Microfluidic chip and method for separating platelet adhesion type CTC

Abstract

The invention discloses a micro-fluidic chip and a method for separating platelet adhesion type CTC. The micro-fluidic chip comprises a porous base material, the porous base material comprises a micro-cavity with the aperture of 30-40 microns, and the micro-cavity comprises micropores with the aperture of 12-18 microns.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a method for obtaining platelet-adherent CTCs, and more particularly to a microfluidic chip and a method for separating platelet-adherent CTCs. Background Art

[0002] Circulating tumor cells (CTCs) play a key role in the process of cancer metastasis. For CTCs to complete the process of tumor metastasis, they must survive in the blood circulation, and CTCs will collide and contact with various blood cells. A large number of studies have shown that platelets, with their numerical advantage, are most likely to interact with CTCs first after they enter the blood and cause corresponding signal activation and functional changes in CTCs. The main biological effects of the interaction between CTCs and platelets include: (1) CTCs can induce platelet activation; (2) platelets protect CTCs from shear stress and anoikis; (3) platelets mediate the immune escape of cancer cells. However, current studies on platelets directly changing the immune phenotype of CTCs to help tumor cells escape immune killing are all carried out based on the co-culture method after separating CTCs and platelets. On the one hand, the separation and enrichment steps are cumbersome and time-consuming, which is not conducive to the rapid presentation of research results and will introduce inevitable errors; secondly, the in-situ interaction of platelets adhering to CTCs is broken by existing separation techniques, and even if co-culture is used later, the original state of platelet-adherent CTCs in the blood microecosystem has been changed. However, so far, there is no technology that can directly separate and enrich platelet-adherent CTCs.

[0003] So far, the sensitivity and specificity of CTC separation techniques are usually designed for detecting single cells, so separating viable platelet-adherent CTCs faces great challenges. Microfiltration technology is widely used in CTC detection due to its simple operation and rapidity, but it is not suitable for separating platelet-adherent CTCs. On the one hand, platelet-adherent CTCs may pass through the pore size of the filter under high filtration pressure, and on the other hand, the high shear force generated during microfiltration may damage platelet-adherent CTCs or break them into single cells, thus affecting the effective enrichment of target cells. Secondly, antibody-based enrichment techniques rely on specific membrane antigens and can only enrich some CTCs, ignoring CTCs that do not express specific antigens. In recent years, separation techniques based on microfluidic chips have shown good sensitivity, but they also face the risk of damage to platelet-adherent CTCs due to high flow rates in narrow channels, and the processing speed is also difficult to meet the current clinical needs.

[0004] Through research on the technology of directly changing the immune phenotype of CTCs by platelets to help tumor cells evade immune killing, it was found that it was carried out based on the co-culture method after separate isolation of CTCs and platelets. However, the co-culture method after separate isolation is cumbersome in terms of procedure steps on the one hand, and fails to achieve the goal of in-situ analysis and detection on the other hand. Therefore, there is an urgent need to invent a new technology for in-situ isolation and detection of platelet-adhered CTCs in whole blood. Summary of the Invention

[0005] To solve the above technical problems, in one aspect, the present application provides a microfluidic chip, which includes a porous substrate. The porous substrate includes microcavities with a pore size of 30-40 microns, and the microcavities include micropores with a pore size of 12-18 microns.

[0006] In another aspect, the present application provides a method for separating platelet-adhered circulating tumor cells (CTCs), the method comprising:

[0007] (a) obtaining a microfluidic chip, which includes a porous substrate. The porous substrate includes microcavities with a pore size of 30-40 microns, and the microcavities include micropores with a pore size of 12-18 microns;

[0008] (b) adding a sample to the microfluidic chip;

[0009] (c) detecting the platelet-adhered CTCs captured in the microfluidic chip; and

[0010] (d) obtaining the platelet-adhered CTCs captured in the microfluidic chip. Brief Description of the Drawings

[0011] The present application will be described in more detail below with reference to the drawings, in which:

[0012] Figure 1 is a schematic diagram of the design of a microfluidic chip according to an embodiment of the present application;

[0013] Figure 2 is a diagram showing the specific dimension markings of the micropores in a microfluidic chip according to an embodiment of the present application;

[0014] Figure 3 is a photo of the SU8-mold;

[0015] Figure 4 is a schematic diagram of step 1 in the fabrication of a filter membrane according to an embodiment of the present application;

