WO2018133617A1 - 一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用 - Google Patents

一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用 Download PDF

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WO2018133617A1
WO2018133617A1 PCT/CN2017/117495 CN2017117495W WO2018133617A1 WO 2018133617 A1 WO2018133617 A1 WO 2018133617A1 CN 2017117495 W CN2017117495 W CN 2017117495W WO 2018133617 A1 WO2018133617 A1 WO 2018133617A1
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graphene
tumor cells
circulating tumor
chip
minutes
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French (fr)
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樊俊兵
王树涛
江雷
李冠男
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北京赛特超润界面科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/182—Graphene
    • C01B32/184—Preparation
    • C01B32/186—Preparation by chemical vapour deposition [CVD]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the field of biological detection technology, and in particular, the invention relates to a graphene chip for specific capture of circulating tumor cells in whole blood, a preparation method and application thereof.
  • Circulating Tumor Cells are tumor cells that fall off from the primary tumor site and enter the blood circulation. Since there are circulating tumor cells in the early stage of cancer, circulating tumor cells are used as biomarkers. It is of great significance to detect and monitor the number of circulating tumor cells in the blood for early detection of cancer.
  • an immunomagnetic separation technique based on specific antibody-modified magnetic beads
  • a microfluidic technology based on increasing the frequency of contact between cells and a substrate
  • a microfiltration technique based on cell size difference
  • Density-based density gradient centrifugation technology and the combination of several technologies.
  • these methods still have the disadvantages of low capture efficiency, low separation purity and long capture time.
  • EpCAM expressed antigens
  • the chip is expected to solve the problems of low detection efficiency and high detection cost in the early detection and postoperative monitoring of cancer.
  • a graphene chip for specific capture of circulating tumor cells in whole blood comprising a graphene-based sheet having a stereoscopic geometric micro-nano structure and a surface of a circulating tumor cell immobilized on a graphene surface Specific antibodies.
  • the graphene core substrate is obtained by spin coating or depositing graphene particles having a three-dimensional geometry on a substrate, wherein the graphene particles have a size of from 1 ⁇ m to 30 ⁇ m.
  • the specific antibody on the surface of the circulating tumor cells is a biotinylated anti-EpCAM antibody.
  • the specific antibody on the surface of the circulating tumor cells is fixed to the surface of the graphene by not less than 0.1 ⁇ g/cm 2 .
  • the present invention also provides a method for preparing a graphene chip for specific capture of circulating tumor cells in whole blood, the method comprising the steps of:
  • the inorganic compound powder is selected from the group consisting of sodium chloride, potassium chloride, copper chloride, copper sulfate, manganese tungstate, vanadium oxide, tungsten oxide and cobalt chloride.
  • the three-dimensional geometric shape comprises one or more of a cube, a rhomboid, an oblique column and an octahedron.
  • the stereocrystalline structure of these inorganic compounds corresponds to a template, graphene is grown on the template, and the template is removed to obtain a graphene particulate material having a three-dimensional structure.
  • the material of the substrate is a non-conductive inorganic non-metallic material, preferably from crystalline silicon, ordinary glass, quartz or microporous filter.
  • the method for immobilizing specific antibodies on the surface of circulating tumor cells on the surface of the graphene-based sheet having a three-dimensional geometric micro-nano structure is specifically:
  • the graphene-based sheet having a three-dimensional geometric micro-nano structure is vacuum-reduced and placed at room temperature; at room temperature, the high-temperature treated graphene-based sheet having a three-dimensional geometric micro-nano structure is immersed at a concentration of 1 to 10%.
  • 1-mer carboxylic acid in methanol solution placed at room temperature; taken out with methanol and dimethyl sulfoxide rinse, blow dry;
  • the method of immobilizing a specific antibody on the surface of a circulating tumor cell on the surface of a graphene-based sheet having a three-dimensional geometric micro/nano structure is:
  • the graphene having a three-dimensional geometric micro-nano structure is heated and reduced by vacuum, and placed at room temperature; the 1-hydrazine carboxylic acid and ethanol are mixed in a volume ratio to prepare a methanol solution of 1-indole carboxylic acid having a concentration of 1 to 10%;
  • the graphene having a three-dimensional geometric micro/nano structure after high temperature treatment is immersed in a methanol solution of 1-indole carboxylic acid having a concentration of 1 to 10% at room temperature, and left at room temperature (preferably left at room temperature for about 45 minutes); Take out, use methanol and two Methyl sulfoxide (DMSO) rinsed and blown dry;
  • DMSO Methyl sulfoxide
  • Streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 5-20 ⁇ g/mL, and N-hydroxysuccinimide and N-(3-dimethyl) are added.
  • a solution of arylamino)-N'-ethylcarbodiimide in dimethyl sulfoxide and then immersing the graphene obtained after drying step a) in the above solution, and placing it at room temperature (preferably at room temperature) Leave for about 30 minutes); remove and wash with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells is a biotinylated anti-EpCAM antibody.
  • the 1-indolecarboxylic acid, N-hydroxysuccinyl, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, streptavidin and biotinylation EpCAM antibodies are commercially available.
  • the present invention further provides the use of the above-described graphene chip for specific capture of circulating tumor cells in whole blood for the capture of circulating tumor cells in whole blood.
  • the surface of the graphene chip of the invention has a three-dimensional geometric micro/nano structure, that is, a solid geometric structure of the replicated inorganic compound single crystal template, which matches the structure of the surface of the circulating tumor cell, and the antibody has a stereo geometric micro-nano structure.
  • the graphene chip has the function of specifically recognizing circulating tumor cells. By using the chip to specifically capture circulating tumor cells, the process of lymphocyte capture circulating tumor cells can be simulated in vitro, and the capture efficiency of circulating tumor cells is greatly enhanced.
  • the stereoscopic geometric micro-nano structure on the surface of the graphene chip prepared by the invention and the recognition of the specific antibody enhance the capture efficiency of the circulating tumor cells by synergistic action.
  • the invention simulates the process of lymphocyte capturing circulating tumor cells, and rapidly enriches and separates circulating tumor cells existing in peripheral blood. Due to the good biocompatibility of the material, the captured circulating tumor cells can be used for further culture, drug detection analysis.
