Capture device suitable for capturing rare cells and method of making same

Abstract

The application discloses a capturing device suitable for capturing rare cells and a preparation method thereof. The capturing device suitable for capturing rare cells comprises a substrate with at least a metal surface layer, a coating layer arranged on the surface of the substrate and a capturing agent connected to the coating layer, the capturing agent can specifically combine with target rare cells, and the coating layer comprises mercapto polyethylene glycol carboxyl and a mercapto compound. The application can reduce non-specific adsorption of background cells and proteins, and improve the purity of captured target cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a capture device suitable for capturing rare cells and a preparation method thereof. BACKGROUND

[0002] Rare cells refer to cells with important biological functions or clinical detection significance in a biological sample. Traditional capture methods of rare cells include Cell Search type immune capture enrichment and physical enrichment. In the enrichment process of rare cells, background / junk cells interfere with the capture and judgment of rare cells, and affect the purity of captured rare cells. At present, some capture devices such as capture screens are added with physical coatings and chemical coatings to improve the capture efficiency of cells. The binding strength of the physical coating is low, and the chemical coating is irreversible, making it difficult to release the captured rare cells. On the other hand, while capturing target cells, background cells are also adsorbed onto the capture screen, and the purity of the captured rare cells is low. In addition, the capture device causes different degrees of damage to the rare cells, which affects the activity of the captured cells and downstream applications.

[0003] The applicant proposed a capture screen in 2020, see CN111175503A. The capture screen includes a screen skeleton and a capture agent arranged on the screen skeleton. The capture agent can specifically bind to target cells or biological molecules. The screen skeleton has metal atoms. The screen skeleton and the capture agent are connected by a thiolated substance. The thiolated substance is combined with the screen skeleton by forming a metal-sulfur bond through reaction, and is combined with the capture agent by forming an amide bond through reaction. The thiolated substance is one or a combination of thiolated sodium alginate and thiolated hyaluronic acid. The capture screen can conveniently elute the captured cells. However, when used for enriching rare cells, background cells are also adsorbed onto the capture screen, and the purity of the captured target rare cells is low. SUMMARY

[0004] In view of at least one of the above technical problems, the purpose of the present application is to provide a capture device suitable for capturing rare cells and a preparation method thereof. The device can reduce the non-specific adsorption of background cells and proteins, and improve the purity of the captured target cells.

[0005] The first aspect of the present application provides a capture device suitable for capturing rare cells, which includes a substrate with at least a metal surface, a coating arranged on the surface of the substrate, and a capture agent connected to the coating. The capture agent can specifically bind to target rare cells. The coating includes thiol polyethylene glycol carboxyl and thiolated substances.

[0006] Preferably, the weight ratio of the mercapto-polyethylene glycol carboxyl and the mercapto-substance is 12:1 to 1:2. More preferably, the weight ratio of the mercapto-polyethylene glycol carboxyl and the mercapto-substance is 10:1 to 1:2, further 5:1 to 1:1, more further 2:1 to 1:1.

[0007] Preferably, the weight average molecular weight of the mercapto-polyethylene glycol carboxyl is 0.11 to 29 kDa. More preferably, the mercapto-polyethylene glycol carboxyl is composed of two or more kinds of mercapto-polyethylene glycol carboxyl with different weight average molecular weights. Further, the weight average molecular weight of the first mercapto-polyethylene glycol carboxyl is 11 to 29 kDa, preferably 15 to 25 kDa; the weight average molecular weight of the second mercapto-polyethylene glycol carboxyl is 1.1 to 2.9 kDa, preferably 1.5 to 2.5 kDa; and the weight average molecular weight of the third mercapto-polyethylene glycol carboxyl is 0.11 to 0.29 kDa, preferably 0.15 to 0.25 kDa.

[0008] In a specific and preferred embodiment, the mercapto-polyethylene glycol carboxyl includes a mercapto-polyethylene glycol carboxyl with a first weight average molecular weight of 11 to 29 kDa and a mercapto-polyethylene glycol carboxyl with a second weight average molecular weight of 1 to 9 kDa. More preferably, the molar ratio of the mercapto-polyethylene glycol carboxyl with the first weight average molecular weight and the mercapto-polyethylene glycol carboxyl with the second weight average molecular weight is (1 to 3):(1 to 3), more preferably (1 to 2):(1 to 2). The molar ratio of the mercapto-polyethylene glycol carboxyl with the first weight average molecular weight and the mercapto-polyethylene glycol carboxyl with the second weight average molecular weight can be 1:2, 1:1, 2:1, 1:3 or 3:1.

