CN103018224B - Based on rare cells separation detecting system and the method for centrifugal microfluidic control techniques - Google Patents

Abstract

The invention discloses a rare cell separation and detection system and method based on centrifugal microfluidic technology. The system includes a microfluidic chip similar to an optical disc, a centrifugal drive module and an optical detection module. Among them, the microfluidic chip includes multiple sets of radially arranged micropipes and microcavities. The overall structure of the chip consists of an elastic micropillar guide rail layer, a deformable film layer, a pipe/cavity layer, a filter membrane layer and a waste liquid collection layer. When in use, first introduce the sample solution and immunomodified microspheres into its liquid storage chamber through the microfluidic chip inlet, and place them on the centrifugal platform of the centrifugal drive module, assemble the elastic microcolumn, and rotate at a low speed. Realize the full mixing and reaction of the sample liquid and the immunomodified microbead liquid in the liquid storage chamber, and then rotate the chip at high speed for separation; then drop the specifically recognized fluorescently labeled antibody solution in the collection area of each separated cell, incubate the reaction, and add the buffer and centrifuged; finally, identified and analyzed by an optical detection module.

Description

Rare cell separation and detection system and method based on centrifugal microfluidic technology

technical field

The invention relates to a rare cell separation and detection system based on centrifugal microfluidic technology and its application method, which can be applied to the fields of non-invasive prenatal diagnosis, early tumor diagnosis and prognosis monitoring, stem cell research and the like.

Background technique

Selective isolation of rare cells from complex mixtures is of great significance in both cell biology and clinical aspects, such as isolation of fetal red blood cells from maternal peripheral blood, isolation of circulating tumor cells (Circulating Tumor Cell, CTC) from peripheral blood of patients, and bone marrow or Separation of stem cells in tissues, etc. The analysis of these cells can often provide very valuable information for clinical diagnosis and treatment of diseases, but the content of such cells in samples is very rare, usually only 1-10 cells/mL, separation and extraction very difficult, thus greatly limiting its clinical application. Traditional rare cell separation and enrichment techniques mainly include density gradient centrifugation, filtration separation, and immunomagnetic bead sorting. Among them, the density gradient centrifugation method and the filtration separation method both use the differences in the physical characteristics of various cells (such as density and size) to screen and enrich target cells, but this type of separation and enrichment method based on the physical characteristics of cells is relatively specific. Poor, the purity of the obtained samples is low, and the risk is that it often leads to errors in disease diagnosis. Immunomagnetic bead sorting is currently the most commonly used method for the separation and enrichment of rare cells. Its basic principle is to use the specific binding of rare cell surface antigens to monoclonal antibodies connected to immunomagnetic beads, and realize the separation of rare cells under the action of an external magnetic field. Separation and enrichment. This method combines the high specificity of antigen-antibody reaction with the enrichment and separation of immunomagnetic beads. It is simple, sensitive and fast, and has high separation purity and high yield without affecting the activity of the separated cells. However, there are still many deficiencies in the application of this method to the enrichment of rare cells. On the one hand, traditional separation methods based on magnetic force often use centrifuge tubes. Compared with the magnetic force on the magnetic beads, the average sedimentation path is longer (several mm~1cm), so the complete sedimentation of the magnetic bead-cell complex It takes a long time, the separation efficiency is low, and because the magnetic field force on the magnetic beads is relatively weak, which is basically the same order of magnitude as the fluid shear force, it is easy to cause some magnetic beads or magnetic bead-cell complexes to be carried by the washing solution. In addition, during the detection process, the isolated and enriched cells often need to be transferred to a new container or plate for detection, which often easily causes some target cells to be missed due to transfer loss. Therefore, in order to increase and improve the specificity and sensitivity of rare cell separation detection, many research institutions and enterprises at home and abroad are committed to researching new rare cell separation and enrichment methods and equipment, especially rare cell separation based on microfluidic technology. The method has developed rapidly in recent years. The most striking of these is the CTC separation method based on microfluidic technology reported by Harvard Medical School, which successfully separated and detected a small amount of CTC in tumor patients using microfluidic chip technology [Nagrath S, Sequist LV, Maheswaran S, et al.Isolation of rare circulating tumor cells in cancer patients by microchip technology.Nature.2007,450:1235-1241.], the capture efficiency of tumor cells is as high as 99%, and the separation purity is about 50%. The microfluidic chip used uses silicon as the substrate, and is processed by photolithography, plasma deep etching, bonding and other processes. The chip contains 78,000 silicon-based microcolumns. , so that when the blood flows through the microcolumn, the CTCs contained in it are immunocaptured, and finally the CTCs are labeled and counted in situ by the immunofluorescence method to realize the detection of the CTCs. Since this method does not require pretreatment of whole blood samples, and the operation process is simple and gentle, the damage to cells is relatively small, and the cumbersome steps of separation and purification and repeated centrifugation and washing are avoided, and the detection sensitivity is high, showing a good application prospect. Nevertheless, this method still has major deficiencies: on the one hand, in order to ensure the capture efficiency of cells, the blood sample must maintain a relatively slow speed (~1mL /h), so the detection process of this method takes a long time (5-7 hours), and the efficiency is low. On the other hand, the chip used is made of opaque silicon base, which is not conducive to optical detection, and the plasma etching process of the micropillars in the chip requires expensive instruments and high manufacturing costs, which limits its large-scale clinical application. Although, some people have tried to improve this method recently [Wang S, Liu K, Liu J, et al.Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers.Angew.Chem.Int.Ed.2011,50: 3084–3088.], such as optimizing the capture efficiency of CTCs in microfluidic chips by adjusting the spacing and arrangement of immune capture microcolumns or adding oblique ridge microstructures in microchannels, but the improvement effect is limited, and it still cannot meet the requirements of efficient capture and fast capture at the same time. Separation requirements. Therefore, it is urgent to develop a system that is easy to operate and can achieve rapid and efficient isolation of rare cells. Thereby become the conception of the present invention.

