Filtering type micro-fluidic chip for separating circulating tumor cells

Abstract

The invention discloses a filter-type micro-fluidic chip for separating circulating tumor cells, and belongs to the technical field of high polymer materials, the filter-type micro-fluidic chip comprises a cover plate and a bottom plate, the cover plate and the bottom plate are of a separable structure, opposite sides of the cover plate and the bottom plate are bonded with PDMS, and the PDMS is provided with a porous filter membrane area; a passage is arranged between the cover plate and the bottom plate and is communicated with the porous filter membrane area; the porous filter membrane area is provided with a porous filter membrane or a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; comprising a basic membrane layer, a membrane middle layer and a zwitterionic polymerization layer which are sequentially arranged from inside to outside, and the zwitterionic polymerization layer has pyridine groups. The capture efficiency of tumor cells can be greatly improved, and the biological pollution resistance of the filter membrane is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer materials, and is a technology combining a polymer porous filter membrane with a microfluidic chip, and specifically relates to a filtering microfluidic chip for separating circulating tumor cells. Background Art

[0002] Circulating tumor cells (CTCs) refer to malignant tumor cells that are shed from primary tumors or metastatic lesions into the blood and are considered to be a necessary condition for tumor metastasis. Clinical studies have shown that patient survival is inversely proportional to the number of CTCs (in cancer patients with high survival rates, the number of CTCs per milliliter of blood sample is less than 5), and that cancer patient survival can be prolonged by reducing or eliminating CTCs through primary or adjuvant therapy. Therefore, the isolation and analysis of CTCs can provide information about tumor metastasis, tumor response after therapeutic intervention, and even provide guidance for personalized treatment of cancer patients.

[0003] Current methods for isolating CTCs include the following: Density gradient centrifugation: There are two methods: Ficoll method and OncoQuick Plus method. Since density gradient centrifugation may lead to a high loss rate of CTCs, it is generally only used for pre-treatment of other enrichment methods.

[0004] Antibody-dependent separation method: This method is mainly divided into positive enrichment and negative enrichment. The principle is to use epithelial cell adhesion factor (EpCAM) and cytokeratin (CK) as detection markers. Antibody magnetic beads-nanoparticles are used to couple epithelial cell surface marker EpCAM, bind tumor cell surface antigens, fluorescently label antibodies, and stain the nucleus to identify CTCs. Of course, the positive enrichment method also has many shortcomings. Not all tumor cells express EpCAM, and cell surface markers may be missing in cell lines, resulting in low enrichment rates. Negative enrichment is a method that does not rely on CTC surface markers. During enrichment, other non-tumor cells in whole blood are first removed, and then tumor cells are enriched. Commonly used gradient methods such as the Ficoll method first remove red blood cells, then use CD45 markers to remove white blood cells, and finally verify CTCs through immunochemical analysis or reverse transcription-polymerase chain reaction (RT-PCR). Similarly, negative enrichment also has many shortcomings. It cannot identify all abnormal cells enriched, and too many white blood cells will interfere with the identification of CTCs.

[0005] Separation method based on cell size: 1. Inertial microfluidics: When the fluid flows in a linear microchannel in laminar flow, the suspended cells therein are affected by the shear gradient lift and the wall effect lift. Under the action of the two forces, cells of different sizes produce different flow characteristics. CTCs can be separated by using different structures inside the chip. However, there is a disadvantage of low flux. 2. Deterministic lateral displacement (DLD) technology: When the direction of fluid flow is at a certain angle to the microcolumn array in the chip, cells of different sizes have different movement trajectories during the flow process. Large cells will be moved sideways to one side, and small cells will move along the original trajectory, thereby achieving the separation of CTCs. Size and deformability are commonly used separation methods in microfluidic cell separation. 3. Membrane filtration technology: Most epithelial CTCs have a diameter of 14-26 µm, while WBCs have a diameter of only 8-20 µm. By integrating the microfluidic chip with various microfilters, when the sample flows through the microfluidic chip, CTCs are retained on the microfilter due to their larger diameter, while blood cells flow out with the buffer. In addition, WBCs are more likely to deform than CTCs. Therefore, the larger white blood cells retained on the microfilter can be washed away by increasing the flow rate, while CTCs, which are less deformable than white blood cells, are still retained on the microfilter.