  • the invention realizes that the stereoscopic geometric micro-nano structure on the graphene chip with different surface micro-nano structures on the surface is matched with the structure of the circulating tumor cell surface, and the surface antigen-antibody specific recognition principle is combined to improve the targeting cell. Efficient and specific capture.
  • the invention utilizes a graphene chip with different stereo geometric micro-nano structures on the surface to perform specific capture of circulating tumor cells, can be used for cancer-related cell capture, can effectively improve the capture efficiency of circulating tumor cells, has low cost, and is simple to operate. For clinical testing.
  • Figure 1 Scanning electron micrograph of the surface of the graphene chip prepared in Example 1;
  • Figure 2 Fluorescence microscopic characterization of cancer cells captured by graphene chips prepared in Example 1;
  • Fig. 3 is a graph showing the capture efficiency of a graphene chip prepared in Example 1 on cancer cells.
  • the average size of the cubic graphene prepared by using the sodium chloride prepared in the present embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by using the surface micro-nano structure of the prepared graphene chip and the specific recognition molecule synergistically includes the following steps:
  • the commercial sodium chloride powder was dissolved in water to prepare a saturated solution of sodium chloride, and the temperature was controlled at 25 degrees.
  • the crystals were allowed to stand, precipitated, and filtered to obtain a cubic sodium chloride single crystal powder having an average length of 20 ⁇ m.
  • a carbon source (ethylene) was introduced at one end (about 20 cm) of the quartz tube, and a carbon source (ethylene) was introduced at the other end; the other end was about 20 cm long and the temperature was 700 degrees and a cubic sodium chloride single crystal powder was placed.
  • the surface of the sodium chloride single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a sodium chloride single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder was dissolved, suction filtered or spin-coated to obtain a graphene film having a cubic micro/nano structure.
  • the scanning electron micrograph of the surface of the prepared graphene chip is shown in Fig. 1. As can be seen from Fig. 1, the obtained graphene chip is mainly composed of cube-shaped graphene particles.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface in which the circulating tumor cells were captured was also carried out as described above, and the capturing efficiency was calculated, and the results are shown in Fig. 3.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface in which the circulating tumor cells were captured was also carried out as described above, and the capturing efficiency was calculated, and the results are shown in Fig. 3.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface in which the circulating tumor cells were captured was also carried out as described above, and the capturing efficiency was calculated, and the results are shown in Fig. 3.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface in which the circulating tumor cells were captured was also carried out as described above, and the capturing efficiency was calculated, and the results are shown in Fig. 3.
  • the graphene chip of the present invention has a capture efficiency of 94% for MCF7 cells, a capture efficiency of prostate cancer cell PC3 cells of 90.5%, and capture of human lymphoid B cell cancer cells Daudi cells.
  • the efficiency was 0.02%
  • the capture efficiency of human lymphoma Jurkat cells was 0.02%
  • the capture efficiency of uterine cancer Hela cells was 0.02%.
  • the average size of the graphene having a cubic structure prepared by using potassium chloride as a template in this example is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and EpCAM non-specific cells as circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial potassium chloride powder was dissolved in water to prepare a saturated solution of potassium chloride, and the temperature was controlled to 25 degrees.
  • the crystal was allowed to stand, precipitated, and filtered to obtain a cubic crystal potassium chloride single crystal powder having an average side length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 600 degrees and a cubic potassium chloride single crystal powder was placed.
  • the surface of the potassium chloride single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a potassium chloride single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder was dissolved, suction filtered or spin-coated to obtain a graphene film having a cubic micro/nano structure.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the graphene stereostructure of the copper chloride prepared by the template in this embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention Further elaboration and verification.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial copper chloride powder was dissolved in water to form a saturated solution of copper chloride, and the temperature was controlled to 25 degrees.
  • the crystal was allowed to stand, precipitated, and filtered to obtain a copper chloride single crystal powder having an average length of 20 ⁇ m.
  • the temperature was maintained at 850 °C, and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 550 degrees, and a rhombohedral copper chloride single crystal powder was placed.
  • the surface of the copper chloride single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a copper chloride single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the adjustment mode is computer display, the exposure time is set to 100ms, and photos are taken 10 times with a Nikon inverted fluorescence microscope (each of which captures 10 different positions in the middle part of the chip that circulates the tumor cells), and the circulating tumor cells are captured.
  • the circulating tumor cells captured on the chip were counted to calculate the capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the oblique column graphene prepared by using the copper sulfate prepared in this embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial copper sulfate powder was dissolved in water to form a saturated solution of copper chloride, and the temperature was controlled at 25 degrees.
  • the crystal was allowed to stand, precipitated, and filtered to obtain a copper sulfate single crystal powder having an average length of 20 ⁇ m.
  • the temperature was maintained at 850 °C, and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 550 degrees, and an orthorhombic copper sulfate single crystal powder was placed.
  • the surface of the copper sulfate single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a copper sulfate single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the adjustment mode is computer display, the exposure time is set to 100ms, and photos are taken 10 times with a Nikon inverted fluorescence microscope (each of which captures 10 different positions in the middle part of the chip that circulates the tumor cells), and the circulating tumor cells are captured.
  • the circulating tumor cells captured on the chip were counted to calculate the capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the cube geometry of the rhombohedral graphene prepared by the manganese tungstate prepared in the present embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the present invention
  • the capture system is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial manganese tungstate powder is dissolved in water to form a saturated solution of manganese tungstate, the temperature is controlled at 25 degrees, and it is allowed to stand still.
  • the crystals were precipitated, filtered, and dried to obtain a manganese tungstate single crystal powder having an average side length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 700 degrees and a copper sulfate single crystal powder was placed.
  • the surface of the tungsten tungstate single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a manganese tungstate single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a rhombic graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the geometrical structure of the rhombohedral graphene prepared by the vanadium oxide prepared in the present embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the present invention
  • the capture system is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial vanadium oxide powder is dissolved in water to form a saturated solution of vanadium oxide, the temperature is controlled at 25 degrees, the crystal is allowed to stand, precipitated, filtered, and dried to obtain a vanadium oxide single crystal powder having an average length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 550 degrees, and a vanadium oxide single crystal powder was placed.