[0016] Figure 5 is a schematic diagram of step 2 in the fabrication of a filter membrane according to an embodiment of the present application;

[0017] Figure 6Schematic diagram of Step 3 in the fabrication of the filter membrane according to an embodiment of the present application;

[0018] Figure 7 PDMS microcolumn effect diagram of Step 6 in the fabrication of the filter membrane according to an embodiment of the present application;

[0019] Figure 8 Schematic diagram of Step 7 in the fabrication of the filter membrane according to an embodiment of the present application;

[0020] Figure 9 Photo of the chip obtained from the fabrication of the filter membrane according to an embodiment of the present application;

[0021] Figure 10 Design diagram of the PMMA fixture and chip according to an embodiment of the present application;

[0022] Figure 11 Schematic diagram of the use of the microfluidic chip filter membrane according to an embodiment of the present application;

[0023] Figure 12 Schematic diagram of the SU8-mold according to an embodiment of the present application;

[0024] Figure 13 Schematic diagram of the overall chip assembly according to an embodiment of the present application;

[0025] Figure 14 Confocal fluorescence microscopy imaging diagram of platelet adhesion type CTC capture according to an embodiment of the present application. Detailed implementation manners

[0026] This application relates to a microfluidic chip, the chip comprising a porous substrate, the porous substrate comprising microcavities with pore sizes of 30 - 40 microns, and the microcavities comprising micropores with pore sizes of 12 - 18 microns. In this application, the porous substrate can comprise any solid substrate of any material suitable for cell filtration. In one embodiment, the porous substrate comprises a membrane. In this application, the porous substrate can have any shape or size suitable for cell filtration. In one embodiment, the porous substrate is circular. In one embodiment, the porous substrate is square. In a preferred embodiment, the diameter of the porous substrate is about 5 cm. In one embodiment, the thickness of the porous substrate is 20 - 100 microns. In one embodiment, the thickness of the porous substrate is 30 - 80 microns. In one embodiment, the thickness of the porous substrate is 40 - 70 microns. In a preferred embodiment, the thickness of the porous substrate is about 60 microns. In one embodiment, the porous substrate comprises a resin. In a preferred embodiment, the porous substrate comprises PDMS. In one embodiment, the porous substrate is produced by lithography. In one embodiment, the microcavity comprises one or more micropores. In a preferred embodiment, one microcavity comprises 4 micropores. In one embodiment, the porous substrate comprises a plurality of microcavities. In one embodiment, the porous substrate comprises 0.25 - 250,000 microcavities. In one embodiment, the porous substrate comprises 20,000 - 100,000 microcavities. In a preferred embodiment, the porous substrate comprises about 40,000 microcavities. In one embodiment, the porous substrate comprises 0.02 - 15,000 microcavities / cm 2 . In one embodiment, the porous substrate comprises 0.1 - 0.5 ten thousand microcavities / cm 2 . In a preferred embodiment, the porous substrate comprises 0.2 - 0.3 ten thousand microcavities / cm 2 . In a more preferred embodiment, the porous substrate comprises about 0.22 ten thousand microcavities / cm 2In the present application, the microcavities can have any size or shape suitable for accommodating the platelet-adherent CTC-containing fluid. In one embodiment, the microcavities are square. In one embodiment, the microcavities are circular. In one embodiment, the aperture diameter of the microcavities is 33 - 37 microns. In a preferred embodiment, the aperture diameter of the microcavities is about 35 microns. In one embodiment, the depth of the microcavities is 20 - 100 microns. In a preferred embodiment, the depth of the microcavities is 30 - 80 microns. In a more preferred embodiment, the depth of the microcavities is 40 - 60 microns. In one embodiment, one side of the microcavity includes an opening for the entry of the platelet-adherent CTC-containing fluid, and the other side of the microcavity includes micropores for retaining the platelet-adherent CTCs in the fluid and passing other cells. In one embodiment, the microcavities are uniformly distributed on the porous substrate. In one embodiment, the microcavities are distributed on the porous substrate in an array. In a preferred embodiment, the microcavities are distributed on the porous substrate in a hexagonal array. In one embodiment, the distance between the microcavities is 20 - 100 microns. In a preferred embodiment, the distance between the microcavities is 30 - 80 microns. In a more preferred embodiment, the distance between the microcavities is 40 - 60 microns. In one embodiment, the aperture diameter of the micropores is 14 - 16 microns. In a preferred embodiment, the aperture diameter of the micropores is about 15 microns. In the present application, the micropores can have any shape suitable for cell filtration. In one embodiment, the micropores are square. In one embodiment, the micropores are circular. In one embodiment, the shapes of the multiple micropores of the porous substrate are the same. In one embodiment, the shapes of the multiple micropores of the porous substrate are different. In one embodiment, the porous substrate includes 10,000 - 1,000,000 micropores. In one embodiment, the porous substrate includes 100,000 - 300,000 micropores. In a preferred embodiment, the porous substrate includes about 150,000 micropores. In one embodiment, the porous substrate includes 1,000 - 50,000 micropores / cm 2 In one embodiment, the porous substrate includes 5,000 - 20,000 micropores / cm 2 In a preferred embodiment, the porous substrate includes 7,000 - 10,000 micropores / cm 2 In a more preferred embodiment, the porous substrate includes about 8,500 micropores / cm 2。In one embodiment, the depth of the micropores is 1 - 100 microns. In a preferred embodiment, the depth of the micropores is 2 - 50 microns. In a more preferred embodiment, the depth of the micropores is 3 - 10 microns. In a more preferred embodiment, the depth of the micropores is about 5 microns. In one embodiment, the micropores are uniformly distributed on the porous substrate. In one embodiment, the micropores are distributed on the porous substrate in the form of an array. In one embodiment, the distance between the micropores is 5 - 10 microns. In one embodiment, the distance between the micropores is 20 - 100 microns. In one embodiment, the distance between the micropores is 30 - 80 microns. In one embodiment, the distance between the micropores is 40 - 60 microns. In one embodiment, the microcavities have the same shape as the micropores. In one embodiment, the microcavities have a different shape from the micropores. In a preferred embodiment, one microcavity includes 4 micropores, both the microcavity and the micropores are square, the aperture of the microcavity is about 35 microns, the aperture of the micropores is about 15 microns, the distance between the micropores is about 5 microns, the depth of the micropores is about 5 microns, and the microcavities are distributed on the porous substrate in the form of a hexagonal array. In one embodiment, when the micropores or microcavities are non-circular, such as square, the aperture / diameter can refer to the distance between opposite inner walls, such as the side length. In one embodiment, when the micropores or microcavities are circular or approximately circular, the aperture / diameter can refer to the inner diameter.