Contents of the invention

The purpose of the present invention is to provide a rare cell separation and detection system based on centrifugal microfluidic technology and its use method. The separation and detection system provided has the characteristics of simple operation, high degree of automation, and rapid and efficient separation of rare cells. It is applied to the efficient separation of rare cells such as fetal red blood cells in maternal peripheral blood, circulating tumor cells in peripheral blood of patients, and stem cells in human tissues.

The present invention provides a rare cell separation and detection system based on centrifugal microfluidic technology, which is characterized in that: the system includes a microfluidic chip 1 similar to an optical disc, a centrifugal drive module 2 and an optical detection system of a separation detection system Module 3. This system utilizes the rotation of the centrifugal platform 4 on the centrifugal module 2, so that the compressed elastic microcolumn 6 fixed on its fixed base 5 periodically squeezes the rubber film 15 on the deformation chamber 9 on the chip that communicates with the liquid storage chamber 7, Fully mixing and rapid reaction of the sample solution and immunomodified microspheres in the liquid storage chamber are realized. At the same time, the centrifugal force generated by the high-speed rotation of the chip is combined with the filter membrane layer 17 integrated in the separation chamber 8 of the chip to achieve efficient separation of immune capture cells.

Specifically, the appearance of the microfluidic chip of the separation detection system is disc-shaped, as shown in Figure 4, and consists of multiple structural layers, including elastic micropillar guide rail layer 14, deformable film layer 15, pipe/cavity Layer 16, filter membrane layer 17 and waste liquid collection layer 18; wherein the pipe/cavity layer 16 of the chip includes a liquid storage chamber 7 and a group of separation chambers 8, and each separation chamber is radially periodic around the liquid storage chamber Arrangement; each separation chamber is fan-shaped, and the end far away from the center of the chip shrinks into a semicircular microcavity, which serves as the separation cell collection area 10. This design of gathering the separation cells in several specific areas avoids the subsequent detection process The tedious and time-consuming large-area microscopic search operation; and each cell collection area has an injection port above it, which is used for loading fluorescently-labeled specific antibody solution during cell identification, during chip rotation and incubation reaction, The sample inlet above each cell collection area is sealed with adhesive tape 13 to prevent the loss of sample solution during chip rotation and incubation. Moreover, this method of directly loading the identification reaction solution in the separation cell collection area can greatly reduce the volume of the precious fluorescently labeled antibody solution required in the identification process and reduce the detection cost; a set of micro The pipe is connected. On the one hand, the micropipe serves as a fluid channel to ensure the transfer of fluid and cells from the liquid storage chamber to the separation chamber. At the same time, it also serves as a capillary valve to temporarily restrict the sample liquid in the liquid storage chamber under low driving pressure; the liquid storage chamber is connected to a set of The through hole includes a sample inlet 11, a vent 12 and at least one deformation chamber 9; the top layer of the deformation chamber is a rubber film 15, which is clamped between the chip elastic microcolumn guide rail layer 14 and the pipeline/cavity layer 16 , elastic deformation can occur under pressure, so the volume of the deformation chamber can be changed by mechanically squeezing the rubber film on the deformation chamber; as shown in Figure 5, the compressive elastic microcolumn 6 installed on the fixed base of the centrifugal module can provide Mechanical pressure, due to the limitation of the elastic micro-column guide layer 14 guide groove