  • the surface of the vanadium oxide single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product is dissolved in water to remove a vanadium oxide single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film was placed in a 1-indolecarboxylic acid solution at room temperature to obtain a carboxylated film; the film was then fixed on a glass piece to obtain a graphene chip composed of rhombic graphene.
  • the adjustment mode is computer display, the exposure time is set to 100ms, and photos are taken 10 times with a Nikon inverted fluorescence microscope (each of which captures 10 different positions in the middle part of the chip that circulates the tumor cells), and the circulating tumor cells are captured.
  • the circulating tumor cells captured on the chip were counted to calculate the capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the octahedral graphene of the oxidized tungsten prepared by the present embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture of the present invention
  • the system is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial tungsten oxide powder is dissolved in water to form a saturated solution of tungsten oxide, and the temperature is controlled to 25 degrees.
  • the crystal is allowed to stand and precipitate, and filtered and dried to obtain a tungsten oxide single crystal powder having an average length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 700 degrees, and a tungsten oxide single crystal powder was placed.
  • the surface of the tungsten oxide single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a tungsten oxide single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the adjustment mode is computer display, the exposure time is set to 100ms, and photos are taken 10 times with a Nikon inverted fluorescence microscope (each of which captures 10 different positions in the middle part of the chip that circulates the tumor cells), and the circulating tumor cells are captured.
  • the circulating tumor cells captured on the chip were counted to calculate the capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic B cell cancer Daudi suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture.
  • the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a human lymphatic cancer cell Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the chip obtained in the step (2) was placed in a six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added and placed in a cell culture incubator. Due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes. As a control experiment, the surface of the flat graphene of the anti-EpCAM antibody immobilized on the surface of the tumor cell was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene surface capturing the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the average size of the cubic graphene of the cubic graphene prepared by using the sodium chloride template in this example is 20 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial sodium chloride powder was dissolved in water to prepare a saturated solution of sodium chloride, and the temperature was controlled at 25 degrees. The crystals were allowed to stand, precipitated, and filtered to obtain a sodium chloride single crystal powder having an average length of 20 ⁇ m.
  • the temperature is maintained at 850 degrees, and a carbon source (ethylene) is introduced;
  • the other end was about 20 cm long and the temperature was 700 degrees and a sodium chloride single crystal powder was placed.
  • the surface of the sodium chloride single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a sodium chloride single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • step (3) placing the chip obtained in step (2) in a microplate, adding 1 mL of blood of a breast cancer patient, and placing it in a cell culture incubator, due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cell and the solid geometry of the graphene. It can specifically capture circulating tumor cells with a capture time of 45 minutes.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • PBS phosphate buffered saline
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the average size of the cubic graphene of the cubic graphene prepared by using the sodium chloride template in this example is 20 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in normal human blood as an example.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial sodium chloride powder was dissolved in water to prepare a saturated solution of sodium chloride, and the temperature was controlled at 25 degrees. The crystals were allowed to stand, precipitated, and filtered to obtain a sodium chloride single crystal powder having an average length of 20 ⁇ m.
  • the temperature was maintained at 850 °C, and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 700 degrees and a sodium chloride single crystal powder was placed.
  • the surface of the sodium chloride single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a sodium chloride single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the average size of the stereoscopic geometric structure of the oblique column graphene prepared by using the copper sulfate prepared in this embodiment is 20 ⁇ m; the capture system of the present invention is further elaborated and verified by taking the breast cancer cell cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial copper sulfate powder was dissolved in water to prepare a saturated solution of copper sulfate, and the temperature was controlled at 25 degrees.
  • the crystal was allowed to stand, precipitated, and filtered to obtain a copper sulfate single crystal powder having an average length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 550 degrees and a copper sulfate single crystal powder was placed.
  • the surface of the copper sulfate single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a copper sulfate single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • step (3) placing the chip obtained in step (2) in a microplate, adding 1 mL of blood of a breast cancer patient, and placing it in a cell culture incubator, due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cell and the solid geometry of the graphene. It can specifically capture circulating tumor cells with a capture time of 45 minutes.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • PBS phosphate buffered saline
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographed separately with a Nikon inverted fluorescence microscope 10 times (each chip capturing the middle part of the circulatory tumor cells with 10 different positions) and captured The circulating tumor cells captured on the chip of the circulating tumor cells were counted, and the capture efficiency was calculated.
  • the average size of the stereoscopic geometric structure of the oblique column graphene prepared by using the copper sulfate prepared in this embodiment is 20 ⁇ m; the capture system of the present invention is further elaborated and verified by taking the breast cancer cell cells in normal human blood as an example.
  • a method for performing specific capture of circulating tumor cells using a graphene surface having a three-dimensional geometry includes the following steps:
  • the commercial copper sulfate powder was dissolved in water to prepare a saturated solution of copper sulfate, and the temperature was controlled at 25 degrees.
  • the crystal was allowed to stand, precipitated, and filtered to obtain a copper sulfate single crystal powder having an average length of 20 ⁇ m.
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 550 degrees and a copper sulfate single crystal powder was placed.
  • the surface of the copper sulfate single crystal powder at the low temperature end is covered with a graphene layer by thermal cracking of ethylene.
  • the obtained product was dissolved in water to remove a copper sulfate single crystal; and dried at a high temperature to obtain a graphene particle powder.
  • the powder is dissolved, suction filtered or spin coated to obtain a graphene film having a three-dimensional geometry.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • step (3) Place the chip obtained in step (2) in a microplate, add 1 mL of normal human blood, and place it in fine In the cell culture chamber, due to the synergistic action of the anti-EpCAM antibody on the surface of the tumor cells and the stereo geometry of the graphene, the circulating tumor cells can be specifically captured, and the capture time is 45 minutes.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • PBS phosphate buffered saline
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the flat graphene chip prepared in the present embodiment is further illustrated and verified by taking the breast cancer cell in the blood of a breast cancer patient as an example.