[0006] Although the existing density gradient centrifugation method can separate some CTCs, the content of CTCs in the blood is extremely small, and the purity of CTCs separated by this method is low; although the antibody-based separation method can separate CTCs with high purity, it is costly and time-consuming; although inertial microfluidics technology and deterministic lateral displacement (DLD) technology can separate CTCs, they have the problem of low flux and low efficiency in capturing CTCs, and the filter membrane is easily contaminated and clogged, which is costly. Summary of the invention

[0007] The purpose of the present invention is to provide a filtering microfluidic chip for separating circulating tumor cells, which can greatly improve the capture efficiency of tumor cells and improve the anti-biological contamination performance of the filter membrane.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A filtering microfluidic chip for separating circulating tumor cells. It comprises a cover plate and a base plate, wherein the cover plate and the base plate are of detachable structure, and PDMS is bonded to opposite sides of the cover plate and the base plate, and the PDMS is provided with a porous filter membrane area; a passage is provided between the cover plate and the base plate, and the passage is connected with the porous filter membrane area; the porous filter membrane area is provided with a porous filter membrane or a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; it comprises a base membrane layer, a membrane intermediate layer and a zwitterionic polymer layer which are arranged in sequence from the inside to the outside, wherein the zwitterionic polymer layer has a pyridine group.

[0009] In the preparation process of the modified porous filter membrane, the hydrophilicity of the filter membrane is enhanced by forming a composite membrane intermediate layer and a zwitterionic polymer layer on the surface of the base membrane, so that when the sample is injected, the surface of the filter membrane first combines with the water in the blood sample to form a water film. In this way, the adhesion between the protein molecules and biological macromolecules such as antibiotics in the blood sample and the filter membrane surface can be reduced, thereby reducing the contamination of the filter membrane by the blood sample and preventing the growth of microorganisms. That is, the anti-biological contamination performance of the filter membrane is improved, and the probability of filter membrane clogging can also be reduced.

[0010] Preferably, the pore size of the modified porous filter membrane in the filtering microfluidic chip is 5-15 μm.

[0011] Preferably, the pyridine groups in the zwitterionic polymeric layer are derived from 2-vinylpyridine and / or 4-butenylpyridine.

[0012] According to one aspect of the present invention, a modified porous filter membrane is provided, wherein the modified porous filter membrane is a composite membrane having a three-layer structure; it includes a base membrane layer, a membrane intermediate layer and a zwitterionic polymer layer arranged in sequence from the inside to the outside, wherein the zwitterionic polymer layer has a pyridine group, and the pyridine group comes from 2-vinylpyridine and / or 4-butenylpyridine.

[0013] The surface of the modified porous filter membrane is a zwitterionic polymer layer and has pyridine groups, which helps to enhance hydrophilicity, thereby reducing the adhesion between the biomacromolecules in the blood sample and the filter membrane surface, thereby preventing filter membrane contamination, that is, improving the anti-biological contamination performance of the filter membrane and reducing the probability of filter membrane clogging. In addition, due to the enhanced hydrophilicity of the modified porous filter membrane, it helps to make the adhesion of tumor cells to the filter membrane low, easy to separate, and less damage to the cells. On the other hand, the presence of amino and sulfonic acid groups helps to improve the uniformity of the water film bound to the surface of composite membrane II; and the presence of amino and sulfonic acid groups helps to extend the side chain of zwitterionic polymers, which plays a positive role in the process of the filter membrane intercepting tumor cells.

[0014] According to one aspect of the present invention, there is provided a method for preparing a modified porous filter membrane, comprising the following steps: S1. Preparation of membrane intermediate layer: immersing a clean and dry base membrane in a polyethyleneimine mixed solution for 6-24 hours to obtain a composite membrane I having a membrane intermediate layer; S2. Preparation of grafted modified microfiltration membrane: The composite membrane I in S1 is treated with a redox initiator and reacted with zwitterionic polymer monomers and 2-vinylpyridine and 4-butenepyridine to obtain a base membrane with a grafted modified microfiltration membrane, which is recorded as composite membrane II.

[0015] Preferably, in step S1, the polyethyleneimine mixed solution is prepared from polyethyleneimine and Tris-HCl buffer solution, which also contains 3-phthalimide propionaldehyde; the mass ratio of polyethyleneimine to 3-phthalimide propionaldehyde is 1:0.5-2. Wherein, the pH of the Tris-HCl buffer solution is 8.5, 50mM; the mass volume ratio of polyethyleneimine, 3-phthalimide propionaldehyde and Tris-HCl buffer solution is 1g:0.5-2g:400-600mL.