on the chip, the compressed elastic micro-column only displaces in the vertical direction, that is, it only exerts vertical pressure when it contacts the chip. When the chip rotates with the centrifugal platform, The compressed elastic micropillars periodically contact the rubber film 15 on each deformation cavity 9, thereby periodically squeezing the deformation cavity, causing the fluid in the liquid storage cavity connected to the deformation cavity to undergo an oscillating motion, and accelerating the fluid mixing reaction process therein; The filter membrane layer 17 of the fluidic chip is a layer of polymer film processed with a micropore array, sandwiched between the chip pipeline/cavity layer and the waste liquid collection layer, with a pore size of 8-12 μm to ensure that it can block the binding immune Target cells behind the microspheres; the waste liquid collection layer 18 of the microfluidic chip includes an annular microcavity processed with a microcolumn array, and the microcolumn array is used as a support structure to prevent the filter membrane from collapsing; in order to ensure that the waste liquid collection chamber can Completely accommodate the separation and reaction waste liquid, the outer diameter of the microcavity should be greater than the distance between the cell collection area of the pipe/cavity layer of the chip and the center of the chip; the elastic microcolumn guide rail layer, pipe/cavity layer and waste liquid The collection layer can be made of glass, PDMS (Polydimethylsiloxane, polydimethylsiloxane), PS (polystyrene, polystyrene), PC (Polycarbonate, polycarbonate), COC (Cyclic olefin copolymer, cycloolefin copolymer ), PMMA (Polymethyl methacrylate), any one of SU-8. In addition, the filter membrane material of the microfluidic chip can be PDMS (Polydimethylsiloxane, polydimethylsiloxane), PS (polystyrene, polystyrene), PC (Polycarbonate, polycarbonate), COC (Cyclic olefin copolymer, Any one of cycloolefin copolymer), PMMA (Polymethyl methacrylate, polymethyl methacrylate), PI (Polyimide, polyimide), PA (Parylene, polyparaxylylene), SU-8.

The centrifugal drive module of the separation detection system consists of a rotary motor 4 , a fixed base 5 and a group of elastic microcolumns 6 . Among them, the rotating motor 4 is a program-controlled speed-regulating motor; the fixed base 5 is used to provide support for a group of elastic micro-column structures 6; the lower end of the elastic micro-column is a smooth spherical shape, which ensures that the elastic micro-column and the guide rail groove and the deformable membrane during the rotation process The contact will not hinder the rotation of the chip; under the action of vertical force, the elastic micro-column can move up and down, and by properly adjusting its height, the pressure generated by the spring deformation of the elastic micro-column can ensure that the lower end of the elastic micro-column is in contact with the groove of the chip guide rail layer. When the chip rotates until the deformation chamber 9 is vertically aligned with the elastic micro-pillar, the compressed elastic micro-pillar will exert pressure on the deformable membrane 15 on the top layer of the deformation chamber to deform it; the elastic micro-pillar passes through a liftable support Fixed on a fixed base, when the support rises, the elastic micro-pillars will not squeeze the deformation cavity of the chip, but when the support descends, the elastic micro-pillars will squeeze the deformation cavity of the chip through elastic force, assisting storage The fluid in the liquid chamber mixes rapidly.

The optical detection module of the separation detection system is composed of a group of lenses 3 that can excite and detect fluorescence. These lenses are respectively located on the top and bottom of the chip. The drive module drives the rotation of the chip to realize in-situ automatic detection of isolated cells, avoiding the loss of captured rare cells during the transfer process from the separation area to the identification area in the traditional method, and further improving the sensitivity of rare cell separation and detection.