[0009] In another specific and preferred embodiment, the mercapto-polyethylene glycol carboxyl further includes a mercapto-polyethylene glycol carboxyl with a third weight average molecular weight of 0.1 to 0.9 kDa. Further, the weight ratio / molar ratio of the mercapto-polyethylene glycol carboxyl with the first weight average molecular weight, the mercapto-polyethylene glycol carboxyl with the second weight average molecular weight, and the mercapto-polyethylene glycol carboxyl with the third weight average molecular weight is (1 to 3):(1 to 3):(1 to 3), for example 1:1:1, 1:2:1, 1:2:2, 2:1:1, 2:2:1 or 2:1:2.

[0010] Preferably, the mercapto-substance includes mercapto-substance sodium hyaluronate or mercapto-substance sodium alginate. The coating can be cleaved, thereby facilitating the elution of the captured cells from the substrate without damaging the cells, with a high elution efficiency.

[0011] Preferably, the mercapto-substance has a mercapto-substance rate of 5 to 45%, and a molecular weight of 5 to 200 kDa.

[0012] More preferably, the thiolation rate of the thiolate is 15-25%, and the molecular weight of the thiolate is 9-39 kDa.

[0013] Optionally, the substrate comprises a mesh, a plate or a microfluidic chip. The substrate is made of metal or a metal alloy.

[0014] In a specific and preferred embodiment, the substrate is a mesh. More preferably, the mesh has a pore size of 10-50 μm. Further, the mesh has a pore size of 10-20 μm, which is suitable for the capture of immune cells.

[0015] In a more preferred embodiment, the mesh is a stainless steel mesh.

[0016] In another more preferred embodiment, the mesh comprises a stainless steel mesh skeleton and a protective layer coated on the mesh skeleton, and the protective layer is a noble metal layer. Further, the protective layer is a gold layer.

[0017] Preferably, the capture agent comprises at least one of a protein and a nucleic acid. More preferably, the protein comprises an antibody. Further, the antibody comprises at least one of a panCK antibody, an EPCAM antibody and a CD3 antibody. Rare cells and the like can be captured by the antibody and the like into the device through specific binding between the capture agent and the antibody, and then eluted from the substrate by lysis of the coating layer for subsequent analysis, downstream application and the like.

[0018] Preferably, the coating layer is formed by incubation of a mixed solution of thiol polyethylene glycol carboxyl and thiolate.

[0019] Preferably, the solid content of the thiolate is 1-20%. More preferably, the solid content of the thiolate is 5-20%, further 8-15%, and more further 10-15%. The solid content of the thiolate can be adjusted to adjust the thickness of the coating layer.

[0020] Preferably, the thiolate is prepared by dissolving sodium hyaluronate and / or sodium alginate in a buffer solution, adding an amino-containing disulfide compound and a carboxyl activator for stirring and reaction, and then adding a disulfide bond reducing agent. More preferably, sodium hyaluronate and / or sodium alginate are dissolved in a 2-morpholinoethanesulfonic acid solution, 3,3'-dithiodi(propionic hydrazine) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added, stirring is performed until complete dissolution and stirring and reaction are performed, and after the reaction is completed, tris(2-carbonyl ethyl) phosphonium hydrochloride is added.

[0021] Preferably, the rare cells are immune cells.

[0022] In a specific and preferred embodiment, the substrate is a sieve net, the sieve net comprises a stainless steel sieve net framework and a gold layer incubated on the surface of the stainless steel sieve net framework, the pore size of the sieve net is 10-50 μm; the coating layer comprises mercapto polyethylene glycol carboxyl and mercapto sodium hyaluronate, the mercapto polyethylene glycol carboxyl comprises mercapto polyethylene glycol carboxyl with a weight average molecular weight of 15-25 kDa, mercapto polyethylene glycol carboxyl with a weight average molecular weight of 1.5-2.5 kDa and mercapto polyethylene glycol carboxyl with a weight average molecular weight of 0.1-0.9 kDa; the capture is an antibody capable of specifically binding to immune cells.

