Preparation method of bionic circulating tumor cell separation chip and kit

Abstract

The invention relates to the technical field of detection, in particular to a preparation method of a bionic circulating tumor cell separation chip and a kit. Comprising the following steps: 1, soaking a hydrophobic modified chip in a polymer solution; 2, rapidly placing the chip soaked in the polymer solution in a nanostructure growth solution for growth; step 3, washing the grown chip with deionized water and drying the chip; 4, the cleaned and dried chip is placed in acid liquor to be treated; and step 5, placing the chip treated by the acid liquor in a modification liquid for specific isolated protein modification. According to the invention, the preparation and post-modification of the micro-nano composite structure chip can be efficiently realized on a large scale, and the chip modified by specific recognition molecules can realize efficient separation of the circulating tumor cells of the panthenic cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a preparation method and a kit for a bionic circulating tumor cell separation chip. Background Art

[0002] Currently, there are two main methods for separating circulating tumor cells:

[0003] First: Separate CTC cells based on size;

[0004] Second: Isolate CTCs after specifically binding to cell surface proteins.

[0005] The first method is fast, but due to problems such as CTC heterogeneity, it is difficult to have a high capture efficiency and the separation purity is not high;

[0006] The second method is the more classic positive separation method, but the capture efficiency of the more commonly used magnetic bead capture method is relatively poor, and the operation process is relatively complicated. In simulated / clinical circulating tumor cell samples, its separation efficiency is relatively low (the simulated circulating tumor cell separation method is to mix a certain amount of cancer cells with a quantitative separation magnetic bead, incubate and wash for a certain period of time, and then calculate the number of cancer cells after separation); and the nanostructure chip separation method, although more efficient, but the initial preparation process is complicated, so it has not formed an effective test kit.

[0007] Therefore, the present application proposes a preparation method and a kit for a bionic circulating tumor cell separation chip. Summary of the invention

[0008] In order to make up for the deficiencies of the prior art and solve the technical problems existing in the background technology, the present invention proposes a preparation method and a kit for a bionic circulating tumor cell separation chip.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for preparing a bionic circulating tumor cell separation chip comprises the following steps:

[0011] Step 1: Soak the hydrophobically modified chip in a polymer solution;

[0012] Step 2: quickly placing the chip soaked in the polymer solution in a nanostructure growth solution for growth;

[0013] Step 3: Rinse the grown chip with deionized water and dry it;

[0014] Step 4: placing the cleaned and dried chip in an acid solution for treatment;

[0015] Step 5: Place the acid-treated chip in a modification solution for specific protein separation modification.

[0016] Preferably, the selection of the chip includes: chip material, chip shape, chip area and chip surface structure;

[0017] Wherein, the chip material includes one of a glass substrate, a silicon substrate, a polystyrene substrate, a polymethyl methacrylate substrate, a polyimide substrate, a polyphenylene ether substrate, a cross-linked polyurethane foam, an iron substrate, a copper substrate, and an aluminum substrate;

[0018] When the chip is made of a hydrophobic material, no hydrophobic modification is required, and when the chip is made of a hydrophilic material, the chip needs to be hydrophobically modified;

[0019] The chip shape includes square, rectangular, circular, trapezoidal or other special shapes;

[0020] Chip area 0.25-100cm 2 ;

[0021] The chip surface structure includes roughness undulations with a roughness of Ra1-200, columnar structure or flat structure;

[0022] Specifically:

[0023] The chip is a rough glass sheet with micron undulations. In the hydrophobic modification of the chip, 1H, 1H, 2H, 2H-perfluorodecyltrimethoxysilane is used and treated in a vacuum oven for 24 hours.

[0024] Preferably, the polymer solution is a block polymer organic solvent solution with a concentration of 0.5-30%wt, the polymer is a combination of multiple polymers, including one or more combinations of block copolymers, random copolymers, and homopolymers, wherein the block polymer is composed of one or more hydrophilic segments and one or more hydrophobic segments, the random copolymer and the homopolymer have the same composition as the hydrophilic segment and the hydrophobic segment, and the organic solvent is a water-insoluble polar organic solvent.