[0016] Preferably, in step S1, after the base membrane is immersed in the polyethyleneimine mixed solution, it is shaken in a constant temperature incubator at 20-35°C and 120 r / min for a reaction of 6-24 h.

[0017] Preferably, in step S2, the redox initiator is ammonium persulfate or sodium pyrosulfite; and the mass concentration of ammonium persulfate and sodium pyrosulfite is 1-5 mg / mL.

[0018] Preferably, in step S2, the redox initiator is dibenzoyl peroxide (BPO) and N,N-dimethylbenzene (DMA); and the mass concentration of DMA is 3-8 mg / mL, and the mass concentration of BPO is 15-30 mg / mL.

[0019] Preferably, in step S2, the zwitterionic polymer monomer is added before 2-vinylpyridine and 4-butenylpyridine; the zwitterionic polymer monomer includes at least one of allyltrimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid, and polymethacrylate carboxybetaine.

[0020] Furthermore, before adding 2-vinylpyridine and 4-butenylpyridine, the mass concentration of the zwitterionic polymer monomer is 3-10 mg / mL.

[0021] Furthermore, in step S2, the zwitterionic polymer monomers include allyltrimethylammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid; and before adding 2-vinylpyridine and 4-butenylpyridine, the mass concentrations of allyltrimethylammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid are both 1.5-5 mg / mL.

[0022] Preferably, in step S2, the volume ratio of 2-vinylpyridine to 4-butenepyridine is 1-2:1-3.

[0023] Furthermore, the mass volume ratio of the zwitterionic polymer monomer to 2-vinylpyridine and 4-butenylpyridine is 3-10 mg: 0.5-2.5 mL: 0.5-2.5 mL.

[0024] Preferably, in step S2, water is added to the composite membrane I and then a redox initiator is added, the treatment temperature is 35-50° C., and the treatment time is 10-30 min.

[0025] Preferably, in step S2, after adding the zwitterionic polymer monomer and 2-vinylpyridine and 4-butenylpyridine, the reaction temperature is 35-50° C. and the reaction time is 1-3 h.

[0026] Preferably, when preparing the modified porous filter membrane, in the process of preparing the grafted modified microfiltration membrane in step S2, butyl acrylate is added to the reaction system while adding 2-vinylpyridine and 4-butenepyridine.

[0027] Preferably, the volume ratio of 2-vinylpyridine, 4-butenylpyridine and butyl acrylate is 1-2:1-3:0.5-2.

[0028] Compared with the prior art, the present invention has the following beneficial effects: 1. In the preparation process of the modified porous filter membrane, the hydrophilicity of the filter membrane is enhanced by grafting a zwitterionic polymer on the surface of the composite membrane I. Therefore, when the sample is injected, the surface of the filter membrane first combines with the water in the blood sample to form a water film. In this way, the adhesion between the protein molecules in the blood sample and biological macromolecules such as antibiotics and the filter membrane surface can be reduced, thereby reducing the contamination of the filter membrane by the blood sample and preventing the growth of microorganisms. In other words, the anti-biological contamination performance of the filter membrane is improved, and the probability of filter membrane clogging can also be reduced.

[0029] 2. Adding 2-vinylpyridine and 4-butenylpyridine in the preparation of composite membrane II can greatly improve the capture efficiency of tumor cells. This may be because the addition of 2-vinylpyridine and 4-butenylpyridine introduces amino and sulfonic acid groups when synthesizing composite membrane II, which helps to improve the uniformity of the water film bound to the surface of composite membrane II, and these groups can also react with related groups on the surface of tumor cells, thereby enhancing the capture effect; in addition, the amino and sulfonic acid groups in 2-vinylpyridine and 4-butenylpyridine may react with zwitterionic allyltrimethylammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid, thereby extending the side chain of the zwitterionic polymer grafted on the surface of composite membrane I, further improving the structure of the modified composite membrane II, which plays a positive role in the process of intercepting tumor cells by the filter membrane.