[0023] The application further provides a preparation method of the above-mentioned capture device suitable for capturing rare cells, comprising the following steps: incubating mercapto on the surface of the substrate first and then incubating mercapto polyethylene glycol carboxyl, or incubating mercapto polyethylene glycol carboxyl on the surface of the substrate first and then incubating mercapto, to form a coating layer on the surface of the substrate; connecting the capture on the coating layer.

[0024] Preferably, the incubation is performed with oscillation, the oscillation rate is 180-220 rpm, and the incubation time is 1-3 h.

[0025] More preferably, the capture is connected to the coating layer by the following steps: freeze-drying the substrate coated with the coating layer, and washing; dissolving N-hydroxysuccinimide in a 2-morpholinoethanesulfonic acid solution to obtain a first solution, dissolving 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in a 2-morpholinoethanesulfonic acid solution to obtain a second solution, mixing the first solution and the second solution to obtain a blended activator solution; contacting the blended activator solution with the substrate, and incubating by standing; preparing an incubation liquid containing the capture and incubating, and incubating the capture on the coating layer.

[0026] Herein, the rare cells refer to cells with a small amount but important in blood and tissues, such as various immune cells in PBMC, T cells, CAR-T cells, circulating fetal cells, nucleated red blood cells, stem cells, circulating tumor cells, etc.

[0027] Herein, the device includes but is not limited to a capture sieve and a capture chip.

[0028] The application adopts the above scheme, and has the following advantages compared with the prior art:

[0029] The capturing device of the present application, on the surface of the metal base layer, is provided with a coating layer including mercapto polyethylene glycol carboxyl and mercapto, which can indirectly improve the purity of the target cells by reducing the non-specific adsorption of background cells and proteins; on the other hand, the functional groups of the coating layer can be further activated, coupled with antibodies, and specifically react with antigens on the target cells to specifically capture the target cells and directly improve the purity of the target cells; through the synergistic effect of the above two aspects, the non-specific adsorption of background cells and proteins is reduced under the premise of ensuring the capture efficiency of the target cells, and the purity of the captured target cells is improved. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1a 、 1b , 1c, 1d respectively show the microscope photos of the background cells captured and adsorbed on the screen of the comparative example, example 1, example 3, example 4.

[0032] Figure 2 The fluorescence and bright field images of region A of the capturing screen of example 11 are shown.

[0033] Figure 3 The fluorescence and bright field images of region A of the capturing screen of comparative example 2 are shown. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application.

[0035] As shown in the specification and claims, the term "comprising" and "including" only indicates the inclusion of the steps and elements explicitly identified, and these steps and elements do not constitute an exclusive list, and the method or device can also include other steps or elements. The term "and / or" used herein includes any combination of one or more related listed items.

[0036] Example 1: Preparation of cell capturing screen

[0037] (1) The cut stainless steel gilded screen was first soaked in ethanol for 2 min, then transferred to a clean glass beaker containing a mixture of water, 25% ammonia water, and 30% hydrogen peroxide (volume ratio 5:1:1) in a cleaning solution. The beaker was sealed and placed in an oil bath at 75°C for 5 min. After cleaning, the screen was sequentially soaked in deionized water, ethanol, and ethanol, then taken out and dried with high-purity nitrogen.

[0038] (2) 3 mL of thiolated sodium hyaluronate (HA, 5 mg / mL) was added to a 5 mL EP tube. Then 18 pieces of the cleaned and treated stainless steel gilded screen were added to the EP tube, and incubated for 2 h on a shaker at 200 rpm. The screen was taken out and reserved for use.

[0039] (3) 3 mL of thiolated polyethylene glycol carboxyl (HS-PEG-COOH (molecular weight Mw=20K Da)) solution (5 mg / mL) was added to a 5 mL EP tube. Then the screen treated in the previous step was added to the EP tube, and incubated for 1 h on a shaker at 200 rpm.