[0027] This application also relates to a method for separating platelet-adherent circulating tumor cells (CTCs), the method comprising: (a) obtaining a microfluidic chip as described in any of the foregoing embodiments.

[0028] The method for separating platelet-adherent circulating tumor cells (CTCs) further comprises: (b) adding a sample to the microfluidic chip. In one embodiment, the sample comprises whole blood. In one embodiment, the flow rate of adding the sample in step (b) is lower than the physiological free flow rate in human capillaries. In one embodiment, the flow rate of adding the sample in step (b) is about 10 times lower than the physiological free flow rate in human capillaries. In one embodiment, the flow rate of adding the sample in step (b) is 30 - 600 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is 60 - 600 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is 60 - 300 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is about 300 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is 60 - 100 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is 30 - 80 μm / s. In one embodiment, the flow rate of adding the sample in step (b) is 60 - 80 μm / s. In a preferred embodiment, the flow rate of adding the sample in step (b) is about 60 μm / s.

[0029] The method for separating platelet-adherent circulating tumor cells (CTCs) further includes: (c) detecting the platelet-adherent CTCs captured in the microfluidic chip. In one embodiment, in step (c), the captured platelet-adherent CTCs are detected by immunolabeling. In one embodiment, the immunolabeling includes reagents for detecting CTC markers, reagents for detecting nuclear markers, and reagents for detecting platelet markers. In one embodiment, the reagents for detecting CTC markers include reagents for detecting EpCAM and / or Pan-CK. In one embodiment, the reagents for detecting nuclear markers include reagents for detecting DAPI. In one embodiment, the reagents for detecting platelet markers include reagents for detecting CD41. In one embodiment, the reagents for detecting CTC markers, the reagents for detecting nuclear markers, and / or the reagents for detecting platelet markers include antibodies. In one embodiment, the immunolabeling includes fluorescence staining. In one embodiment, the detection in step (c) includes fluorescence microscopy imaging. In a preferred embodiment, the detection in step (c) includes inverted fluorescence microscopy imaging.