During use, the sample solution and immunomodified microspheres are first introduced into the storage chamber through the microfluidic chip inlet. Due to the capillary valve action of the microchannel, the sample solution and immunomodified microspheres will be confined to the storage solution. It will not directly enter the separation chamber through connecting micropipes; after the sample injection is completed, place the chip on the centrifugal platform, and lower the elastic microcolumn support so that the lower end of the microcolumn touches the groove of the guide rail, and the elastic microcolumn spring is in the Compressed state: After the assembly of the elastic microcolumns is completed, the centrifugal platform is rotated at a low speed, and the elastic microcolumns are used to periodically squeeze the deformed film on the upper surface of the chip deformation cavity during the rotation process, so that the fluid in the liquid storage chamber oscillates and realizes the liquid storage chamber. Rapid and sufficient mixing and reaction of the sample solution and immunomodified microspheres; after the reaction, the centrifugal platform is rotated at high speed, and the large centrifugal force will drive the fluid and cell mixture in the liquid storage chamber to overcome the action of the micropipe capillary valve and enter the separation chamber. In the separation chamber area, due to centrifugal force, the fluid will pass through the filter membrane and flow to the lower waste liquid chamber, and cells with smaller particle sizes will also enter the waste liquid chamber through the membrane pores together with the fluid, while generally rare cells (such as fetal red blood cells) , CTC and stem cells) have a larger diameter, and after combining with immune microspheres, the scale of the complex is larger, so it will be blocked by the filter membrane on the upper layer of the separation chamber, thereby achieving the separation of rare cells from most other cells. Under the action of centrifugal force, the cells in the upper layer of the separation chamber (including immunocaptured cells) finally gather in the collection area of the chip. After the separation is completed, the sealing tape on the corresponding liquid inlet of each collection area is removed, the fluorescently labeled antibody solution that can specifically recognize the cells to be detected is added, and the sealing tape is affixed again, and the reaction is incubated. After the reaction is over, add buffer to the sample inlet of the chip and centrifuge at high speed, use the buffer to wash to remove unreacted fluorescently labeled antibody molecules, and finally control the centrifuge platform through automatic rotation and positioning, and use the optical detection module to detect cells in each collection area. bit identification and analysis.

This shows that the present invention is characterized in that:

① The end of each separation chamber away from the center of the chip shrinks into a separation cell collection area, and each cell collection area corresponds to a sample inlet loaded with identification reaction solution. When the chip rotates, each identification reaction solution injection port is covered with transparent tape. seal;

②The elastic microcolumn guide rail layer of the microfluidic chip is a concave ring structure, which is used to limit the longitudinal displacement of the elastic microcolumn during the rotation of the chip; there is a through hole in the corresponding deformation cavity area, and the elastic microcolumn compressed through the through hole The micropillars can exert pressure on the deformable membrane on the top layer of the deformation chamber;

③The deformable film layer of the microfluidic chip is clamped between the chip elastic micropillar guide rail layer and the pipe/cavity layer, and can be elastically deformed under pressure;

④ The system includes a microfluidic chip similar to an optical disc, a centrifugal drive module and an optical detection module of a separation detection system; wherein the microfluidic chip is composed of multiple structural layers, which are sequentially composed of elastic microcolumn guide rail layers, which can be Deformable membrane layer, pipe/cavity layer, filter membrane layer and waste liquid collection layer; the centrifugal drive module consists of a rotating motor, a fixed base and a group of elastic micropillars; the optical detection module of the separation detection system consists of a group of movable Lens composition for excitation and detection of fluorescence;

⑤ When the system provided by the present invention is used, firstly, the sample liquid and immunomodified microspheres are introduced into the liquid storage chamber through the microfluidic chip inlet, and placed on the centrifugal platform of the centrifugal drive module, and the elastic microspheres are assembled. The column rotates at a low speed. During the rotation process, the elastic micro-column periodically squeezes the deformed film on the surface of the chip to realize the full mixing and reaction of the sample liquid and the immunomodified micro-bead liquid in the liquid storage chamber, and then rotates the chip at a high speed. Combined with the filter membrane to realize the rapid and efficient separation of immune capture cells; after separation, drop the fluorescently labeled antibody solution that can specifically recognize the cells to be detected in each separated cell collection area, incubate the reaction, then add buffer and centrifuge to remove unreacted cells Fluorescently labeled antibody molecules; finally, identified and analyzed by an optical detection module.