[0030] 3. In the process of separating and enriching tumor cells using a microfluidic chip assembled with a modified porous filter membrane, the adhesion of tumor cells to the filter membrane is low, making it easy to separate and the cells less damaged. This may be because allyltrimethylammonium chloride and 2-acrylamide-2-methylpropanesulfonic acid groups were introduced on the surface of the base membrane when preparing composite membrane II, which enhanced the hydrophilicity of the filter membrane, making it easier for tumor cells to fall off during backwashing and less damaged. In addition, the addition of 2-vinylpyridine and 4-butenylpyridine introduced pyridine groups when synthesizing composite membrane II, which may help to further reduce the force of positive and negative atoms in zwitterions on the cell surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an infrared spectrum of the composite film II and PET obtained according to Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] Example 1 Preparation of modified porous filter membrane 1. Preparation of membrane intermediate layer The base membrane was soaked in acetone for 12 hours to remove impurities adsorbed on its surface, and then dried in a vacuum drying oven at 40°C for 1 hour. The base membrane was a membrane chip in a microporous filter purchased from Hangzhou Baimai Medical Technology Co., Ltd., made of polycarbonate polymer material, with a thickness of 11μm±1μm and a micropore size of 10μm.

[0034] Polyethyleneimine (PEI) was added to Tris-HCl buffer solution (pH=8.5, 50 mM), and after being stirred evenly, 3-phthalimide propionaldehyde was added, wherein the mass volume ratio of PEI, 3-phthalimide propionaldehyde and Tris-HCl buffer solution was 1 g:1 g:500 mL.

[0035] The dried base membrane was completely immersed in the above PEI mixed solution, and reacted for 12 hours in a constant temperature shaker at 25°C, 120r / min, to form a membrane intermediate layer on the surface of the base membrane, which was recorded as composite membrane I; after the composite membrane I was rinsed with water three times, water was added according to a mass volume ratio of 10g:500mL, and it was shaken for 30 minutes in a constant temperature shaker at 25°C, 150r / min, to remove unstable PEI and 3-phthalimide propionaldehyde. After the shaking was completed, the obtained composite membrane I was dried in a vacuum drying oven at 40°C for 1 hour and set aside.

[0036] 2. Preparation of grafted modified microfiltration membrane Take the above-mentioned dried composite membrane I and add it to water, then add N, N-dimethylbenzene (DMA) and dibenzoyl peroxide (BPO) as redox initiators under argon atmosphere, and heat it in an oil bath at 40°C for 20 minutes. Among them, the mass concentration of DMA is 5 mg / mL, and the mass concentration of BPO is 20 mg / mL. Then, zwitterionic polymer monomers allyltrimethylammonium chloride and 2-acrylamide-2-methylpropanesulfonic acid are added to the reaction system, wherein the mass concentrations of allyltrimethylammonium chloride and 2-acrylamide-2-methylpropanesulfonic acid are both 3 mg / mL. The above system is heated in an oil bath at 40°C for 2 hours to obtain a base membrane with a grafted modified microfiltration membrane, which is recorded as composite membrane II.

[0037] After the composite membrane II was rinsed with water for 3 times, water was added according to the mass volume ratio of 10g:500mL, and the composite membrane II was shaken at 150r / min for 30min in a constant temperature shaker at 25°C. After the shaking, the composite membrane II was dried in a vacuum drying oven at 40°C for 1h to obtain a usable modified porous filter membrane. The modified porous filter composite membrane II is a composite membrane with a three-layer structure, including a base membrane layer, a membrane intermediate layer, and a zwitterionic polymer layer arranged in sequence from the inside to the outside.

[0038] Fourier transform infrared spectroscopy was used to characterize the base membrane and the composite membrane II obtained by the reaction. The samples were prepared by potassium bromide tableting method with a scanning wavelength range of 500 cm -1 ~4000cm -1 ; Scan line number is 32; Resolution is 4cm -1 The infrared spectrum obtained is shown in Figure 1 As can be seen from the figure, composite membrane II has -1 ~3400cm -1 A strong and broad absorption peak appears near 1750cm -1 The absorption peak near 1040cm is still retained, which is the absorption peak of C=O; -1 The change in the absorption peak near 1040cm -1 ~1260cm -1 The absorption peak near 1400cm-1 changes, which may be attributed to the stretching vibration of sulfonate. -1 The absorption peak near changes, which may be attributed to the role of CN bonds. The above can verify that the reaction formed composite film II on the surface of composite film I.