[0030] The method for separating platelet-adherent circulating tumor cells (CTCs) further includes: (d) obtaining the platelet-adherent CTCs captured in the microfluidic chip. In one embodiment, step (d) includes obtaining the captured platelet-adherent CTCs by micromanipulation. In one embodiment, step (d) includes obtaining the captured platelet-adherent CTCs based on the results of immunolabeling. In one embodiment, the results of immunolabeling include positive immunolabeling. In one embodiment, the results of immunolabeling include that the reagents for detecting CTC markers, the reagents for detecting nuclear markers, and the reagents for detecting platelet markers are all positive. In one embodiment, the results of immunolabeling include EpCAM+ and / or Pan-CK+, DAPI+, and CD41+. In one embodiment, the micromanipulation includes using an Eppendorf TransferMan 4r micromanipulator.

[0031] Examples

[0032] This application will be described in detail through the following exemplary specific examples. The following examples are only used to help those skilled in the art better understand various inventions of this application. It should be pointed out that the spirit of this application and the protection scope of the claims are not limited by the following specific examples.

[0033] Table 1. List of Reagents and Equipment Used

[0034]

[0035]

[0036]

[0037] Example 1

[0038] Fabrication of microfluidic chip

[0039] The CAD drawing of the microfluidic chip fabrication is presented in the attachment. In this invention, 150,000 microholes are evenly distributed on a membrane with a diameter of 5 cm, achieving gentle treatment of platelet-adherent CTCs. Figure 1 It is a schematic diagram of the microfluidic chip design. Figure 2 It is a diagram showing the specific dimension markings of the microholes in the microfluidic chip.

[0040] The specific steps for mold making are as follows:

[0041] Step 1: Clean the silicon wafer. Measure the thickness of the silicon wafer, soak it in a mixed reagent of concentrated sulfuric acid and hydrogen peroxide at a ratio of 3:1 for 10 - 15 min; rinse with ultrapure water, put it into anhydrous ethanol for cleaning for 1 min; take it out and put it into acetone for cleaning for 1 min; rinse with ultrapure water; blow dry the surface water residue with a nitrogen gun; place it on a 200 °C hot plate to dry the water film; the time is 20 - 30 min.

[0042] Step 2: Coating of the first layer of photoresist. Modify the surface of the wafer with HDMS reagent to increase the adhesion between the photoresist and the silicon wafer substrate; pour about 15 ml of SU-8-2005 photoresist on the surface of the silicon wafer, manually level the surface of the silicon wafer; place it on the wafer holder of the spin coater; let it stand for 1 - 2 min; rotate at a low speed of 500 rpm for 10 s and at a high speed of 800 rpm for 18 s.

[0043] Step 3: Pre-baking ①. Place the spin-coated wafer on the baking table at 95 °C for 4 min; after heating is completed, take out the wafer and let it cool naturally, and measure the thickness to be 5 μm.

[0044] Step 4: First layer exposure ①. Use an ultraviolet irradiator to measure the irradiation intensity of the lithography machine at 20 mj / cm 2 , according to the irradiation energy required for a 5 μm thick photoresist is 100 mj / cm 2 Set the exposure time of the lithography machine to 5 s;

[0045] Step 5: Post-baking ①. Place the exposed wafer at 95 °C for 10 min to promote the cross-linking reaction in the exposed area of the lithography machine.

[0046] Step 6: Coating of the second layer of photoresist. After post-baking and cooling; pour SU-8-2025 photoresist on the surface, manually level the surface of the silicon wafer, place it on the wafer holder of the spin coater, let it stand for 1 - 2 min, rotate at a low speed of 500 rpm for 10 s, and at a high speed of 2000 rpm for 19 s.

[0047] Step 7: Soft Bake ②. Place the spin-coated wafer on the hotplate at 65°C for 3 min and then at 95°C for 16 min. After heating is complete, remove the wafer and let it cool naturally, then measure the thickness, which is 65 μm.

[0048] Step 8: Second Layer Exposure ②. Use an ultraviolet irradiator to measure the irradiation intensity of the lithography machine; according to the required irradiation energy for a 65-μm-thick photoresist, which is 180 mj / cm 2 Set the exposure time of the lithography machine to 9 s.

[0049] Step 9: Post-Bake ②. Place the exposed wafer at 65°C for 3 min and then at 95°C for 14 min.

[0050] Step 10: Development. Place the cooled wafer in the developer propylene glycol monomethyl ether acetate (PGMEA) for development. Note to shake the wafer to accelerate the removal of the photoresist. After completely removing the photoresist, repeat the process 2 - 3 times with fresh developer and then blow dry with a nitrogen gun.

