Preparation method and kit for biomimetic circulating tumor cell separation chip

Abstract

The present invention relates to the field of detection technology, and in particular to a method for preparing a biomimetic circulating tumor cell separation chip and a kit; the method comprises the following steps: step 1: soaking a 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: rinsing the grown chip with deionized water and drying it; step 4: placing the cleaned and dried chip in an acid solution for treatment; and step 5: placing the acid-treated chip in a modification solution for specific protein separation modification. The present invention can efficiently and large-scale realize the preparation and post-modification of micro-nano composite structure chips. After modification with specific recognition molecules, the chip can achieve efficient separation of pan-cancer circulating tumor cells.

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 isolating 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 rapid, but due to issues such as CTC heterogeneity, it is difficult to achieve high capture efficiency and the separation purity is not high;

[0006] The second, more classic, method is positive separation. However, the commonly used magnetic bead capture method has relatively poor capture efficiency and a complex operation process. Its separation efficiency is relatively low in simulated / clinical circulating tumor cell samples (the simulated circulating tumor cell separation method involves mixing a certain amount of cancer cells with a quantitative separation magnetic bead, incubating for a certain period of time, washing, and then calculating the number of separated cancer cells). While the nanostructured chip separation method is more efficient, the initial preparation process is complex, and therefore has not yet formed an effective test kit.

[0007] Therefore, the present application proposes a preparation method and a kit for a biomimetic 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 biomimetic 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: Place 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] 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] If the chip is made of a hydrophobic material, no hydrophobic modification is required. If the chip is made of a hydrophilic material, a hydrophobic modification is required.

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

[0020] Chip area is 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. 1H, 1H, 2H, 2H-perfluorodecyltrimethoxysilane is used to hydrophobize the chip and the chip is 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, and 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 composition as the hydrophilic segment and the hydrophobic segment, and the organic solvent is a non-water-soluble 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 segment includes PCL, PLGA, PTMC, PLA, PS, PMMA, etc.; the hydrophilic segment includes PEG, PVP, P4VP, PEI, etc.; the random copolymer includes PLGA, etc., and the homopolymer includes 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% by weight, and the block ratio of the block polymer is hydrophobic:hydrophilic 1-100:1;

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

[0029] Specifically:

[0030] A PCL-PEG-PCL triblock polymer consisting of a hydrophobic segment PCL and a hydrophilic segment PEG was dissolved in ethyl acetate at a concentration of 2-10% wt and dissolved by shaking on a shaker at a rate of 200 r / min. 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 cosolvent 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, cyclopentane 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 liquid is prepared from 2% wt sodium lauryl 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, the chip needs to be grown in an environment heated to 50° C. for 6 hours in the nanostructure growth liquid.

[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, for 3 hours, then wash with PBS; place in SA 10μg / mL PBS solution, for 3 hours, then wash with PBS; place in specific recognition protein solution, for 3 hours, then wash with PBS, and store at -20°C;

[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, single recognition protein or a combination of multiple recognition proteins.

[0040] A chip kit suitable for the preparation method of the biomimetic circulating tumor cell separation chip described above, wherein 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;

[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 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 sequentially 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 incubated at 37°C for 1 hour; in M2, the cells are incubated with the cell fixative at room temperature for 20 minutes, with the cell permeabilization solution at room temperature for 10 minutes, with the cell blocking solution at room temperature for 1 hour, with the cancer cell stain at room temperature for 30 minutes, with the leukocyte stain at room temperature for 2 hours, with the cell nuclear stain at room temperature for 10 minutes, and with the 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 nanoscale burr structures on the surface of a chip with a micron-scale structure and constructs a micro-nano composite structure. This allows for rapid, efficient, and large-scale preparation of micro-nano composite structure chips, which can then be chemically modified 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 modification with 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 efficient and specific separation of circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is 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 described in Example 1 of the present invention is prepared;

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

[0053] Figure 4 This is a schematic diagram of the combination of cells and chips described in Example 2 of the present invention;

[0054] Figure 5 This is a SEM photograph of cells bound to 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] Figure 9 Graph showing the cell separation efficiency compared with the prior art in Example 4 of the present invention. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0060] Unless otherwise defined, all professional and scientific terms used herein have the same meaning 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 with reference to the accompanying drawings and specific embodiments. The preferred embodiments and materials described in the present invention are for demonstration purposes only.