  • a method for performing specific capture of circulating tumor cells with a flat graphene chip surface includes the following steps:
  • a temperature of 850 degrees was maintained at one end of the quartz tube (about 20 cm), and a carbon source (ethylene) was introduced; the other end was about 20 cm long and the temperature was 600 degrees, and a glass piece was placed.
  • Graphene powder was obtained by thermal cracking of ethylene. The powder is dissolved, suction filtered or spin coated to obtain a flat graphene film.
  • the above film was placed in a vacuum box and heated under 100 degrees for about 12 hours.
  • the film is immersed in a methanol solution of 1-indole carboxylic acid at a concentration of 1 to 10% at room temperature, and left at room temperature; it is taken out by washing with methanol and dimethyl sulfoxide, and dried to obtain a carboxylated film.
  • the film was then mounted on a glass slide to obtain a carboxylated graphene film chip.
  • step (3) The chip obtained in step (2) is placed in a microplate culture dish, and 1 mL of breast cancer patient blood is added and placed in a cell culture incubator, due to the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cell and the solid geometry of the graphene. Role, can specifically capture circulating tumor cells, the capture time is 45 minutes.
  • the chip in which the circulating tumor cells were captured was washed 3 times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • PBS phosphate buffered saline
  • the 2 ⁇ g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.

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Abstract

本发明公开了一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用。所述石墨烯芯片的制备是利用无机化合物粉末重结晶得到的具有立体几何形状的单晶结构做为模板，通过引入碳源，使其高温热解得到具有立体结构的石墨烯颗粒溶液，将该溶液通过抽滤或旋涂得到表面具有立体几何微纳结构的石墨烯基片，最后在石墨烯基片上修饰特异识别性抗体得到针对循环肿瘤细胞特异性捕获的石墨烯芯片。该抗体修饰的立体几何状微纳结构的石墨烯芯片可显著增强对循环肿瘤细胞的捕获效率。

Description

一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用 技术领域
本发明属于生物检测技术领域，具体地，本发明涉及一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用。
背景技术
循环肿瘤细胞(Circulating Tumor Cells,CTCs)是指从肿瘤原发病灶部位脱落，进入到血液循环的肿瘤细胞。由于在癌症发生的早期，就有循环肿瘤细胞的出现，因此将循环肿瘤细胞做为生物标记物，通过检测，监视以及分析血液中循环肿瘤细胞的数量，对癌症进行早期检测意义重大。目前捕获循环肿瘤细胞的方法主要有以下几种，分别是基于特异性抗体修饰的磁珠的免疫磁性分离技术；基于增加细胞与基底接触频率的微流体技术；基于细胞尺寸差别的微过滤技术；基于密度的密度梯度离心技术；以及将几种技术结合联合起来使用。然而这些方法仍然存在着捕获效率低，分离纯度低以及捕获时间长等缺点。
2009年，中科院理化所王树涛研究员团队(Angew.Chem.Int.Ed.,2009,48,8970-8973)利用三维的硅纳米线阵列，从全血样品中实现了对循环肿瘤细胞的有效分离。该方法是利用三维的纳米结构与靶向细胞的表面的纳米结构(纤毛/伪足)之间的相互作用，即结构识别作用，以及阵列表面修饰的抗体(anti-EpCAM)与肿瘤细胞表面高表达的抗原(EpCAM)之间特异性的分子识别作用，增强了三维的硅纳米线对于循环肿瘤细胞的亲和力，显著提高了对于循环肿瘤细胞的捕获效率。此后，利用材料表面微纳结构来分离循环肿瘤细胞受到了广泛的关注，大量的材料如PEDOT导电聚合物(Adv.Mater.2011,23,4788-4792)、PDMS(Cancer 2012,118,1145-1154)、TiO2纳米纤维(Adv.Mater.2012,24,2756-2760)，石英纳米线阵列(Nano Lett.2012,12,2697-2704)以及Fe3O4纳米粒子修饰的基片(Small2012,8,1657-1663)等都是利用三维纳米结构的构建从而增强循环肿瘤细胞的捕获效率。但上述方法仍存在着一定的不足，比如材料制备成本高，制备方法较为复杂，制备工艺较为复杂。同时，缺少一个确实存在的理论模型来指导我们进行进一步的研究。因此，开发新型的循环肿瘤细胞检测技术，实现低成本、高效率的检测已成为当前癌症检测所需亟待解决的重要问题。
发明内容
本发明的目的在于，提供一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片制备新方法，该方法制备工艺简单，成本低廉，适于工业化批量生产。该芯片有望解决当前在癌症早期检测、术后监测等方面存在的检测效率低和检测费用高等难题。
为达到上述目的，本发明采用了如下的技术方案：
一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片，所述石墨烯芯片包括具有立体几何微纳结构的石墨烯基片以及在石墨烯表面固定的循环肿瘤细胞表面 的特异性抗体。所述石墨烯芯基片是由具有立体几何结构的石墨烯颗粒通过在基片上旋涂或沉积得到，其中石墨烯颗粒的大小为1μm～30μm。
优选地，所述循环肿瘤细胞表面的特异性抗体为生物素化抗EpCAM抗体。
本发明中，所述循环肿瘤细胞表面的特异性抗体在石墨烯表面的固定量不少于0.1μg/cm2。
本发明还提供了一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，所述方法包括以下步骤：
1)利用无机化合物粉末重结晶得到具有立体几何形状的单晶，所述立体几何形状是由经过结晶过程而形成的无机化合物晶体所具有规则的几何外形结构；
2)利用化学气相沉积，在步骤1)得到的单晶表面上构筑具有立体几何结构的石墨烯层；将石墨烯层覆盖的单晶溶解得到含石墨烯的溶液，去除单晶并干燥得到石墨烯粉末，石墨烯粉末溶解并将溶解后得到的溶液添加到基片上抽滤或者旋涂得到具有立体几何微纳结构的石墨烯基片；
3)通过在具有立体几何微纳结构的石墨烯基片表面上固定循环肿瘤细胞表面的特异性抗体。
优选地，所述的无机化合物粉末选自氯化钠、氯化钾、氯化铜、硫酸铜、钨酸锰、氧化钒、氧化钨和氯化钴中的一种。
优选地，所述的立体几何形状包括立方体、斜方锥、斜方柱和八面体中的一种或多种。这些无机化合物的立体晶体结构相当于做模板，在此模板上生长石墨烯，再除去模板，既得到具有立体结构的石墨烯颗粒材料。
优选地，在步骤2)中，所述的基片的材料是不导电的无机非金属材料，优选自晶体硅、普通玻璃、石英或微孔滤膜。
优选地，具有立体几何微纳结构的石墨烯基片表面上固定循环肿瘤细胞表面的特异性抗体的方法具体为：