[0039] Assembling the filtration microfluidic chip A 1mm thick glass is used as the bottom plate, and a 2mm thick glass is used as the cover plate. The bottom plate and the cover plate are respectively permanently bonded with 500μm PDMS by plasma, and the cover plate is connected and matched with the bottom plate, and the PDMS is contained inside. The two layers of PDMS in the middle correspond to the porous filter membrane area, and the modified porous filter membrane composite membrane II is arranged therein. The shape of the composite membrane II is adapted to the porous filter membrane area to form the main structure of the filtering microfluidic chip.

[0040] The cover plate is provided with an upper flow channel, an inlet and an outlet of the chip, and the inlet is connected to the upper flow channel of the chip; the lower glass cover plate is provided with a lower flow channel of the chip, and one end of the lower flow channel is connected to the outlet; and the end of the upper flow channel away from the inlet is connected to the porous filter membrane area, and the end of the lower flow channel away from the outlet is also connected to the porous filter membrane area. In this way, the inlet-upper flow channel-porous filter membrane area-lower flow channel-outlet forms a passage, in which the cell suspension can flow under the action of the external pump body, and the modified porous filter membrane can be used to separate tumor cells, etc.

[0041] Furthermore, the filtering microfluidic chip is 55 mm long and 25 mm wide. The cover plate and the bottom plate bonded with PDMS are set to an open structure, and the two can be separated, so as to facilitate the replacement of the filter membrane.

[0042] Furthermore, the modified porous filter membrane is circular, and its radius is slightly larger than the radius of the porous filter membrane area to prevent the cell suspension from leaking out.

[0043] In this embodiment, a base membrane with a pore diameter of 10 μm is selected for modification, and the resulting modified microporous filter membrane is used to assemble a filtering microfluidic chip. In addition, as required, the pore diameter of the selected base membrane can also be 6 μm, 10 μm, 15 μm or other specifications.

[0044] Example 2 Compared with Example 1, the difference between this embodiment and Example 1 is that in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, after adding the zwitterionic polymer monomer to the reaction system, 2-vinylpyridine and 4-butenylpyridine are added thereto, and the volume ratio of 2-vinylpyridine to 4-butenylpyridine is 1:1; and the mass volume ratio of allyltrimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid to 2-vinylpyridine and 4-butenylpyridine is 3 mg:3 mg:1 mL:1 mL.

[0045] Example 3 Compared with Example 1, the difference between this embodiment and Example 1 is that in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, after adding the zwitterionic polymer monomer to the reaction system, 2-vinylpyridine and 4-butenylpyridine are added thereto, and the volume ratio of 2-vinylpyridine to 4-butenylpyridine is 1:1; and the mass volume ratio of allyltrimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid to 2-vinylpyridine and 4-butenylpyridine is 3 mg:3 mg:1.5 mL:1.5 mL.

[0046] Other conditions and steps are the same.

[0047] Example 4 Compared with Example 1, the difference between this embodiment and Example 1 is that in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, after adding the zwitterionic polymer monomer to the reaction system, 2-vinylpyridine and 4-butenylpyridine are added thereto, and the volume ratio of 2-vinylpyridine to 4-butenylpyridine is 1:1; and the mass volume ratio of allyltrimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid to 2-vinylpyridine and 4-butenylpyridine is 3 mg:3 mg:2 mL:2 mL.

[0048] Other conditions and steps are the same.

[0049] Example 5 Compared with Example 1, the present embodiment is different in that: in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, when adding 2-vinylpyridine and 4-butenepyridine to the reaction system, butyl acrylate is added at the same time; the volume ratio of 2-vinylpyridine, 4-butenepyridine and butyl acrylate is 1:1:0.5.

[0050] Other conditions and steps are the same.

[0051] Example 6 Compared with Example 1, the present embodiment is different in that: in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, butyl acrylate is added when 2-vinylpyridine and 4-butenepyridine are added to the reaction system; the volume ratio of 2-vinylpyridine, 4-butenepyridine and butyl acrylate is 1:1:1.

[0052] Other conditions and steps are the same.

[0053] Comparative Example 1 The difference between this comparative example and Example 1 is that the filtering microfluidic chip is directly assembled using the base membrane.

[0054] Comparative Example 2 The difference between this comparative example and Example 2 is that in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, 4-butenylpyridine is replaced by an equal mass of 2-vinylpyridine.

[0055] Other conditions and steps are the same.