[0025] Preferably, the hydrophobic segment comprises a polymer segment with high biocompatibility, and the hydrophilic segment comprises a polymer segment with strong hydrophilicity and good flexibility;

[0026] Among them, the hydrophobic segments include PCL, PLGA, PTMC, PLA, PS, PMMA, etc.; the hydrophilic segments include PEG, PVP, P4VP, PEI, etc.; the random copolymers include PLGA, etc., and the homopolymers include PCL, PTMC, PLA, PS, PMMA, PEG, PVP, P4VP, PEI, etc.;

[0027] The polymer is dissolved in an organic solvent at a concentration of 0.5-30%wt, and the block ratio of the block polymer is 1-100:1 hydrophobic:hydrophilic;

[0028] Organic solvents include ethyl acetate, dichloromethane, chloroform, dimethoxyethane, etc.;

[0029] Specifically:

[0030] The PCL-PEG-PCL triblock polymer composed of a hydrophobic segment PCL and a hydrophilic segment PEG was dissolved in ethyl acetate with a concentration of 2-10% wt, and dissolved by shaking on a shaker at a rate of 200 r / min, and the block ratio of the triblock polymer PCL:PEG was 20:1.

[0031] Preferably, the nanostructure growth liquid is prepared from a surfactant with a concentration of 0-5%wt and a water-soluble organic solvent aqueous solution, wherein the surfactant is used to maintain a uniform spreading state of the polymer solution, and the water-soluble organic solvent is used as a co-solvent to assist the organic solvent in the polymer solution to diffuse out of the water.

[0032] Preferably, the surfactant of the nanostructure growth liquid includes sodium dodecyl sulfate, polyvinyl alcohol, etc., and the surfactant concentration range is 0-10%wt, and the optimal range is: 0.5-2%wt;

[0033] The water-soluble organic solvents include methanol, ethanol, propanol, tetrahydrofuran, dioxane, cyclopentyl ether, ethylene glycol dimethyl ether, etc. The concentration range of the co-solvent is 0-70%wt, and the optimal range is: 0-10%wt.

[0034] Specifically:

[0035] The nanostructure growth solution is prepared from 2% wt sodium dodecyl sulfate and 5% wt tetrahydrofuran aqueous solution.

[0036] Preferably, the specific steps of step five are as follows: in step one, the chip is immersed for 20 minutes, and in step two, when the chip is in the nanostructure growth liquid, it needs to be grown in an environment heated to 50° C. for 6 hours.

[0037] Preferably, the specific steps of step five are as follows:

[0038] Treat with 1M dilute hydrochloric acid for 12 hours, then wash with PBS; place in EDC / NHS 10mg / mL MES solution, pH=6.0, treat for 3 hours, then wash with PBS; place in SA 10μg / mL PBS solution, treat for 3 hours, then wash with PBS; place in specific recognition protein solution, treat for 3 hours, then wash with PBS, and store at -20℃;

[0039] For different cancers, the specific recognition protein recognition sites are different, including EpCAM site, PSMA site, HER-2 site, Vimentin site, EGFR site, NsE site, a single recognition protein or a combination of multiple recognition proteins.

[0040] A chip kit suitable for the preparation method of the bionic circulating tumor cell separation chip described above, wherein the kit contains a cell fixative, a cell membrane permeation solution, a cell blocking solution, a PBS cleaning solution, and a stain; the stain includes a cancer cell stain, a leukocyte stain, a cell nucleus stain, and a cell immune stain PD-L1 stain;

[0041] The steps to isolate cells are as follows:

[0042] M1: First, incubate the blood sample with the modified chip. After a period of time, wash it with PBS for 3-5 times.

[0043] M2: Then, incubate with cell fixative, cell permeabilization solution, and cell blocking solution for a period of time, and wash with PBS three times each time;

[0044] M3: Then three to four stains are incubated in sequence for a period of time, and each time they need to be washed three times with PBS;

[0045] M4: The chip is then placed under a fluorescence microscope for reading to determine the number of circulating tumor cells in the blood sample.

[0046] Preferably, in M1, the cells are co-incubated at 37°C for 1 hour; in M2, the cells are incubated with a cell fixative at room temperature for 20 minutes, with a cell permeabilization solution at room temperature for 10 minutes, with a cell blocking solution at room temperature for 1 hour, with a cancer cell stain at room temperature for 30 minutes, with a leukocyte stain at room temperature for 2 hours, with a cell nucleus stain at room temperature for 10 minutes, and with a cell immunostaining PD-L1 stain at room temperature for 30 minutes.

[0047] The beneficial effects of the present invention are:

[0048] 1. The present invention grows a nanometer-level burr structure on the surface of a chip with a micrometer-level structure and constructs a micro-nano composite structure, so as to quickly and efficiently prepare a micro-nano composite structure chip on a large scale and perform chemical modification to facilitate subsequent CTC separation and capture.