[0040] (4) The polymer screen after HA and HS-PEG-COOH incubation was cleaned by repeated rinsing (3 times of replacement washing) with 3 mL of PBS solution. Then the liquid on the surface of the screen was removed, and the screen was placed in the center of a 24-well plate.

[0041] (5) For the activation of one piece of polymer screen, 0.348 mg of Sulfo-NHS (Chinese name: N-hydroxysulfosuccinimide) was dissolved in 20 μL of 2-morpholinoethanesulfonic acid solution (0.05 M, pH=6), and 0.609 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was dissolved in 35 μL of 2-morpholinoethanesulfonic acid buffer. After complete dissolution, the two were mixed to obtain 55 μL of mixed activator solution. Then, the mixed activator solution was added dropwise to the surface of the screen, and incubated for 30 min in a light-shielded room at room temperature. The activated chip was cleaned by 2 times of replacement washing with 500 μL of phosphate buffer solution. Then the liquid on the surface of the screen after washing was removed, and the screen was placed in a new 24-well plate.

[0042] (6) 2 μL of CD3 antibody was mixed with 50 μL of MES solution (0.05 M, pH=6) to prepare an antibody incubation solution. The prepared antibody incubation solution was added to the well plate containing the chip, and a total of 52 μL was added. Incubation was carried out at 37°C for 1.5 h.

[0043] Example 2: Preparation of cell capture screen

[0044] This example is basically the same as Example 1, except that steps (2) and (3) are different, which are as follows:

[0045] (2) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0046] (3) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0047] Example 3: Preparation of cell capture sieve

[0048] This example is essentially the same as Example 1, except that steps (2) and (3) are different, and these two steps are as follows:

[0049] (2) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0050] (3) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0051] Example 4: Preparation of cell capture sieve

[0052] This example is essentially the same as Example 1, except that steps (2) and (3) are different, and these two steps are as follows:

[0053] (2) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0054] (3) Take 3 mL of HS-PEG-COOH (Mw = 2K Da) solution (5 mg / mL) and add to a 5 mL EP tube. Then add 12 pieces of the washed sieve to the EP tube and incubate for 1 h with blending at 200 rpm on a shaker. Remove the sieves for later use.

[0055] Example 5: Preparation of cell capture sieve

[0056] This example is substantially the same as Example 1, except that steps (2) and (3) are different, which are as follows:

[0057] (2) Take 3 mL of the synthesized and verified sodium hyaluronate thiol, add to a 5 mL EP tube. Then add 18 pieces of the washed and treated screen mesh to the EP tube, and incubate for 2 h on a shaker at 200 rpm. Take out the screen mesh for standby.

[0058] (3) Take 1 mL of HS-PEG-COOH (molecular weight Mw=20K Da) solution (5 mg / mL), 1 mL of HS-PEG-COOH (molecular weight Mw=2K Da) solution (5 mg / mL), and 1 mL of HS-PEG-COOH (molecular weight Mw=0.2K Da) solution (5 mg / mL) for a total of 3 mL, and add to a 5 mL EP tube. Then add the screen mesh treated in the previous step to the EP tube, and incubate for 1 h on a shaker at 200 rpm.

[0059] Example 6: Preparation of a cell capture screen

[0060] This example is substantially the same as Example 1, except that steps (2) and (3) are different, which are as follows:

[0061] (2) Take 3 mL of the synthesized and verified sodium hyaluronate thiol, add to a 5 mL EP tube. Then add 18 pieces of the washed and treated screen mesh to the EP tube, and incubate for 2 h on a shaker at 200 rpm. Take out the screen mesh for standby.

[0062] (3) Take 1 mL of HS-PEG-COOH (molecular weight Mw=20K Da) solution (5 mg / mL), 1 mL of HS-PEG-COOH (molecular weight Mw=2K Da) solution (0.5 mg / mL), and 1 mL of HS-PEG-COOH (molecular weight Mw=0.2K Da) solution (0.005 mg / mL) for a total of 3 mL, and add to a 5 mL EP tube. Then add the screen mesh treated in the previous step to the EP tube, and incubate for 1 h on a shaker at 200 rpm.