[0056] Comparative Example 3 The difference between this comparative example and Example 2 is that in the preparation of the modified porous filter membrane, when preparing the grafted modified microfiltration membrane, 2-vinylpyridine is replaced with an equal mass of 4-butenylpyridine.

[0057] Other conditions and steps are the same.

[0058] Test example 1. Capture efficiency of different tumor cells by filtration microfluidic chip Breast cancer MDA-MB-231 cells, lung cancer A549 cells and liver cancer HepG2 cells were subcultured in RPMI1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator.

[0059] Fresh peripheral blood was collected from healthy volunteers, and breast cancer MDA-MB-231 cells, lung cancer A549 cells, and liver cancer HepG2 cells were added to the blood, respectively, so that the final concentration of tumor cells in the circulating fluid was 10 3 / mL, after oscillation and mixing, the filter-type microfluidic chips assembled in Examples 1-6 and Comparative Examples 1-3 were respectively used for filtration separation. When the cell fluid was filtered and separated, 3mL of blood sample containing tumor cells was injected into the filter-type microfluidic chip at a flow rate of 0.1mL / min using an external injection pump. After the blood sample was completely filtered, the injection pump was turned off. Each blood sample was subjected to 10 parallel tests when each filter-type microfluidic chip was used for filtration separation.

[0060] After filtration, 5 parallel tests were randomly selected from each filter microfluidic chip, and the filter membrane was rinsed with 2% Triton X-100 buffer for 10 minutes, and then blocked with PBS buffer containing 10% FBS at room temperature for 1 hour. PancytoKeratin Alexa488 antibody and anti-CD45 antibody (PerCP / Cy5.5) were added and incubated in a wet box for 1 hour, and then hydrated with PBS buffer for 5 minutes. Then, DAPI fluorescent staining was added for 10 minutes, anti-quenching sealing agent was added to seal the slice, and the cells were observed and counted under a fluorescence microscope to calculate the capture efficiency. For the remaining 5 parallel tests in each filter microfluidic chip, the filter membrane was rotated 180º, 0.5 mL PBS was added from the inlet of the microfluidic chip for reverse flushing, and the tumor cells captured on each filter membrane were collected at the inlet of the microfluidic chip using a basic membrane with a pore size of 6 μmd. Fluorescence staining and counting were performed using the above method, and the cell loss rate was calculated. Cell loss rate = number of tumor cells collected by reverse flushing / total number of tumor cells captured by the filter membrane × 100%.

[0061] The results of calculating the capture efficiency of the filtration microfluidic chip obtained in each embodiment and comparative example for different tumor cells are shown in Table 1, and the results of the cell loss rate are shown in Table 2.

[0062] Table 1 Capture efficiency of different tumor cells by filtration microfluidic chip (%)

[0063] Referring to the data in Table 1, compared with Comparative Example 1, the capture efficiency of the microfluidic chips obtained in Examples 1-6 and Comparative Examples 2-3 for different tumor cells is improved.

[0064] Among them, the capture efficiency of the microfluidic chip obtained in Examples 1-4 for MDA-MB-231 cells is increased by 5.17%, 11.02%, 11.97%, and 13.06% respectively compared with Comparative Example 1; the capture efficiency of the microfluidic chip obtained in Examples 1-4 for A549 cells is increased by 5.46%, 11.34%, 12.46%, and 13.30% respectively compared with Comparative Example 1; the capture efficiency of the microfluidic chip obtained in Examples 1-4 for HepG2 cells is increased by 5.96%, 10.35%, 11.20%, and 13.90% respectively compared with Comparative Example 1. It can be seen that compared with the basic membrane, the microfluidic chip assembled with the modified porous filter membrane can greatly improve the capture efficiency of different tumor cells, that is, the modified porous filter membrane has a better enrichment effect on tumor cells. That is, in the preparation process of the modified porous filter membrane, by grafting zwitterionic polymers on the surface of composite membrane I, not only can the adhesion between protein molecules in blood samples and biological macromolecules such as antibiotics and the filter membrane surface be reduced, but also the modified composite membrane II has an increased hydrophilicity, which enhances the interception effect on tumor cells. This may be because when preparing composite membrane II, allyl trimethylammonium chloride and 2-acrylamide-2-methylpropane sulfonic acid groups were introduced on the surface of the base membrane, which helps to adsorb tumor cells, especially the presence of methyl ammonium chloride groups and sulfonic acid groups, which helps to react with related groups on the surface of tumor cells, thereby enhancing the capture effect.