[0051] Step 11: Hard Bake. Place the SU-8 mold (as Figure 3 shown) on the hotplate at 160°C for 20 min for high-temperature hard baking to ensure the influence of temperature on the mold within the 160°C operating range of the SU8 photoresist.

[0052] Step 12: Modification. Seal the container, place the fabricated SU-8 mold inside, and drop 2 - 3 drops of trimethylchlorosilane (TMCS) for volatile modification (hydrophobicity) to facilitate subsequent PDMS casting and demolding.

[0053] The specific steps for fabricating the filter membrane are as follows:

[0054] Step 1: Prepare the glue. Turn on the electronic scale; Mix A glue and B glue at a ratio of 10:1, with a weight of approximately 50 g. This process is carried out in a beaker, as Figure 4 shown.

[0055] Step 2: Mix the glue. Manually stir for 3 - 5 min, then place it in a vacuum drying oven and only evacuate the air to defoam for 10 - 15 min, as Figure 5 shown.

[0056] Step 3: Prepare the PDMS casting fixture. Check if the fixture is in good condition and place the processed SU-8 mold in the fixture, as Figure 6 shown.

[0057] Step 4: Equipment preparation. Set the temperature of the constant-temperature forced-air drying oven to 85°C. Try to use the forced-air type for higher curing efficiency.

[0058] Step 5: Cure the PDMS. Pour the degassed PDMS into the jig and let it stand for 1 min. Wait until the surface bubbles disappear, then place it in a thermostatic forced-air drying oven, ensuring the equipment is level. The curing time is 30 - 40 min.

[0059] Step 6: Demold. After the PDMS is cured, take out the jig; cool it to room temperature; then demold. The effect diagram of the PDMS micro-columns is as Figure 7 shown.

[0060] Step 7: Assemble and seal the chip. After the PDMS is poured, it forms the internal column cavity; bond it with the PET film (the PDMS is cut and punched with holes of 1 mm). Subsequently, place the bonded chip in the fixture, fix it properly to enhance the sealing effect, as Figure 8 shown.

[0061] Inject the photosensitive resin into the internal cavity of the chip, heat and cure it, and then cure it under ultraviolet light. This step uses an oven for heating.

[0062] After curing is completed, remove the fixture, tear off the PC film, and carefully remove the resin film with tweezers. The film is very brittle and requires great care. The obtained chip is as Figure 9 shown.

[0063] The specific steps for fabricating the filter are as follows:

[0064] Step 1: Design the PMMA fixture and chip as Figure 10 shown.

[0065] The upper cavity upper plate, the lower cavity lower plate, and the middle resin filter membrane are sealed by fixing and tightening with screws. E and F are the upper and lower threaded inlet and outlet connectors, with the upper one being the inlet and the lower one being the outlet, as Figure 11 shown.

[0066] The specific steps for fabricating the mold are as follows:

[0067] Step 1: Clean the silicon wafer. Immerse it in a mixed reagent of concentrated sulfuric acid and hydrogen peroxide at a ratio of 3:1 for 10 - 15 min, rinse it with ultrapure water, put it in anhydrous ethanol for cleaning for 1 min, take it out and put it in acetone for cleaning for 1 min, then rinse it with ultrapure water again, and dry the surface water residue with a nitrogen gun. Finally, place it on a 200 °C hot plate to dry the water film for 20 - 30 min.

[0068] Step 2: Coating the photoresist. Use the HDMS reagent to modify the surface of the wafer to increase the adhesion between the photoresist and the silicon wafer substrate. Pour about 15 ml of SU-8-2075 photoresist onto the surface of the silicon wafer, manually level the surface of the silicon wafer, place it on the wafer holder of the spin coater, let it stand for 1 - 2 min, rotate at a low speed of 500 rpm for 5 s, and then at a high speed of 2100 rpm for 20 s.

[0069] Step 3: Pre-baking ①. Place the spin-coated wafer on the baking table at 65°C for 7 minutes and then at 95°C for 45 minutes. After heating is complete, remove the wafer and let it cool naturally.

[0070] Step 4: Exposure. Measure the actual SU8 coating thickness with a film thickness gauge, which is 100 μm. Use an ultraviolet irradiator to measure the irradiation intensity of the lithography machine, which is 20 mj / cm 2 , and set the exposure time of the lithography machine to 12 s according to the required irradiation energy of 240 mj / cm for the photoresist with a thickness of 100 μm. 2

[0071] Step 5: Post-baking. Place the exposed wafer on the baking table at 65°C for 5 minutes and then at 95°C for 22 minutes to promote the cross-linking reaction in the exposed area of the lithography machine.