[0063] Example 7: Preparation of a cell capture screen

[0064] This example is substantially the same as Example 1, except that step (6) is different, which is as follows:

[0065] (6) 7 μL of CD3 antibody is mixed with 50 μL of MES solution (0.05 M, pH=6) to prepare an antibody incubation solution. Add 57 μL of the prepared antibody incubation solution to the well plate where the chip is located, and incubate at 37°C for 2 h.

[0066] Comparative Example 1

[0067] (1) The cut stainless steel gold-plated screen was first soaked in ethanol for 2 min, then transferred to a clean glass beaker containing a mixture of water, 25% ammonia water, and 30% hydrogen peroxide (cleaning solution prepared fresh) in a volume ratio of 5:1:1. The beaker was sealed and placed in an oil bath at 75°C for timed heating for 5 min. After cleaning, the screen was sequentially soaked in deionized water, ethanol, and ethanol, then taken out and dried with high-purity nitrogen.

[0068] (2) 3 mL of sulfhydrylated sodium hyaluronate (HA, 2.5 mg / mL) was added to a 5 mL EP tube. Then 18 pieces of the cleaned and treated stainless steel gold-plated screen were added to the EP tube, and incubated for 2 h on a shaker at 200 rpm.

[0069] (3) The HA-incubated polymer screen was cleaned by repeatedly rinsing (3 times of replacement washing) with 3 mL of PBS solution. Then the liquid on the surface of the screen was removed and placed in the center of a 24-well plate.

[0070] (5) 0.348 mg of Sulfo-NHS (Chinese name: N-hydroxysulfosuccinimide) was dissolved in 20 μL of 2-morpholinoethanesulfonic acid solution (0.05 M, pH = 6), and 0.609 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) was dissolved in 35 μL of 2-morpholinoethanesulfonic acid buffer. After complete dissolution, the two were mixed to obtain 55 μL of mixed activator solution. Then, the mixed activator solution was added dropwise to the surface of the screen, and incubated in a light-shielded room at room temperature for 30 min. The activated chip was cleaned by 2 times of replacement washing with 500 μL of phosphate buffer solution. Then the liquid on the surface of the screen after washing was removed and placed in a new 24-well plate.

[0071] (6) 2 μL of CD3 antibody was mixed with 50 μL of MES solution (0.05 M, pH = 6) to prepare an antibody incubation solution. The prepared antibody incubation solution was added to the well plate containing the chip, a total of 52 μL, and incubated at 37°C for 1.5 h.

[0072] Rare cell capture experiment 1

[0073] (1) Provide background cells

[0074] Jurkat cells (Ca. 2400 W / mL) were filtered with a 400-mesh screen, and the cell filtrate was collected and re-counted for use.

[0075] (2) Provide target rare cells

[0076] MCF7 (Ca.10W / 1mL) cells were incubated with carboxyfluorescein diacetate succinimidyl ester (CFSE, 1 μL / 10 mM) at 37℃ for 10 min, and then 5 mL of cold medium (DMEM) was added to neutralize the dye at room temperature for 5 min. The cells were centrifuged at 400g for 5 min, and the supernatant was discarded. The cells were resuspended with 5 mL of fresh medium, centrifuged at 400g for 5 min, and repeated once. Finally, the cells were resuspended with 1 mL of fresh medium and stored for use.

[0077] (3) Capture screen processing

[0078] The capture screens prepared in Examples 1, 3, 4, and Comparative Examples were first soaked with 600 μL of phosphate buffer solution for 1 min, and then the capture screens were clamped with tweezers and fixed on the capture clamp. The capture screen was connected to the assembly tool pipeline.

[0079] (4) Capture

[0080] 3 μL of diluted (1:20 dilution) MCF7 was taken and counted under a microscope at 477 nm. The counted MCF7 cell solution was poured into a centrifuge tube with 100 μL of phosphate buffer solution, 200W / 200 μL of Jurkat cells were added, and a total of about 303 μL of mixed cell solution was added to the tool at a speed of 50 μL / min for filtration and capture.