a)将具有立体几何微纳结构的石墨烯基片真空加热还原，放至室温；室温下，将高温处理后的具有立体几何微纳结构的石墨烯基片浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干；
b)将5～20μg/mL的链霉亲和素的磷酸盐溶液，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，然后将步骤a)吹干后的石墨烯基片浸泡在上述溶液中，室温下放置，取出用磷酸盐缓冲液洗涤；
c)将循环肿瘤细胞表面的特异性抗体用磷酸盐缓冲液稀释至浓度为5～20μg/mL，然后滴加到步骤b)用磷酸盐缓冲液洗涤后得到的石墨烯的表面上，室温下放置，得到用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片。
更进一步优选地，具有立体几何微纳结构的石墨烯基片表面上固定循环肿瘤细胞表面的特异性抗体的方法为：
a)将具有立体几何微纳结构的石墨烯真空加热还原，放至室温；将1-芘羧酸与乙醇按体积比混合配制成浓度为1～10％的1-芘羧酸的甲醇溶液；室温下，将高温处理后的具有立体几何微纳结构的石墨烯浸泡在上述浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置(优选室温下放置45分钟左右)；取出，分别用甲醇和二 甲基亚砜(DMSO)润洗，吹干；
b)将链霉亲和素用磷酸盐缓冲液稀释为浓度为5～20μg/mL的链霉亲和素的磷酸盐溶液，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，然后将步骤a)吹干后得到的石墨烯浸泡在上述溶液中，室温下放置(优选室温下放置30分钟左右)；取出，用磷酸盐缓冲液洗涤；
c)将循环肿瘤细胞表面的特异性抗体用磷酸盐缓冲液稀释至浓度为5～20μg/mL，然后滴加到步骤b)用磷酸盐缓冲液洗涤后得到的石墨烯的表面上，室温下放置，得到用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片。
所述的循环肿瘤细胞表面的特异性抗体为生物素化抗EpCAM抗体。
所述的1-芘羧酸、N-羟基琥珀酰亚、N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺、链霉亲和素与生物素化抗EpCAM抗体均为市售产品。
本发明还另外提供了上述用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片在全血中循环肿瘤细胞的捕获中的应用。
本发明所述石墨烯芯片的表面具有立体几何微纳结构，即复制的无机化合物单晶模板的立体几何结构，与循环肿瘤细胞表面的结构相匹配，同时该抗体修饰的具有立体几何微纳结构的石墨烯芯片具有特异性识别循环肿瘤细胞功能。利用该芯片特异性捕获循环肿瘤细胞，可以在体外模拟淋巴细胞捕获循环肿瘤细胞的过程，极大的增强了对循环肿瘤细胞的捕获效率。本发明所制备的石墨烯芯片表面的立体几何微纳结构和特异性抗体的识别通过协同作用，提高对循环肿瘤细胞捕获效率。
本发明模拟淋巴细胞捕获循环肿瘤细胞的过程，对外周血中存在的循环肿瘤细胞进行快速的富集和分离。由于该材料具有良好的生物相容性，可将捕获的循环肿瘤细胞用于进一步的培养，药物检测分析。
本发明的实现是利用表面具有不同表面微纳结构的石墨烯芯片上的立体几何微纳结构与循环肿瘤细胞表面的结构相匹配作用，结合表面抗原-抗体特异性识别原理，提高对靶向细胞的高效特异性捕获。
本发明利用表面具有不同立体几何微纳结构的石墨烯芯片进行循环肿瘤细胞的特异性捕获，可用于癌症相关的细胞的捕获，可以有效提高循环肿瘤细胞的捕获效率，成本低廉，操作简单，可用于临床检测。
附图说明
图1：实施例1中所制备的石墨烯芯片表面扫描电镜图；
图2：实施例1中所制备的石墨烯芯片捕获癌细胞的荧光显微镜表征图；
图3：实施例1中所制备的石墨烯芯片对癌细胞的捕获效率图。
具体实施方式
以下结合实施例及附图对本发明进行进一步的说明，然而，本发明可以以不同形式进行体现，并不应理解成受限于文中所述实施方式。相反，提供这些实施方式可以使得本发明公开彻底而完整，并完整地将本发明的范围传达给本领域的技术人 员。
实施例1
本实施例所制备的氯化钠为模板制备的立方体石墨烯平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用所制备的石墨烯芯片表面微纳结构和特异性识别分子协同作用，进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氯化钠为模板的立方体石墨烯的制备
(a)制备立方体氯化钠单晶
将商业用氯化钠粉末溶于水中配成氯化钠饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的立方体氯化钠单晶粉末。
(b)制备由立方体石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为700度并放置立方体氯化钠单晶粉末。通过乙烯的热裂解，使得低温端的氯化钠单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氯化钠单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立方体微纳结构的石墨烯薄膜。所制备的石墨烯芯片表面扫描电镜图如图1所示，从图1可以看出，所得到的石墨烯芯片主要是有立方体形的石墨烯颗粒组成。
(2)由立方体石墨烯组成的薄膜的特异性抗体修饰
(a)羧酸化石墨烯
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在上述石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(i)将(a)中所述石墨烯薄膜芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺、N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟，然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，结果如图2所示，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率，结果如图3所示。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为94％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率，结果如图3所示。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循 环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率，结果如图3所示。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率，结果如图3所示。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率，结果如图3所示。
(8)从图2和图3可以得到，本发明的石墨烯芯片对MCF7细胞的捕获效率为94％，前列腺癌细胞PC3细胞的捕获效率为90.5％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.02％，人淋巴癌细胞Jurkat细胞的捕获效率为0.02％，子宫癌细胞Hela细胞的捕获效率为0.02％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例2
本实施例所制备的氯化钾为模板的立方体结构的石墨烯平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氯化钾为模板的石墨烯芯片的制备
(a)制备立方体氯化钾单晶
将商业用氯化钾粉末溶于水中配成氯化钾饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的立方体结构的氯化钾单晶粉末。
(b)制备由立方体石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为600度并放置立方体氯化钾单晶粉末。通过乙烯的热裂解，使得低温端的氯化钾单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氯化钾单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立方体微纳结构的石墨烯薄膜。