[0049] 2. The present invention can realize the preparation and post-modification of micro-nano composite structure chips efficiently and on a large scale. After the modification of specific recognition molecules, the chip surface has a composite structure of micron-scale undulations and nano-scale burrs, wherein the micron-scale undulations can match the cell body, the nano-burrs can match the cell pseudopodia, and the specific recognition molecules can match the cell surface recognition objects. The triple matching synergy can realize the separation of circulating tumor cells with high efficiency and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A diagram of a reaction vessel for preparing the chip described in Example 1 of the present invention;

[0051] Figure 2 This is a photo of the chip after the chip preparation in Example 1 of the present invention is completed;

[0052] Figure 3 This is a SEM photo of the chip described in Example 1 of the present invention;

[0053] Figure 4 This is a schematic diagram of combining cells with a chip in Example 2 of the present invention;

[0054] Figure 5 This is a SEM photo of the cells combined with the Wiener structure on the chip surface after cell separation in Example 2 of the present invention;

[0055] Figure 6 This is a comparison chart of the separation efficiency of the kit at different separation times in Example 2 of the present invention;

[0056] Figure 7 This is a comparison chart of the separation efficiency of different cells by the kit in Example 2 of the present invention;

[0057] Figure 8 This is a comparison chart of the effects of different structures on cell separation efficiency in Example 3 of the present invention;

[0058] Fig. 9 This is a graph of cell separation efficiency in Example 4 of the present invention compared with the prior art. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0060] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The present invention is further described according to the accompanying drawings and specific embodiments of the present invention, and the preferred implementation methods and materials described in the present invention are only for demonstration purposes.

[0061] like Figure 1-Figure 9 As shown:

[0062] Embodiment 1:

[0063] Circulating tumor cell separation chip preparation process:

[0064] The single preparation in the laboratory has a chip surface area of ​​8cm2 and a preparation efficiency of 100 chips / hour. The graphene sheet structure chip developed in the early stage of the laboratory has a chip surface area of ​​1cm2 and a preparation efficiency of 10 chips / hour. The preparation process is simple, with two glass cylinders and a chip rack. 50 chips can be placed on the chip rack. The preparation reaction container is as follows Figure 1 As shown. A polymer solution is added to one glass jar and a nanostructure growth solution is added to another. The chip rack filled with chips is immersed in the polymer solution first, then taken out and put into the nanostructure growth solution. After growth, it can be rinsed with deionized water.

[0065] Dissolve the triblock polymer PCL-PEG-PCL (block ratio PCL:PEG is 20:1) in ethyl acetate (mass fraction 2%). Shake on a shaker at 200r / min to dissolve. The rough glass sheet with micron undulations was hydrophobically modified using 1H,1H,2H,2H-perfluorodecyltrimethoxysilane and treated in a vacuum oven for 24h. Insert the hydrophobically modified micron undulations of the rough glass sheet into the ethyl acetate solution of the triblock polymer PCL-PEG-PCL, soak for 20min, take it out, and quickly soak it in the nanostructure growth solution (2%wt sodium dodecyl sulfate and 5%wt tetrahydrofuran aqueous solution), then heat it to 50°C, grow for 6h, take it out, wash and dry it with deionized water. The chip photo after preparation is as follows Figure 2 As shown, the chip surface structure is as follows Figure 3 shown.

[0066] Circulating tumor cell separation chip modification process:

[0067] After being treated with 1M dilute hydrochloric acid for 12 hours, the cells were washed with PBS and placed in EDC / NHS 10 mg / mL MES solution, pH = 6.0. After being treated with PBS for 3 hours, the cells were washed with PBS and placed in SA 10 μg / mL PBS solution. After being treated with PBS for 3 hours, the cells were washed with PBS and placed in biotin-anti-EpCAM 1 μg / mL PBS solution. After being treated with PBS for 3 hours, the cells were washed with PBS and stored at -20°C.

[0068] Embodiment 2:

[0069] Circulating tumor cell separation chip separation and staining process:

[0070] The samples to be tested were directly incubated with the modified chip, incubated at 37°C for different time periods, rinsed with PBS, then incubated with cell fixative at room temperature for 20 min, rinsed with PBS, incubated with cell permeabilization solution at room temperature for 10 min, rinsed with PBS, incubated with cell blocking solution at room temperature for 1 h, rinsed with PBS, incubated with cancer cell stain at room temperature for 30 min, rinsed with PBS, incubated with leukocyte stain at room temperature for 2 h, rinsed with PBS, incubated with cell nuclear stain at room temperature for 10 min, rinsed with PBS, incubated with cell immunostaining PD-L1 stain at room temperature for 30 min, and rinsed with PBS. The separation effects at different times are as follows Figure 6 shown.