[0081] After capture was completed, the capture screen was removed and the background cell adsorption was observed with a fluorescence microscope. The photos were counted. Figure 1a The capture screen shown in the comparative example was not blocked, and the adsorbed background cells were more; Figure 1b The capture screen shown in Example 1 was not blocked, and the adsorbed background cells were more; Figure 1c The capture screen shown in Example 3 was not blocked, and the adsorbed background cells were more; Figure 1d The capture screen shown in Example 4 was not blocked, and the adsorbed background cells were more;

[0082] Examples 8-11

[0083] The capture screens of Examples 8-11 were prepared differently from Example 1:

[0084] (1) The molecular weight and proportion of HS-PEG-COOH are shown in Table 1 below:

[0085] (2) The antibody incubated on the chip surface was a panCK antibody, not a CD3 antibody, and the fluorescent dye was connected to the target cells through the panCK antibody. Specifically, the panCK antibody was incubated and dyed with Alexa 488 at 37℃ for 10 min.

[0086] The other steps were basically the same as in Example 1.

[0087] Table 1

[0088]

[0089] Comparative Example 2

[0090] The preparation of the capture screen of Comparative Example 2 is different from that of Example 1 in that:

[0091] (1) No HS-PEG-COOH, i.e. no HA coating on the gold-plated stainless steel screen mesh, and the Sulfo-NHS activation is performed immediately after washing;

[0092] (2) The antibody incubated on the chip surface is a panCK antibody instead of a CD3 antibody, and the fluorescent dye is linked to the target cells through the panCK antibody. Specifically, the panCK antibody is incubated with Alexa 488 at 37°C for 10 min for staining.

[0093] The other procedures are basically the same as those of Example 1.

[0094] Rare Cell Capture Experiment 2

[0095] The capture screens prepared in Examples 8-11 and Comparative Example are used to perform cell capture experiments according to the following procedures:

[0096] (1) Provide background cells

[0097] The Jurkat cells (Ca. 2400 W / mL) are filtered with a 400-mesh screen, and the cell filtrate is collected and re-counted for standby.

[0098] (2) Provide target rare cells

[0099] The SKBR3 (Ca. 10 W / 1 mL) cells are resuspended with 5 mL of culture medium, centrifuged at 400 g for 5 min, and repeated once. Finally, resuspended with 1 mL of fresh culture medium for standby.

[0100] (3) Capture screen treatment

[0101] The capture screens prepared in Examples 8-11 and Comparative Example are first soaked with 600 μL of phosphate buffer solution for 1 min, and then stored in a freeze-dried state. The capture screen is clamped with tweezers, placed on the capture clamp, clamped and fixed, and connected to the assembly tool pipeline.

[0102] (4) Capture

[0103] Take 3 μL of diluted (1:20 dilution) SKBR3 cells under a microscope to count, and use 100 μL of phosphate buffer solution to flush the counted SKBR3 cell liquid into a centrifuge tube, add 500 W / 200 μL of Jurkat cells, a total of about 303 μL of mixed cell liquid, and add it to the device at a speed of 50 μL / min for filtration capture.

[0104] After the capture is completed, remove the capture screen and transfer it to a fluorescence inverted microscope. First, use the 488 nm channel to observe the counted and captured SKBR3 cells, and take a photo for counting. After counting is completed, use 60 μL of 4', 6-diamidino-2-phenylindole (DAPI, 1 μg / mL) to stain the capture screen in situ at room temperature for 10 min. Then, transfer the DAPI-stained capture screen to the fluorescence inverted microscope, use the 385 nm channel to observe the background cell adsorption, and take a photo for counting.

[0105] The fluorescence images of the capture screen of Example 11 and the fluorescence and bright field images of the capture screen of Comparative Example 2 are shown in Figure 2 and Figure 3 . Figure 2 In FIG. 1, I-1 and I-2 are green fluorescence images of a certain area (i.e., area A) of the capture screen of Example 11 magnified by 10 times and 20 times, respectively, and there is a target cell in the area A; II-1 and II-2 are blue fluorescence images of the area A of the capture screen of Example 11 magnified by 10 times and 20 times, respectively, and all nucleated cells (including target cells and background cells) in the area A are marked by blue fluorescence; III-1 and III-2 are bright field images of the area A of the capture screen of Example 11 magnified by 10 times and 20 times, respectively. Figure 3 In FIG. 1, I-1 and I-2 are green fluorescence images of a certain area (i.e., area A) of the capture screen of Example 11 magnified by 10 times and 20 times, respectively, and there is a target cell in the area A; II-1 and II-2 are blue fluorescence images of the area A of the capture screen of Example 11 magnified by 10 times and 20 times, respectively, and all nucleated cells (including target cells and background cells) in the area A are marked by blue fluorescence; III-1 and III-2 are bright field images of the area A of the capture screen of Example 11 magnified by 10 times and 20 times, respectively. Figure 2 Figure 3 By comparing the blue fluorescence images (i.e., II-1 and II-2) of FIG. 1 and FIG. 2, the number of blue fluorescence points in the blue fluorescence image of the capture screen of Comparative Example 2 is more than that in the blue fluorescence image of the capture screen of Example 11; it can be known that there are a large number of background cells on the capture screen of Comparative Example 2, and the purity of the target cells on the capture screen is low, while the number of background cells on the capture screen of Example 11 is small and the purity of the target cells is high.