(2)由立方体石墨烯组成的薄膜芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟，然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个 捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为94％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为94％，前列腺癌细胞PC3细胞的捕获效率为88.6％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.01％，人淋巴癌细胞Jurkat细胞的捕获效率为0.02％，子宫癌细胞Hela细胞的捕获效率为0.01％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例3
本实施例所制备的氯化铜为模板的石墨烯立体几何结构平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氯化铜为模板的的由斜方锥石墨烯组成的薄膜芯片的制备
(a)制备斜方锥氯化铜单晶
将商业用氯化铜粉末溶于水中配成氯化铜饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的氯化铜单晶粉末。
(b)制备由斜方锥石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为550度并放置斜方锥氯化铜单晶粉末。通过乙烯的热裂解，使得低温端的氯化铜单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氯化铜单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由斜方锥石墨烯组成的薄膜芯片表面的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备 得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为94.4％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液 浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为94.4％，前列腺癌细胞PC3细胞的捕获效率为90.6％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为 0.02％，人淋巴癌细胞Jurkat细胞的捕获效率为0.02％，子宫癌细胞Hela细胞的捕获效率为0.02％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例4
本实施例所制备的硫酸铜为模板的斜方柱石墨烯的平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于硫酸铜为模板的由斜方柱石墨烯组成的薄膜芯片的制备
(a)制备斜方柱硫酸铜单晶
将商业用硫酸铜粉末溶于水中配成氯化铜饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的硫酸铜单晶粉末。
(b)制备由斜方柱石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为550度并放置斜方柱硫酸铜单晶粉末。通过乙烯的热裂解，使得低温端的硫酸铜单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除硫酸铜单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由斜方柱石墨烯组成的薄膜芯片表面的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度， 取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为93.4％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照 (每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为93.4％，前列腺癌细胞PC3细胞的捕获效率为90.1％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.01％，人淋巴癌细胞Jurkat细胞的捕获效率为0.01％，子宫癌细胞Hela细胞的捕获效率为0.01％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例5
本实施例所制备的钨酸锰为模板的斜方锥石墨烯立体几何结构平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于钨酸锰为模板的由斜方锥石墨烯组成的薄膜芯片的制备
(a)制备斜方锥钨酸锰单晶
将商业用钨酸锰粉末溶于水中配成钨酸锰饱和溶液，控制温度为25度，静置、 析出晶体，过滤、干燥得到边长平均为20μm的钨酸锰单晶粉末。
(b)制备由斜方锥石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为700度并放置硫酸铜单晶粉末。通过乙烯的热裂解，使得低温端的钨酸锰单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除钨酸锰单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的斜方锥石墨烯薄膜。
(2)由斜方锥石墨烯组成的薄膜芯片表面特异性抗体的修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个 捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为92.7％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为92.7％，前列腺癌细胞PC3细胞的捕获效率为88.8％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.01％，人淋巴癌细胞Jurkat细胞的捕获效率为0.01％，子宫癌细胞Hela细胞的捕获效率为0.01％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例6
本实施例所制备的氧化钒为模板的斜方柱石墨烯立体几何结构平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氧化钒为模板的由斜方柱石墨烯组成的薄膜芯片的制备
(a)制备斜方柱氧化钒单晶
将商业用氧化钒粉末溶于水中配成氧化钒饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的氧化钒单晶粉末。
(b)制备由斜方柱石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为550度并放置氧化钒单晶粉末。通过乙烯的热裂解，使得低温端的氧化钒单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氧化钒单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由斜方柱石墨烯组成的薄膜芯片特异性抗体的修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜放入1-芘甲酸溶液中，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到由斜方柱石墨烯组成石墨烯芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为90.2％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细 胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为90.2％，前列腺癌细胞PC3细胞的捕获效率为89.1％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.01％，人淋巴癌细胞Jurkat细胞的捕获效率为0.01％，子宫癌细胞Hela细胞的捕获效率为0.01％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现 极低的非特异性吸附。
实施例7
本实施例所制备的氧化钨为模板的八面体石墨烯立体几何结构平均尺寸为20μm；以循环肿瘤细胞EpCAM特异性细胞和EpCAM非特异性细胞为待捕获的循环肿瘤细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氧化钨为模板的由八面体石墨烯组成的薄膜芯片的制备
(a)制备八面体氧化钨单晶
将商业用氧化钨粉末溶于水中配成氧化钨饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的氧化钨单晶粉末。