[0071] Calculation and simulation of circulating tumor cell separation efficiency: Obtain a laboratory-cultured cancer cell dispersion, count a certain number of cancer cells under a microscope, and after the separation and staining process, determine the number of cancer cells on the chip, so as to calculate the separation efficiency through the ratio.

[0072] By adjusting the specific recognition protein types, such as the PSMA site for prostate cancer, the EpCAM site for liver cancer, and the Vimentin site for kidney cancer, the kit achieves efficient separation of pan-cancer CTCs and has been extended to intestinal cancer, single-negative, double-negative, and triple-negative breast cancer, prostate cancer, lung cancer, high-metastasis lung cancer, bile duct cancer, kidney cancer, gastric cancer, liver cancer, esophageal cancer, thyroid cancer, pancreatic cancer, and neuroblastoma, all of which can achieve a separation efficiency of about 90%. Separation effect such as Figure 7 shown.

[0073] Embodiment three:

[0074] Preparation of separation chips with different structures

[0075] The micro-nano structure is as described in Example 1. The microstructure is prepared using a triblock polymer PCL-PEG-PCL (block ratio PCL: PEG is 5:1) and a chip, the nanostructure is prepared using a flat glass substrate and a triblock polymer PCL-PEG-PCL (block ratio PCL: PEG is 20:1), and the flat structure is prepared using a flat glass substrate and a triblock polymer PCL-PEG-PCL (block ratio PCL: PEG is 5:1). The other preparation processes are the same as those described in Example 1. After that, the cell separation and staining process is the same as in Example 2, and the separation efficiency is calculated. The separation effect is shown in FIG. Figure 8 shown.

[0076] Embodiment 4:

[0077] Comparison with CellSearch magnetic beads on the market

[0078] Prostate cancer tumor cell 22RV1 cells were selected as cancer cells in the simulated circulating tumor cell separation experiment. After culture, digestion and counting were performed to obtain a cell suspension of 10^5 cells / mL for separation. 1 mL of cell suspension was separated using this kit using the method of Example 2, and only the nuclear stain was used for staining by incubation at room temperature. CellSearch micron magnetic beads were used for cell separation, and the subsequent fixation and staining reagents needed to be configured on-site in the laboratory. Obtain CellSearch micron magnetic beads. According to the instructions, take 100 μL of magnetic bead dispersion and incubate it with 10 μg / mL biotin-anti-EpCAM specific antibody protein. After 45 minutes, wash it with PBS and adsorb it on the magnet three times. After discarding the supernatant, add 1 mL of cell suspension and let it stand for 45 minutes. After adsorbing it on the magnet for 10 minutes, wash it with PBS and adsorb it on the magnet three times to complete the separation. Then add 1 mL of fixative paraformaldehyde and treat it for 20 minutes. Wash it with PBS and adsorb it on the magnet three times. Add 1 mL of Triton X-100 and treat it for 10 minutes. Wash it with PBS and adsorb it on the magnet three times. Add cell blocking solution BSA solution and treat it for 120 minutes. Wash it with PBS and adsorb it on the magnet three times. Add cell nuclear stain and treat it for 10 minutes. Wash it with PBS and adsorb it on the magnet three times. Then count the separated cancer cells to calculate the separation efficiency. Fig. 9 As shown in the figure, the separation efficiency of this kit for prostate cancer tumor cells 22RV1 is much higher than that of the existing product CellSearch magnetic beads. At the same time, because the magnetic bead method needs to wait for the magnetic bead adsorption process, the overall operation time is much longer than the kit. Therefore, this kit has significant advantages in separation efficiency and operation efficiency.

[0079] Any matters not described in the present invention are applicable to the prior art.

Claims

1. A method for preparing a bionic circulating tumor cell separation chip, characterized in that: The following steps are involved: Step 1: Soak the hydrophobically modified chip in a polymer solution; Step 2: quickly placing the chip soaked in the polymer solution in a nanostructure growth solution for growth; Step 3: Rinse the grown chip with deionized water and dry it; Step 4: placing the cleaned and dried chip in an acid solution for treatment; Step 5: Place the acid-treated chip in a modification solution for specific protein separation modification.

2. The method for preparing a bionic circulating tumor cell separation chip according to claim 1, characterized in that: The selection of the chip includes: chip material, chip shape, chip area and chip surface structure; Wherein, the chip material includes one of a glass substrate, a silicon substrate, a polystyrene substrate, a polymethyl methacrylate substrate, a polyimide substrate, a polyphenylene ether substrate, a cross-linked polyurethane foam, an iron substrate, a copper substrate, and an aluminum substrate; When the chip is made of a hydrophobic material, no hydrophobic modification is required, but when it is made of a hydrophilic material, the chip needs to be hydrophobically modified; The chip shape includes square, rectangular, circular, trapezoidal or other special shapes; Chip area is 0.25-100cm2; The chip surface structure includes rough undulations with a roughness of Ra1-200, columnar structure or flat structure.