[0106] ​The counting results of the target cells and the background cells on the capture screen of examples 8-11 and comparative example 2 after the capture experiment are shown in Table 2 below.

[0107] Table 2

[0108]

[0109] As shown in Table 2, in the case that the number of the target cells is much more than that of the background cells, the capture screen of example 11 can ensure a high capture rate of the target cells, and the number of the background cells remaining on the capture screen is less, so that the purity of the target cells after capture is improved, and the capture screen is suitable for capturing rare cells. The capture efficiency of the capture screen of comparative example 2 is low, and the number of the background cells adsorbed on the capture screen is more, so that the purity of the target cells is low.

[0110] The above examples are only for illustrating the technical concept and characteristics of the present application, and are a preferred embodiment, and the purpose is that the person skilled in the art can understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent transformation or modification according to the principle of the present application should be covered in the protection scope of the present application.

Claims

1. A capture device suitable for capturing rare cells, comprising a substrate having at least a surface layer of metal, a coating disposed on the surface of the substrate, and a capture agent attached to the coating, the capture agent being capable of specifically binding to a target rare cell, characterized in that, The coating is formed by incubating a mixture of thiol polyethylene glycol carboxyl and thiolate, the thiol polyethylene glycol carboxyl is composed of thiol polyethylene glycol carboxyl with a first weight average molecular weight, thiol polyethylene glycol carboxyl with a second weight average molecular weight and thiol polyethylene glycol carboxyl with a third weight average molecular weight, the first weight average molecular weight is 20 kDa, the second weight average molecular weight is 1.5-2.5 kDa, and the third weight average molecular weight is 0.15-0.25 kDa, and the molar ratio of thiol polyethylene glycol carboxyl with the first weight average molecular weight, thiol polyethylene glycol carboxyl with the second weight average molecular weight, and thiol polyethylene glycol carboxyl with the third weight average molecular weight is 1:1:1; the thiolate is thiolated sodium hyaluronate or thiolated sodium alginate, the thiolation rate of the thiolate is 5-45%, and the molecular weight of the thiolate is 5-200 kDa; the rare cells are immune cells, and the capture is an antibody capable of specifically binding to immune cells.

2. The capture device of claim 1, wherein, The feeding weight ratio of the thiol polyethylene glycol carboxyl and the thiolate is 12:1-1:

2.

3. The capture device of claim 1, wherein, The substrate includes a screen, a plate or a microfluidic chip.

4. The apparatus of claim 1, wherein, The substrate is a screen, and the screen includes a stainless steel screen skeleton and a coating on the surface of the stainless steel screen skeleton, and the pore size of the screen is 10-50 µm.

5. The apparatus of claim 1, wherein, The thiol polyethylene glycol carboxyl is composed of three thiol polyethylene glycol carboxyls with weight average molecular weights of 20 kDa, 2 kDa and 0.2 kDa respectively in a molar ratio of 1:1:

1.

6. A method of producing a capture device suitable for capturing rare cells as claimed in any one of claims 1 to 5, characterised in that, The method comprises the following steps: incubating the thiolate on the surface of the substrate first and then incubating the thiol polyethylene glycol carboxyl, or incubating the thiol polyethylene glycol carboxyl on the surface of the substrate first and then incubating the thiolate, to form a coating on the surface of the substrate; and connecting the capture on the coating.

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