The invention combines immune microspheres, microporous membrane filtration and a centrifugal platform to construct a rare cell separation and detection system, which greatly improves the separation purity of rare cells and accelerates the speed and efficiency of separation and detection of rare cells. Compared with the existing rare cell separation and detection system based on microfluidic technology, the rare cell separation and detection system provided by the present invention has obvious advantages in terms of separation and detection speed and cost. The specific separation of rare cells avoids the complicated processing and expensive cost of making integrated immune-modified microcolumn microfluidic chips; Homogeneous reaction, compared with the existing microfluidic rare cell separation detection system based on microcolumn array (its immune reaction is a heterogeneous reaction system), it has a faster reaction rate, and periodic mechanical extrusion produces The fluid oscillating motion can further improve the reaction binding efficiency of the immune microspheres and the target cells in the sample liquid. In addition, the rare cell separation and detection system provided by the present invention can gather immune-separated cells in a specific micro-area, avoiding the tedious and time-consuming large-area microscopic search operation in the detection process, and greatly reducing the experimental operation cost of rare cell separation and detection. Labor intensity, improving the efficiency of rare cell separation and detection.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the rare cell separation and detection system based on the centrifugal microfluidic technology of the present invention.

FIG. 2 is a schematic diagram of the structure and assembly of the separation and detection system shown in FIG. 1 .

Fig. 3 is a schematic top view of the microfluidic chip structure of the rare cell separation and detection system of the present invention.

Fig. 4 is a schematic diagram of the structure and assembly of the microfluidic chip of the rare cell separation and detection system of the present invention.

Fig. 5 is a schematic cross-sectional view of the chip deformation chamber area when the rare cell separation and detection system of the present invention performs a mixed reaction.

Detailed ways

Further illustrate substantive characteristics and remarkable progress of the present invention below in conjunction with embodiment.

Example 1

Collect about 5mL of peripheral blood from tumor patients, add EpCAM-labeled immune microspheres that can bind to tumor cells into the blood sample, and then inject it into the liquid storage cavity of the microfluidic chip of the rare cell separation detection system; place the chip after adding the sample Put it on the centrifugal platform, and assemble the elastic micro-column, rotate the centrifugal platform at 60~100 rpm to realize automatic and rapid mixing reaction; after 5 minutes, increase the rotational speed of the rotating centrifugal platform to 500 rpm to achieve the separation of target cells; 10 After 10 minutes, turn off the centrifugal platform, tear off the sealing tape of the sample port above each cell collection area, and add CD45-FITC that can identify blood cells and CK-PE antibody that specifically marks tumor cells into the cell collection area in turn, and the liquid addition is complete. Then seal each sample port with tape again; incubate for 20 minutes, inject phosphate buffered solution (PBS) into the sample port of the chip, and rotate the centrifuge platform at 600 rpm to wash and remove unbound fluorescently labeled antibody molecules in the cell collection area . Finally, adjust the rotating platform, place each cell collection area of the chip at the lens of the optical detection module in turn, and identify and analyze the isolated cells.

Example 2

Collect about 100mL of human bone marrow, add CD71-labeled immune microspheres that can bind to fetal red blood cells into the blood sample, and then inject it into the liquid storage chamber of the microfluidic chip of the rare cell separation detection system; place the chip after adding the sample in a centrifuge On the platform, assemble the elastic micro-column, rotate the centrifuge platform at 60~100 rpm to realize automatic and rapid mixing reaction; after 5 minutes, increase the speed of the rotating centrifugal platform to 500 rpm to achieve the separation of target cells; after 10 minutes , turn off the operation of the centrifugal platform, and tear off the sealing tape of the sample port above each cell collection area, add the fluorescently labeled antibody solution that can recognize fetal red blood cell hemoglobin into the cell collection area, and seal the sample port again with tape after the liquid addition is completed; Incubate for 20 minutes, inject phosphate buffered solution (PBS) into the chip inlet, and rotate the centrifuge platform at 600 rpm to wash and remove unbound fluorescently labeled antibody molecules in the cell collection area. Finally, adjust the rotating platform, place each cell collection area of the chip at the lens of the optical detection module in turn, and identify and analyze the isolated cells.