3. The method for preparing a bionic circulating tumor cell separation chip according to claim 1, characterized in that: The polymer solution is a polymer organic solvent solution with a concentration of 0.5-30%wt. The polymer is a combination of multiple polymers, including one or more combinations of block copolymers, random copolymers, and homopolymers, wherein the block polymer is composed of one or more hydrophilic segments and one or more hydrophobic segments, and the random copolymer and the homopolymer have the same components as the hydrophilic segment and the hydrophobic segment, and the organic solvent is a water-insoluble polar organic solvent.

4. The method for preparing a bionic circulating tumor cell separation chip according to claim 3, characterized in that: The hydrophobic segments include polymer segments with high biocompatibility, and the hydrophilic segments include polymer segments with strong hydrophilicity and good flexibility; The polymer is dissolved in an organic solvent at a concentration of 0.5-30% by weight, and the block ratio of the block polymer is 1-100:1, hydrophobic:hydrophilic.

5. The method for preparing a bionic circulating tumor cell separation chip according to claim 3, characterized in that: The nanostructure growth liquid is composed of a surfactant with a concentration of 0-5%wt and a water-soluble organic solvent aqueous solution, wherein the surfactant is used to maintain a uniform spreading state of the polymer solution, and the water-soluble organic solvent is used as a co-solvent to assist the organic solvent in the polymer solution to diffuse out of the water.

6. The method for preparing a bionic circulating tumor cell separation chip according to claim 5, characterized in that: The nanostructure growth liquid surfactant includes sodium dodecyl sulfate and polyvinyl alcohol, the surfactant concentration range is 0-10%wt, the water-soluble organic solvent includes methanol, ethanol, propanol, tetrahydrofuran, dioxane, cyclopentane ether, ethylene glycol dimethyl ether, and the cosolvent concentration range is 0-70%wt.

7. The method for preparing a bionic circulating tumor cell separation chip according to claim 1, characterized in that: The specific steps of step five are as follows: in step one, the chip immersion time is 0-60 minutes, and in step two, when the chip is in the nanostructure growth liquid, it needs to grow in an environment with a temperature of 0-80° C. for 0-12 hours.

8. The method for preparing a bionic circulating tumor cell separation chip according to claim 1, characterized in that: The specific steps of step five are as follows: After treating with 1M dilute hydrochloric acid for 12 hours, wash with PBS buffer; put into 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) 0-50 mg / mL 2-morpholineethanesulfonic acid (MES) solution, pH=6.0, treat for 0-6 hours, and wash with PBS; put into streptavidin (SA) 0-100 μg / mL PBS solution, treat for 0-6 hours, and wash with PBS; Place in specific recognition protein solution, treat for 0-6 hours, wash with PBS, and store at -20℃; For different cancers, the specific recognition protein recognition sites are different, including EpCAM site, PSMA site, HER-2 site, Vimentin site, EGFR site, NsE site, a single recognition protein or a combination of multiple recognition proteins.

9. A chip kit suitable for the method for preparing the bionic circulating tumor cell separation chip according to any one of claims 1 to 8, characterized in that: The kit contains a cell fixative, a cell membrane permeabilization solution, a cell blocking solution, a PBS cleaning solution, and a stain; the stain includes a cancer cell stain, a leukocyte stain, a cell nucleus stain, and a cell immune stain PD-L1 stain; The steps to isolate cells are as follows: M1: First, incubate the blood sample with the modified chip. After a period of time, wash it with PBS for 3-5 times. M2: Then, incubate with cell fixative, cell permeabilization solution, and cell blocking solution for a period of time, and wash with PBS three times each time; M3: Then three to four stains are incubated in sequence for a period of time, and each time they need to be washed three times with PBS; M4: The chip is then placed under a fluorescence microscope for reading to determine the number of circulating tumor cells in the blood sample.

10. The chip kit according to claim 9, characterized in that: In M1, the cells were incubated at 37°C for 0-3 h. In M2, the cells were incubated with the cell fixative at room temperature for 0-90 min, with the cell permeabilization solution at room temperature for 0-60 min, with the cell blocking solution at room temperature for 0-4 h, with the cancer cell stain at room temperature for 0-2 h, with the leukocyte stain at room temperature for 0-4 h, with the cell nuclear stain at room temperature for 0-90 min, and with the cell immunostaining PD-L1 stain at room temperature for 0-90 min.

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