[0072] Step 6: Development. Place the cooled wafer in the developer propylene glycol monomethyl ether acetate (PGMEA) for development. Shake it to accelerate the removal of the photoresist. After completely removing the photoresist, repeat the process 2 - 3 times with fresh developer, and then blow it dry with a nitrogen gun.

[0073] Step 7: Hard baking. Place the SU-8 mold on the baking table at 160°C for high-temperature hard baking for 20 minutes.

[0074] Step 8: Modification. Seal the container; place the fabricated SU-8 mold inside; drop 2 - 3 drops of trimethylchlorosilane (TMCS) for volatile modification (hydrophobic removal); to facilitate the subsequent demolding of PDMS casting. The mold is as Figure 12 shown.

[0075] The specific steps for the overall chip assembly are as follows:

[0076] As Figure 13 shown,

[0077] a) Drive the sample at end a of the chip to b;

[0078] b) Connect the catheter at end b and connect it to end E;

[0079] c) The fluid at end E passes through the resin filter membrane and flows out from end F;

[0080] d) Connect the catheter at end F and connect it to end C;

[0081] e) Use negative pressure extraction to make the fluid at end C flow to end d.

[0082] Example 2

[0083] The whole blood of liver cancer patients (derived from the remaining whole blood after the patients' clinical tests in Zhongshan Hospital, with the informed consent of the patients and ethical review) was injected into the microfluidic chip designed as described in Example 1 at a low flow rate (60 μm / s) using a syringe pump. After washing the chip with PBS at the same flow rate, the chip was taken out and placed in a culture dish. Before immunofluorescence staining, the microfluidic chip was incubated with a blocking buffer of 5% BSA (prepared by dissolving 1 mg of BSA in 20 ml of TBS / Tween-20 solution) for 30 minutes. Reagents for detecting CTC markers EpCAM and Pan-CK, reagents for detecting nuclear marker DAPI, and reagents for detecting platelet marker CD41 (a mixture of 0.1 mg / mL Alexa Fluor 555-labeled Pan-ck, 0.1 mg / mL PE-labeled EpCAM primary antibody, and Alexa Fluor 647-labeled CD41 primary antibody) were incubated overnight, and then the excess antibodies were washed off with 1×PBS. The nuclei were stained with 8.4 μM nuclear stain DAPI for 10 minutes, and the chip was washed with 1×PBS. Subsequently, it was fixed with 4% paraformaldehyde for 10 minutes, and then permeabilized with 1% Triton-X PBS solution for 10 minutes. Finally, the chip was placed between two glass slides for inverted fluorescence microscopy imaging. Using an Eppendorf TransferMan 4r micromanipulator, platelet-adherent CTCs identified by micromanipulation directly from the chip (identification criteria: CTCs were identified by EpCAM, Pan-CK, and DAPI staining, platelets were identified by CD41 staining, and only platelet-adherent CTCs closely adhering to the CTC cell membrane were captured) were transferred to PBS buffer for secondary verification with a confocal fluorescence microscope. The confocal fluorescence microscopy imaging is shown as Figure 14 shown (EpCAM / CK staining appears red, CD41 staining appears yellow, DAPI staining appears blue, and the merged image shows platelet-adherent CTCs presenting all three colors simultaneously).

[0084] Example 3

[0085] The performance of the chip was verified using a simulated blood sample. To test and optimize the operating performance of the microfluidic chip designed as described in Example 1, we injected human liver cancer cell line (MHCC-97L)-platelet adhesion clusters generated artificially into a healthy donor blood sample, which was derived from the remaining whole blood after the patients' clinical tests in Zhongshan Hospital, with the informed consent of the patients. This process was approved by the Ethics Committee of Zhongshan Hospital, Fudan University.

[0086] The low-metastatic human liver cancer cell line MHCC-97L was purchased from the Chinese Academy of Sciences.

[0087] Preparation process of artificially generated human liver cancer cell line (MHCC-97L)-platelet adhesion clusters is as follows:

[0088] 1. Cell line preparation:

[0089] Resuscitate MHCC-97L cells from liquid nitrogen and culture them to the logarithmic growth phase according to the conventional culture method.

[0090] Culture medium: DMEM containing 10% fetal bovine serum (FBS), supplemented with 1% double antibiotics (penicillin and streptomycin).