Example 3

Collect about 1mL of human umbilical cord blood, add immune microspheres that can selectively bind to hematopoietic stem cells into the blood sample, and then inject it into the liquid storage chamber of the microfluidic chip of the rare cell separation detection system; place the added chip in a centrifuge On the platform, assemble the elastic micro-column, rotate the centrifuge platform at 60~100 rpm to realize automatic and rapid mixing reaction; after 5 minutes, increase the speed of the rotating centrifugal platform to 500 rpm to achieve the separation of target cells; after 10 minutes , turn off the operation of the centrifugal platform, and tear off the sealing tape of the sample port above each cell collection area, add the CD45-FITC solution that can identify hematopoietic stem cells into the cell collection area, and seal the sample port again with tape after the liquid addition is completed; For 20 minutes, inject phosphate buffer solution (PBS) into the chip inlet, and rotate the centrifuge platform at 600 rpm to wash and remove unbound fluorescently labeled antibody molecules in the cell collection area. Finally, adjust the rotating platform, place each cell collection area of the chip at the lens of the optical detection module in turn, and identify and analyze the isolated cells.

Claims (10)

1. A rare cell separation detection system based on centrifugal microfluidic technology, characterized in that: the system includes a microfluidic chip similar to an optical disc, a centrifugal drive module and an optical detection module of a separation detection system; The microfluidic chip of the optical disk is composed of multiple structural layers, which are elastic microcolumn guide rail layer, deformable film layer, pipe/cavity layer, filter membrane layer and waste liquid collection layer; the centrifugal drive module consists of a rotating motor, a It is composed of a fixed base and a group of elastic micropillars; the optical detection module of the separation detection system is composed of a group of lenses that can excite and detect fluorescence; The pipeline/cavity layer of the microfluidic chip includes a liquid storage cavity and a group of separation cavities, and each separation cavity is arranged radially and periodically around the liquid storage cavity; between the liquid storage cavity and each separation cavity The space is connected by a group of micro-pipes; the liquid storage chamber is connected with a group of through holes, and the liquid storage chamber includes a sample inlet, a vent hole and at least one deformation chamber; the end of each separation chamber away from the center of the chip shrinks into a Separate the cell collection area, and each cell collection area corresponds to a sample inlet loaded with the identification reaction solution. When the chip rotates, each identification reaction solution inlet is sealed with transparent tape; The system utilizes the rotation of the centrifugal platform on the centrifugal drive module, so that the compressed elastic microcolumn fixed on its fixed base periodically squeezes the deformable film on the deformation cavity on the chip that communicates with the liquid storage cavity. 2. The system according to claim 1, characterized in that the appearance of the microfluidic chip similar to an optical disc is disc-shaped. 3. The system of claim 1, wherein: ①The guide rail layer of the elastic microcolumn of the microfluidic chip is a concave annular structure, which is used to limit the longitudinal displacement of the elastic microcolumn during the rotation of the chip; there is a through hole in the corresponding deformation cavity area, and the elastic microcolumn compressed through the through hole The micropillars exert pressure on the deformable membrane on the top layer of the deformation chamber; ② The deformable film layer of the microfluidic chip is clamped between the elastic micropillar guide rail layer and the pipe/cavity layer of the chip, and can be elastically deformed under pressure. 4. The system of claim 2, wherein: ① The separation chamber is fan-shaped, and the end away from the center of the chip shrinks into a semicircular microcavity as a collection area for separated cells (10); and each cell collection area is provided with a sample inlet for cell identification Load fluorescently labeled specific antibody solution during the process, and the sample inlet above each cell collection area is sealed with adhesive tape (13) to prevent the loss of sample solution during chip rotation and incubation; ② On the one hand, the micropipe serves as a fluid channel to ensure the transfer of fluid and cells from the liquid storage chamber to the separation chamber, and also serves as a capillary valve to temporarily restrict the sample liquid in the liquid storage chamber under low driving pressure; ③The top layer of the deformation cavity is a rubber film (15), which is clamped between the chip elastic micro-column guide rail layer (14) and the pipe/cavity layer (16); ④ The compressed elastic microcolumn (6) installed on the fixed base of the centrifugal drive module provides mechanical pressure, and the