[0061] like Figures 1-9 As shown:

[0062] Example 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 laboratory has a chip surface area of ​​1cm2 and a preparation efficiency of 10 chips / hour. The preparation process is simple, two glass cylinders, a chip rack, and the chip rack can hold 50 chips. 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 placed in the nanostructure growth solution. After growth, the chips are taken out and 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 and dissolve on a shaker at 200r / min. 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 undulation rough glass sheet into the ethyl acetate solution of the triblock polymer PCL-PEG-PCL, soak for 20min, take out, and quickly soak in the nanostructure growth solution (2%wt sodium dodecyl sulfate and 5%wt tetrahydrofuran aqueous solution), then heat to 50°C, grow for 6h, take out, wash with deionized water, and dry. 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 treatment 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 treatment for 3 hours, the cells were washed with PBS and placed in SA 10 μg / mL PBS solution. After treatment for 3 hours, the cells were washed with PBS and placed in biotin-anti-EpCAM 1 μg / mL PBS solution. After treatment for 3 hours, the cells were washed with PBS and stored at -20°C.

[0068] Example 2:

[0069] Circulating tumor cell separation and staining process:

[0070] The sample to be tested was 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 minutes, rinsed with PBS, incubated with cell permeabilization solution at room temperature for 10 minutes, rinsed with PBS, incubated with cell blocking solution at room temperature for 1 hour, rinsed with PBS, incubated with cancer cell stain at room temperature for 30 minutes, rinsed with PBS, incubated with leukocyte stain at room temperature for 2 hours, rinsed with PBS, incubated with cell nuclear stain at room temperature for 10 minutes, rinsed with PBS, incubated with cell immunostaining PD-L1 stain at room temperature for 30 minutes, rinsed with PBS. The separation effect at different times is 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, and then 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 has achieved efficient separation of pan-cancer CTCs and has been expanded to intestinal cancer, single-negative, double-negative, and triple-negative breast cancer, prostate cancer, lung cancer, highly metastatic 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 is as follows Figure 7 shown.

[0073] Example 3:

[0074] Preparation of separation chips with different structures

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

[0076] Example 4:

[0077] Comparison with commercially available CellSearch magnetic beads

[0078] Prostate cancer 22RV1 cells were selected as the cancer cells for the simulated circulating tumor cell isolation experiment. After culture, digestion and counting were performed to obtain a cell suspension of 10^5 cells / mL for isolation. 1 mL of the cell suspension was isolated 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 isolation, and subsequent fixation and staining reagents must be prepared on-site in the laboratory. Obtain CellSearch micron magnetic beads. According to the instructions, absorb 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 10 minutes of adsorption on the magnet, wash it with PBS and adsorb it 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 three times. Add 1 mL of mold-penetrating liquid Triton X-100 and treat it for 10 minutes. Wash it with PBS and adsorb it three times. Add cell blocking liquid BSA solution and treat it for 120 minutes. Wash it with PBS and adsorb it three times. Add cell nuclear stain and treat it for 10 minutes. Wash it with PBS and adsorb it three times. Then count the separated cancer cells to calculate the separation efficiency. Figure 9 As shown, this kit achieves significantly higher separation efficiency for prostate cancer 22RV1 cells than the existing CellSearch magnetic beads. Furthermore, because the magnetic bead method requires waiting for the beads to adsorb, the overall operation time is significantly longer than with the kit. Therefore, this kit offers significant advantages in both separation and operational efficiency.

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

Claims

1. A method for preparing a biomimetic circulating tumor cell separation chip, characterized in that: The chip is a rough glass sheet with micron undulations. The specific steps are as follows: The triblock polymer PCL-PEG-PCL was dissolved in 2% ethyl acetate by mass and shaken at 200 r / min. The block ratio of PCL:PEG was 20:

1. 1H,1H,2H,2H-perfluorodecyltrimethoxysilane was used and vacuum oven treated for 24 hours to hydrophobize a rough glass sheet with micron undulations. A hydrophobically modified micron-undulated rough glass sheet was inserted into an ethyl acetate solution of the triblock polymer PCL-PEG-PCL, soaked for 20 minutes, removed, and immersed in a nanostructure growth solution. The sheet was then heated to 50°C, grown for 6 hours, removed, rinsed with deionized water, and dried. The nanostructure growth solution was a 2% wt sodium dodecyl sulfate and 5% wt tetrahydrofuran aqueous solution. After treatment with 1M dilute hydrochloric acid for 12 hours, the cells were washed with PBS. The cells were placed in a 10 mg / mL 2-morpholineethanesulfonic acid (MES) solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS), pH 6.0, for 3 hours, and then washed with PBS. The cells were placed in a 10 μg / mL streptavidin (SA) solution in PBS for 3 hours, and then washed with PBS. The cells were placed in a 1 μg / mL biotin-anti-EpCAM solution in PBS for 3 hours, and then washed with PBS and stored at -20°C.

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