(b)制备由八面体石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为700度并放置氧化钨单晶粉末。通过乙烯的热裂解，使得低温端的氧化钨单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氧化钨单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由八面体石墨烯组成的石墨烯芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)捕获并处理待测样品中的循环肿瘤细胞
(a)分别取乳腺癌细胞MCF7细胞悬浮液10μL，用细胞计数器计数并计算其浓度，取一定量上述细胞悬浮液，用RPMI1640细胞培养基稀释至1×105个细胞/mL，混合均匀，室温下保存。
(b)分别将混合均匀后得到的细胞悬浮液3mL滴加到放置在细胞培养板(直径 3.5cm)中的步骤(2)得到的表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片，然后置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。
(c)将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。
(d)倒置放置在载玻片上，放置在荧光显微镜样品台上进行观测，先通过眼睛可观测的模式，在目镜处观测，通过调节焦距找到肿瘤细胞所在的平面。调节模式为电脑显示，曝光时间设置为100ms，用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(e)实验结果表明，该芯片对MCF7细胞的捕获效率为95.3％；对照实验中平整表面的MCF7细胞的捕获效率仅为0.9％，这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获。
(4)作为对照组1，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的前列腺癌细胞PC3悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(5)作为对照组2，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴B细胞癌细胞Daudi悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(6)作为对照组3，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的人淋巴癌细胞Jurkat悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(7)作为对照组4，将步骤(2)得到的芯片置于六孔板中，加入3mL浓度为1×105个细胞/mL的子宫癌细胞Hela悬浮液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。作为对照实验，固定了肿瘤细胞表面的抗EpCAM抗体的平整石墨烯表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。捕获了循环肿瘤细胞的平整石墨烯表面也按上述步骤进行，并计算捕获效率。
(8)实验结果表明，本发明的石墨烯芯片对MCF7细胞的捕获效率为95.3％，前列腺癌细胞PC3细胞的捕获效率为91.7％，人淋巴B细胞癌细胞Daudi细胞的捕获效率为0.01％，人淋巴癌细胞Jurkat细胞的捕获效率为0.02％，子宫癌细胞Hela细胞的捕获效率为0.02％。这些数据表明该方法可以实现循环肿瘤细胞的高效特异性捕获，并实现极低的非特异性吸附。
实施例8
本实施例所制备的氯化钠为模板的立方体石墨烯立体几何结构平均尺寸为20μm；以乳腺癌病人血液中乳腺癌细胞的捕获为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氯化钠为模板的由立方体石墨烯组成的薄膜芯片的制备
(a)制备立方体氯化钠单晶
将商业用氯化钠粉末溶于水中配成氯化钠饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的氯化钠单晶粉末。
(b)制备由立方体石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)； 另一端约20cm长温度为700度并放置氯化钠单晶粉末。通过乙烯的热裂解，使得低温端的氯化钠单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氯化钠单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由立方体石墨烯组成的薄膜芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)将步骤(2)得到的芯片置于芯片培养皿中，加入1mL乳腺癌病人的血液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。
(4)实验结果表明，本发明的石墨烯芯片应用于全血捕获癌细胞的捕获个数为3。这些结果表明本发明的石墨烯芯片具有高效灵敏的捕获性能和极低的非特异性吸附。临床实验结果显著。
实施例9
本实施例所制备的氯化钠为模板的立方体石墨烯立体几何结构平均尺寸为20μm；以正常人血液中乳腺癌细胞的捕获为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于氯化钠为模板的由立方体石墨烯组成的薄膜芯片的制备
(a)制备立方体氯化钠单晶
将商业用氯化钠粉末溶于水中配成氯化钠饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的氯化钠单晶粉末。
(b)制备由立方体石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为700度并放置氯化钠单晶粉末。通过乙烯的热裂解，使得低温端的氯化钠单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除氯化钠单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由立方体石墨烯组成的薄膜芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)将步骤(2)得到的芯片置于芯片培养皿中，加入1mL正常人的血液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。
(4)实验结果表明，本发明的石墨烯芯片应用于全血捕获癌细胞的捕获个数为0。这些结果表明本发明的石墨烯芯片具有高效灵敏的捕获性能和极低的非特异性吸附。临床实验结果显著。
实施例10
本实施例所制备的硫酸铜为模板的斜方柱石墨烯立体几何结构平均尺寸为20μm；以乳腺癌病人血液中乳腺癌细胞细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于硫酸铜为模板的由斜方柱石墨烯组成的薄膜芯片的制备
(a)制备硫酸铜单晶
将商业用硫酸铜粉末溶于水中配成硫酸铜饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的硫酸铜单晶粉末。
(b)制备由斜方柱石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为550度并放置硫酸铜单晶粉末。通过乙烯的热裂解，使得低温端的硫酸铜单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除硫酸铜单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由斜方柱石墨烯组成的薄膜芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)将步骤(2)得到的芯片置于芯片培养皿中，加入1mL乳腺癌病人的血液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获 了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。
(4)实验结果表明，本发明的石墨烯芯片应用于全血捕获癌细胞的捕获个数为3。这些结果表明本发明的石墨烯芯片具有高效灵敏的捕获性能和极低的非特异性吸附。临床实验结果显著。
实施例11
本实施例所制备的硫酸铜为模板的斜方柱石墨烯立体几何结构平均尺寸为20μm；以正常人血液中乳腺癌细胞细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有立体几何结构的石墨烯表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于硫酸铜为模板的由斜方柱石墨烯组成的薄膜芯片的制备