[0091] 2. Platelet preparation:

[0092] Extract platelets from the anticoagulated blood of healthy volunteers (using sodium citrate as anticoagulant).

[0093] Use centrifugation to prepare platelet-rich plasma (PRP), namely 200g, 15 minutes.

[0094] Further centrifuge PRP at 1000g for 10 minutes, discard the supernatant, and obtain platelet precipitate.

[0095] Resuspend platelets in the culture medium containing 0.5% FBS, and adjust the concentration to 1×10^8 / mL.

[0096] Experimental procedures

[0097] 3. Co-culture of cells:

[0098] (1) Cell seeding:

[0099] Inoculate 97L cells into a 6-well plate at a density of 1×10^5 cells / mL.

[0100] Place it in an incubator at 37°C and 5% CO 2 and culture until the cells adhere to the wall.

[0101] (2) Addition of platelets:

[0102] Add the platelet suspension to 97L cells according to the ratio of platelets to tumor cells (such as 50∶1 or 100∶1).

[0103] Gently mix evenly, avoiding shear force on the cells.

[0104] (3) Co-culture:

[0105] Co-culture in an incubator at 37°C and 5% CO 2 for 12 hours. During this period, gently shake the culture plate to promote the adhesion of platelets to tumor cells.

[0106] 4. Subsequent treatment

[0107] (1) Removal of non - adherent platelets:

[0108] Gently wash 2 - 3 times with PBS to remove platelets that have not adhered to tumor cells.

[0109] (2) Detection of platelet - adherent CTCs:

[0110] Microscopic observation: Observe the adhesion of platelets to tumor cells through an optical microscope or a fluorescence microscope.

[0111] (3) Prepare the platelet - adherent CTCs after digestion

[0112] Digest the cells in the six - well plate with 1% trypsin to prepare the platelet - adherent CTCs for standby.

[0113] Then, the platelet - adherent cell clusters are labeled with fluorescent dyes (specifically as described in Example 2) before or after treatment for positive identification. The whole blood containing the platelet - adherent cell clusters is passed through the microfluidic chip placed in the filter by an injection pump, rinsed with phosphate - buffered saline (PBS), and the cells on the device are subjected to immunofluorescent staining. The captured platelet - adherent cell clusters are identified through a fluorescence microscope in the microwells.

[0114] To determine the capture efficiency of the microfluidic chip at different flow rates (60μm / s, 300μm / s, and 600μm / s), we imaged and compared the platelet - adherent cell clusters entering and exiting the device. We constructed a two - channel microfluidic interface to image each treated platelet - adherent cell cluster under a microscope, which enabled us to simultaneously track the entry and exit of fluorescent platelet - adherent cell clusters in whole blood in the same field of view. In these experiments, we chose to stain the cell nuclei rather than the cell membranes or cytoplasm to more clearly distinguish the cells and more accurately count the number of cells in the cell clusters.

[0115] Table 2. Statistical table of the capture efficiency of the microfluidic chip at different flow rates

[0116]

[0117]

[0118] Secondly, the influence of pore size in the microfluidic chip on the separation of platelet-adherent cell clusters was further optimized. We designed pore sizes of 12 μm, 15 μm, and 18 μm, and prepared the microfluidic chip as described in Example 1. In the test, we still used the whole blood sample of the human hepatocellular carcinoma cell line (MHCC-97L)-platelet adhesion clusters for the experiment. The results showed that the adjusted opening size presented a trade-off between cluster capture sensitivity and specificity. The test was carried out at the optimal flow rate of 60 μm / s, and the test results are shown in the following table. It can be found that some CTC-platelet adhesion structures were missed when the pore size was 18 μm. When the pore size was 12 μm, although the capture efficiency was high, it would cause the pore to be easily blocked or contaminated by white blood cells. Considering the data of purity, clogging degree, and capture efficiency, we confirmed that the 15-μm mesh opening was the best design choice.