compressed elastic microcolumn only displaces in the vertical direction; ⑤ The filter membrane layer (17) of the microfluidic chip is a layer of polymer film processed with a micropore array, sandwiched between the chip pipeline/cavity layer and the waste liquid collection layer, and the pore size is 8-12 μm; ⑥The waste liquid collection layer (18) of the microfluidic chip includes an annular microcavity processed with a microcolumn array, and the microcolumn array is used as a support structure; ⑦ The outer diameter of the ring-shaped microcavity should be greater than the distance between the channel/cavity layer cell collection area of the chip and the center of the chip. 5. The system of claim 1, wherein: ① The elastic microcolumn guide rail layer, pipe/cavity layer and waste liquid collection layer of the microfluidic chip are made of glass, polydimethylsiloxane, polystyrene, polycarbonate, cycloolefin copolymer Any one of , polymethyl methacrylate and SU-8; ②Filtration membrane material is any one of polydimethylsiloxane, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, polyimide, parylene and SU-8 kind. 6. The system according to claim 1, wherein the rotary motor included in the centrifugal drive module is a programmable speed-regulating motor; the elastic micro-column can move up and down under the action of vertical force, and the elastic micro-column can move up and down. The lower end is a smooth spherical shape, and the lower end is in direct contact with the groove surface of the chip guide rail layer through the pressure generated by its own spring; the elastic micro-column is fixed on the fixed base through a liftable bracket. 7. The system according to claim 6, characterized in that the optical detection module of the separation detection system is composed of a group of lenses (3) that can excite and detect fluorescence, the lenses (3) are respectively positioned on the upper and lower sides of the chip, and the distance between the lens and the centrifugal platform The central axis distance is equal to the distance between the chip separation cell collection area and the central axis of the centrifugal platform. 8. use according to the method for the system described in any one in claim 1-7, it is characterized in that at first the microsphere of sample liquid and immunomodification is imported in its liquid storage cavity by microfluidic chip inlet, and It is placed on the centrifugal platform of the centrifugal drive module, assembled with elastic microcolumns, and rotated at a low speed. During the rotation process, the elastic microcolumns periodically squeeze the deformable film on the surface of the chip to realize the separation of sample liquid and immunomodification in the liquid storage chamber. The microsphere liquid is fully mixed and reacted, and then the chip is rotated at high speed, and the immunocaptured cells are quickly and efficiently separated by centrifugal force combined with the filter membrane; after separation, a fluorescently labeled antibody solution that can specifically recognize the cells to be detected is added dropwise to each separated cell collection area , incubate the reaction, then add buffer and centrifuge to remove unreacted fluorescently labeled antibody molecules; finally, identify and analyze through the optical detection module. 9. The method according to claim 8, characterized in that it comprises the following steps: a) import the sample liquid and immunomodified microspheres into the liquid storage cavity through the microfluidic chip inlet, and place them on the centrifugal platform of the centrifugal drive module; b) Lower the elastic micro-column support on the centrifugal drive module, make the lower end of the micro-column contact the guide rail groove, and make the elastic micro-column spring in a compressed state, rotate the centrifugal platform at a low speed, and use the elastic micro-column to deform the upper surface of the chip deformation cavity during the rotation process. The periodic extrusion of the deformed film realizes the full mixing and reaction of the sample liquid and the immunomodified microspheres in the liquid storage chamber; c) After the reaction, the centrifugal platform is rotated at high speed, and the centrifugal force combined with the filter membrane is used to realize the rapid and efficient separation of immune capture cells, and the separated target cells are gathered in the collection area of the chip; d) After the separation is completed, remove the sealing tape on the corresponding liquid inlet of each collection area, add a fluorescently labeled antibody solution that can specifically recognize the cells to be detected, and paste the sealing tape again, and incubate the reaction; e) After the reaction is over, add buffer to the chip inlet and centrifuge at high speed to remove unreacted fluorescently labeled antibody molecules, and finally identify and analyze the cells in the collection area through the optical detection module. 10. The method of claim 9, wherein: ① The speed of the low-speed rotating centrifugal platform is 60-300 rpm; ② The speed of the high-speed rotating centrifugal platform is 500-5000 rpm.