(a)制备硫酸铜单晶
将商业用硫酸铜粉末溶于水中配成硫酸铜饱和溶液，控制温度为25度，静置、析出晶体，过滤、干燥得到边长平均为20μm的硫酸铜单晶粉末。
(b)制备由斜方柱石墨烯组成的薄膜芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为550度并放置硫酸铜单晶粉末。通过乙烯的热裂解，使得低温端的硫酸铜单晶粉末表面覆盖石墨烯层。将得到的产物溶于水中，去除硫酸铜单晶；高温干燥，得到石墨烯颗粒粉末。将粉末溶解，抽滤或旋涂，得到具有立体几何结构的石墨烯薄膜。
(2)由斜方柱石墨烯组成的薄膜芯片的特异性抗体修饰
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的具有立体几何结构的石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的石墨烯芯片。
(3)将步骤(2)得到的芯片置于芯片培养皿中，加入1mL正常人的血液，置于细 胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。
(4)实验结果表明，本发明的石墨烯芯片应用于全血捕获癌细胞的捕获个数为0。这些结果表明本发明的石墨烯芯片具有高效灵敏的捕获性能和极低的非特异性吸附。临床实验结果显著。
实施例12
本实施例所制备的平整石墨烯芯片，以乳腺癌病人血液中乳腺癌细胞细胞为例，对本发明的捕获体系作进一步阐述和验证。利用具有平整石墨烯芯片表面进行循环肿瘤细胞的特异性捕获的方法包括以下步骤：
(1)基于平整石墨烯芯片的制备
(a)制备石墨烯芯片
在管式炉中，在石英管的一端(约20cm)保持温度为850度，通入碳源(乙烯)；另一端约20cm长温度为600度并放置玻璃片。通过乙烯的热裂解，得到石墨烯粉末。将粉末溶解，抽滤或旋涂，得到具有平整石墨烯薄膜。
(2)基于平整石墨烯芯片的化学修饰特异性抗体
(a)羧酸化石墨烯芯片
将上述薄膜置于真空箱中在100度以上加热还原约12小时。室温条件下，将该薄膜浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干，得到羧酸化的薄膜；然后将该薄膜固定在玻璃片上，得到羧酸化的石墨烯薄膜芯片。
(b)将肿瘤细胞表面的生物素化抗EpCAM抗体固定在石墨烯芯片表面上，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的平整石墨烯芯片。
(i)将(a)中所述芯片置于六孔板内，加入含有N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，室温下反应2小时。
(ii)将该芯片置于链霉亲和素用磷酸盐缓冲液稀释为浓度为20μg/mL的链霉亲和素的磷酸盐溶液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤；
(iii)取20μL 20μg/mL生物素化的抗EpCAM抗体的PBS溶液，滴加到步骤(ii)得到的芯片表面，室温放置30分钟,然后用PBS润洗三次，洗去未连接的生物素化的抗EpCAM抗体，制备得到表面固定有循环肿瘤细胞表面的特异性抗体的平整石墨烯芯片。
(3)将步骤(2)得到的芯片置于芯片培养皿中，加入1mL乳腺癌病人血液，置于细胞培养箱中，由于肿瘤细胞表面的抗EpCAM抗体与石墨烯立体几何结构的协同 作用，可以特异性捕获循环肿瘤细胞，捕获时间是45分钟。将捕获了循环肿瘤细胞的芯片用磷酸盐缓冲液(PBS)清洗3次，然后用质量浓度为4％的多聚甲醛水溶液浸泡20分钟，质量浓度为0.4％的Triton-X100水溶液浸泡10分钟，2μg/mL DAPI水溶液浸泡15分钟，从而达到染色的目的。用Nikon倒置荧光显微镜10倍下分别拍照(每个捕获了循环肿瘤细胞的芯片选取中间部分10个不同的位置)，并对捕获了循环肿瘤细胞的芯片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。
(4)实验结果表明，本发明的石墨烯芯片应用于全血捕获癌细胞的捕获个数为0。这些结果表明本发明的石墨烯芯片具有高效灵敏的捕获性能和极低的非特异性吸附。临床实验结果显著。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制。尽管参照实施例对本发明进行了详细说明，本领域的普通技术人员应该理解，对本发明的技术方案进行修改或者等同替换，都不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片，其特征在于，所述石墨烯芯片包括具有立体几何微纳结构的石墨烯基片以及在石墨烯基片表面固定的循环肿瘤细胞表面的特异性抗体。
  2. 根据权利要求1所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片，其特征在于，所述的石墨烯基片是通过将具有立体几何微纳结构的石墨烯颗粒通过抽滤或者旋涂得到，其中石墨烯颗粒的大小为1μm～30μm。
  3. 根据权利要求1所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片，其特征在于，所述循环肿瘤细胞表面的特异性抗体为生物素化抗EpCAM抗体。
  4. 根据权利要求1所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片，其特征在于，所述循环肿瘤细胞表面的特异性抗体在石墨烯表面的固定量不少于0.1μg/cm2。
  5. 一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，所述方法包括以下步骤：
    1)利用无机化合物粉末重结晶得到具有立体几何形状的单晶，所述立体几何形状是由经过结晶过程而形成的无机化合物晶体所具有规则的几何外形结构；
    2)利用化学气相沉积，在步骤1)得到的单晶表面上构筑具有立体几何结构的石墨烯层；将石墨烯层覆盖的单晶溶解得到含石墨烯的溶液，去除单晶并干燥得到石墨烯粉末，石墨烯粉末溶解并将溶解后得到的溶液添加到基片上抽滤或者旋涂得到表面具有立体几何微纳结构的石墨烯的基片；
    3)通过在立体几何微纳结构的石墨烯基片表面固定对循环肿瘤细胞具有特异性的抗体，得到循环肿瘤细胞的特异性捕获的石墨烯芯片。
  6. 根据权利要求5所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，其特征在于，所述的无机化合物粉末选自氯化钠、氯化钾、氯化铜、硫酸铜、钨酸锰、氧化钒、氧化钨和氯化钴中的一种。
  7. 根据权利要求5所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，其特征在于，步骤1)中，所述立体几何形状包括立方体、斜方锥、斜方柱和八面体中的一种或多种。
  8. 根据权利要求5所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，其特征在于，在步骤2)中，所述的基片选自晶体硅、普通玻璃、石英或微孔滤膜。
  9. 根据权利要求5所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片的制备方法，其特征在于，具有立体几何微纳结构的石墨烯基片表面上固定循环肿瘤细胞表面的特异性抗体的方法具体为：
    a)将具有立体几何微纳结构的石墨烯真空加热还原，放至室温；室温下，将高温处理后的具有立体几何微纳结构的石墨烯浸泡在浓度为1～10％的1-芘羧酸的甲醇溶液中，室温下放置；取出用甲醇和二甲基亚砜润洗，吹干；
    b)将该石墨烯基片置于5～20μg/mL链霉亲和素用磷酸盐缓冲液中，室温下放置(优选室温下放置30分钟左右)；取出基片，用磷酸盐缓冲液洗涤，并加入含有 N-羟基琥珀酰亚胺和N-(3-二甲基胺基丙基)-N'-乙基碳二亚胺的二甲基亚砜溶液中，然后将步骤a)吹干后的石墨烯浸泡在上述溶液中，室温下放置，取出用磷酸盐缓冲液洗涤；
    c)将循环肿瘤细胞表面的特异性抗体用磷酸盐缓冲液稀释至浓度为5～20μg/mL，然后滴加到步骤b)用磷酸盐缓冲液洗涤后得到的石墨烯的表面上，室温下放置，得到用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片。
  10. 权利要求1-4任一项所述的一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片在全血中循环肿瘤细胞的捕获中的应用。
PCT/CN2017/117495 2017-01-20 2017-12-20 一种用于全血中循环肿瘤细胞的特异性捕获的石墨烯芯片及其制备方法和应用 WO2018133617A1 (zh)

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