[0119] Table 3. Statistical table of capture efficiency of microfluidic chips with different pore sizes

[0120]

[0121] To address the current limitations in the separation and research of platelet-adherent CTCs, the above-mentioned platelet-adherent CTC separation chip was developed. The chip captures CTC-other cell complexes in micropores through physical dimensions, while single cells in the blood will pass through the micropores. Once the platelet-adherent CTCs enter the pores, they will be restricted on the grid. The micron-wide grid lines ensure that platelet-adherent CTCs enter different pores; a flow rate 10 times lower than the physiological free flow rate in human capillaries can prevent the dissociation of platelet-adherent CTCs; the chip design adopts a hexagonal arrangement to ensure the maximum number of micropores. In the present invention, 150,000 micropores are evenly distributed on a membrane with a diameter of 5 cm, realizing gentle treatment of platelet-adherent CTCs and providing a high processing rate, meeting the throughput requirements of clinical applications. After separation, multi-color immunofluorescence imaging of platelet-adherent CTCs is performed to obtain platelet-adherent CTCs for subsequent research on functions and molecular detection. The present invention can also be extended to the separation of homotypic CTC clusters or heterotypic CTC clusters, having good clinical application value and practical significance.

[0122] In summary, compared with the prior art, the present invention has the following advantages:

[0123] 1. The microfluidic chip of the present application realizes the in-situ separation of platelet-adherent CTCs in blood and integrates the sorting and detection of platelet-adherent CTCs.

[0124] 2. The microfluidic chip of the present application does not require targeting tumor-specific antigens, but instead physically identifies platelet-adherent CTCs from untreated whole blood specimens based on a micro-well design, with a relatively low cost.

[0125] 3. The present application passes through the chip within a specific flow rate range, which can not only prevent the dissociation of platelet-adherent CTCs caused by too high a flow rate but also avoid the clogging of micro-wells caused by too low a flow rate, ensuring the detection accuracy while not affecting the high throughput of the experiment.

[0126] 4. Through multi-color immunofluorescence staining, precise capture of target platelet-adherent CTCs can be achieved under a microscope, with simple operation, providing technical support for subsequent mechanism and function research.

[0127] 5. The chip design of the present application adopts a hexagonal arrangement to ensure the maximum number of micro-wells. The present invention evenly distributes 150,000 micro-wells on a membrane with a diameter of 5 cm, achieving gentle treatment of platelet-adherent CTCs while providing a high processing rate and meeting the throughput requirements of clinical applications.

[0128] 6. Without changing the existing structure, the chip of the present application can also be extended to other CTC adhesion structures, such as CTC-CTC clusters, CTC-neutrophil adhesion structures, or CTC-red blood cell adhesion structures, etc., simply by changing the pore size.

[0129] 7. The CTC adhesion structure captured by the chip of the present application can enrich target cells from whole blood under short-term and gentle conditions, ensuring cell viability to the greatest extent and solving the current clinical problem of inability to obtain highly viable CTCs.

Claims

1. A microfluidic chip, the chip comprising a porous substrate, the porous substrate comprising a microcavity with a pore size of 30-40 microns, and the microcavity comprising a micropore with a pore size of 12-18 microns.

2. The microfluidic chip of claim 1, wherein the porous substrate comprises a membrane.

3. The microfluidic chip as claimed in claim 1, wherein the porous substrate comprises a resin, preferably PDMS.

4. The microfluidic chip as claimed in claim 1, wherein the depth of the micropores is 1-100 microns, preferably 2-50 microns, more preferably 3-10 microns; and / or the depth of the microcavity is 20-100 microns, preferably 30-80 microns, more preferably 40-60 microns.

5. The microfluidic chip as claimed in claim 1, wherein the pore size of the microcavity is 33-37 microns, preferably 35 microns; and / or the pore size of the micropore is 14-16 microns, preferably 15 microns.

6. A method for isolating platelet-adherent circulating tumor cells (CTCs), the method comprising: (a) obtaining a microfluidic chip as described in any one of claims 1 to 5; (b) adding a sample to the microfluidic chip; (c) detecting platelet-adherent CTCs captured in the microfluidic chip; and (d) Obtaining platelet-adherent CTCs captured in the microfluidic chip.

7. The method of claim 6, wherein the sample comprises whole blood.

8. The method of claim 6, wherein the flow rate of adding the sample in step (b) is 30-600 μm / s, preferably 60-300 μm / s, more preferably 60 μm / s.

9. The method of claim 6, wherein in step (c), the captured platelet adhesion CTC is detected by immunolabeling; preferably, the immunolabeling comprises reagents for detecting CTC markers, such as reagents for detecting EpCAM, Pan-CK, reagents for detecting cell nuclear markers, such as reagents for detecting DAPI, and reagents for detecting platelet markers, such as reagents for detecting CD41.

10. The method of claim 6, wherein step (d) comprises obtaining the captured platelet-adherent CTCs by micromanipulation.

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