[0065] In addition, compared with Example 1, the capture efficiency of the microfluidic chips obtained in Examples 2-3 for the above three types of tumor cells was significantly improved. It can be seen that adding 2-vinylpyridine and 4-butenylpyridine when preparing composite membrane II can greatly improve the capture efficiency of tumor cells.

[0066] In addition, the capture efficiency of the microfluidic chip obtained in Comparative Examples 2-3 for MDA-MB-231 cells, A549 cells, and HepG2 cells is better than that in Comparative Example 1, but worse than that in Example 2; it can be seen that when 2-vinylpyridine and 4-butenepyridine are added simultaneously in the preparation of composite membrane II, the obtained modified porous filter membrane has a better capture effect on tumor cells than adding 2-vinylpyridine or 4-butenepyridine alone. This may be because the amino and sulfonic acid groups in 2-vinylpyridine and 4-butenepyridine react during the mixing process, so that the side chain of the obtained zwitterionic polymer is extended, and the structure of the modified composite membrane II is further improved to form an attachment site that is easy for tumor cells to fix, thereby improving the capture efficiency of tumor cells.

[0067] Compared with Example 2, the capture efficiency of the microfluidic chips obtained in Example 5 and Example 6 for MDA-MB-231 cells increased by 4.66% and 5.64%, for A549 cells increased by 4.03% and 4.53%, and for HepG2 cells increased by 4.24% and 4.88%. It can be seen that in the process of preparing composite membrane II, the simultaneous addition of 2-vinylpyridine, 4-butenepyridine and butyl acrylate helps to further enhance the capture effect of the modified filter membrane on tumor cells, and the addition amount of butyl acrylate is positively correlated with the capture effect of the filter membrane on tumor cells within a certain range.

[0068] Table 2 Cell loss rate (%) of different tumor cells captured by filtration microfluidic chip

[0069] Referring to the data in Table 2, compared with Comparative Example 1, the cell loss rates of the microfluidic chips obtained in Examples 1-6 and Comparative Examples 2-3 for different tumor cells in the backwashing step were all reduced.

[0070] Among them, in the backwashing step, the cell loss rate of the microfluidic chip obtained in Examples 1-4 for MDA-MB-231 cells is reduced by 8.3%, 13.9%, 14.8%, and 16.7% respectively compared with Comparative Example 1; the cell loss rate of the microfluidic chip obtained in Examples 1-4 for A549 cells is reduced by 7.1%, 11.6%, 15.2%, and 17.0% respectively compared with Comparative Example 1; the cell loss rate of the microfluidic chip obtained in Examples 1-4 for HepG2 cells is reduced by 7.3%, 11.9%, 14.7%, and 17.4% respectively compared with Comparative Example 1. It can be seen that compared with the basic membrane, the microfluidic chip assembled with the modified porous filter membrane can greatly reduce the cell loss rate, that is, in the process of separating and enriching tumor cells with the microfluidic chip assembled with the modified porous filter membrane, the obtained tumor cells have low adhesion to the filter membrane, are easy to separate, and the cells are less damaged.

[0071] This may be because allyl trimethylammonium chloride and 2-acrylamide-2-methylpropanesulfonic acid groups were introduced into the surface of the base membrane during the preparation of composite membrane II, which enhanced the hydrophilicity of the filter membrane. When the sample was injected, a water film was formed on the surface of the filter membrane, thereby reducing the interaction between the tumor cells and the filter membrane, so that the tumor cells were easier to fall off during the backwashing process and were less damaged. In addition, since the surface of the modified porous filter membrane was a grafted zwitterionic polymer layer, the interaction between it and the tumor cells was more due to the interaction between the positive and negative atoms in the zwitterions on the cell surface, which was small and easy to separate.

[0072] In addition, compared with Example 1, the cell loss rate of the microfluidic chip obtained in Examples 2-3 for the above three tumor cells in the backwashing step was greatly reduced, which shows that the addition of 2-vinylpyridine and 4-butenylpyridine in the preparation of composite membrane II can reduce it. This may be because the addition of 2-vinylpyridine and 4-butenylpyridine introduces pyridine groups when synthesizing composite membrane II, which may help to further reduce the force of the positive and negative atoms in the zwitterions on the cell surface.

