluid.
5. disposable separating chips module according to claim 3, is characterized in that, described micro-fluidic chip runner height is 30-100 μm; The length of side of equilateral triangle is 20-40 μm.
6. disposable separating chips module according to claim 3, it is characterized in that, described microtrabeculae is divided into multirow, is projected as 30-50 μm with the spacing of the adjacent microtrabeculae of a line in x-axis, the spacing of adjacent rows microtrabeculae is projected as 45-70 μm, the wherein middle line parallel of x-axis and described pipeline in y-axis.
7. disposable separating chips module according to claim 3, is characterized in that, the pitch angle of described micro-pillar array is 1-5 degree.
8. disposable separating chips module according to claim 1, is characterized in that, described micro-fluidic chip adopts two entrance structure, is blood sample entrance and PBS buffer inlet respectively; Described micro-fluidic chip adopts two outlet liquid collecting structure, is the outlet after intermediate flow road junction is connected with chip both sides respectively, and be separated the pregnant solution obtained and flow out from the enriching and recovering liquid outlet of intermediate flow channel, the waste liquid port after waste liquid then connects from chip both sides flows out.
9. the using method of the disposable separation module described in the arbitrary claim of claim 1-8, is characterized in that, comprise the following steps:
(1) blood sample to be measured and PBS damping fluid are added blood sample liquid storage tank and PBS sample liquid storage tank respectively, the outlet of enriching and recovering liquid is placed 1.5ml sterile centrifugation tube and is received enriching and recovering liquid;
(2) disposable separating chips module is put into the Module-embedding mouth of separometer, air pressure control interface respectively with blood sample liquid storage tank and PBS sample liquid storage tank Butt sealing;
(3) instrument is opened, micro-fluidic chip starts processing blood, realize the concentration and separation of circulating tumor cell according to the size of cell dia, concentration and separation liquid flows out from concentration and separation outlet and accesses 1.5ml sterile centrifugation tube, and the waste liquid after separation can be stored in waste liquid liquid storage tank.
10. the using method of disposable separating chips module according to claim 9, is characterized in that, adopt air pressure sample introduction control blood sample and PBS damping fluid to pass into the mode of micro-fluidic chip, inlet pressure is 1000-2000mbars.
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