[0073] In addition, the cell loss rate of the microfluidic chip obtained in Comparative Examples 2-3 for MDA-MB-231 cells, A549 cells, and HepG2 cells in the backwashing step was lower than that in Comparative Example 1, but higher than that in Example 2. It can be seen that the addition of 2-vinylpyridine and 4-butenepyridine simultaneously in the preparation of composite membrane II is better than the addition of 2-vinylpyridine or 4-butenepyridine alone, and the resulting modified porous filter membrane has a better protective effect on tumor cells. This may be because the simultaneous presence of amino groups and sulfonic acid groups helps to improve the uniformity of the water film attached to the surface of composite membrane II.

[0074] Compared with Example 2, the cell loss rates of the microfluidic chips obtained in Examples 5 and 6 for MDA-MB-231 cells in the backwashing step were reduced by 8.6% and 9.7%, respectively, for A549 cells in the backwashing step were reduced by 8.1% and 10.1%, respectively, and for HepG2 cells in the backwashing step were reduced by 8.3% and 12.5%, respectively. It can be seen that in the process of preparing composite membrane II, the simultaneous addition of 2-vinylpyridine, 4-butenepyridine and butyl acrylate helps to further reduce the adhesion between tumor cells and the obtained modified filter membrane pair.

[0075] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0076] The above-described embodiments provide a detailed description of the technical solution of the present invention. It should be understood that the above-described embodiments are only specific embodiments of the present invention and are not intended to limit the present invention. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A filtering microfluidic chip for separating circulating tumor cells, comprising a cover plate and a bottom plate, characterized in that: The cover plate and the base plate are of detachable structure, and PDMS is bonded to opposite sides of the cover plate and the base plate, and the PDMS is provided with a porous filter membrane area; a passage is provided between the cover plate and the base plate, and the passage is connected with the porous filter membrane area; the porous filter membrane area is configured with a porous filter membrane or a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; it includes a base membrane layer, a membrane middle layer and a zwitterionic polymer layer which are arranged in sequence from the inside to the outside, wherein the zwitterionic polymer layer has a pyridine group.

2. The filtering microfluidic chip for separating circulating tumor cells according to claim 1, characterized in that: The pore size of the modified porous filter membrane is 5-15 μm.

3. A modified porous filter membrane, characterized in that: It comprises a base film layer, a film middle layer and a zwitterionic polymer layer which are arranged in sequence from the inside to the outside; the zwitterionic polymer layer has a pyridine group which is derived from 2-vinylpyridine and / or 4-butenepyridine.

4. A method for preparing a modified porous filter membrane, characterized in that: The following steps are involved: S1. Preparation of membrane intermediate layer: immersing a clean and dry base membrane in a polyethyleneimine mixed solution for 6-24 hours to obtain a composite membrane I having a membrane intermediate layer; S2. Preparation of grafted modified microfiltration membrane: The composite membrane I in S1 is treated with a redox initiator and reacted with zwitterionic polymer monomers and 2-vinylpyridine and 4-butenepyridine to obtain a base membrane with a grafted modified microfiltration membrane, which is recorded as composite membrane II.

5. The method for preparing a modified porous filter membrane according to claim 4, characterized in that: In step S1, the polyethyleneimine mixed solution includes polyethyleneimine and Tris-HCl buffer solution, and also contains 3-phthalimide propionaldehyde; the mass ratio of polyethyleneimine to 3-phthalimide propionaldehyde is 1:0.5-2.

6. The method for preparing a modified porous filter membrane according to claim 4, characterized in that: In step S2, the zwitterionic polymer monomer is added before 2-vinylpyridine and 4-butenylpyridine; The zwitterionic polymer monomer includes at least one of allyltrimethylammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid and polymethacrylate carboxybetaine, and before adding 2-vinylpyridine and 4-butenylpyridine, the mass concentration of the zwitterionic polymer monomer is 3-10 mg / mL.

7. The method for preparing a modified porous filter membrane according to claim 4, characterized in that: In step S2, the volume ratio of 2-vinylpyridine to 4-butenepyridine is 1-2:1-3.

8. The method for preparing a modified porous filter membrane according to claim 4, characterized in that: In step S2, the mass volume ratio of the zwitterionic polymer monomer to 2-vinylpyridine and 4-butenylpyridine is 3-6 mg: 0.5-2.5 mL: 0.5-2.5 mL.

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