WO2014036951A1 - Method and device for specifically capturing circulating tumor cell by utilizing surface of fractal structure and application thereof - Google Patents

Method and device for specifically capturing circulating tumor cell by utilizing surface of fractal structure and application thereof Download PDF

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WO2014036951A1
WO2014036951A1 PCT/CN2013/082982 CN2013082982W WO2014036951A1 WO 2014036951 A1 WO2014036951 A1 WO 2014036951A1 CN 2013082982 W CN2013082982 W CN 2013082982W WO 2014036951 A1 WO2014036951 A1 WO 2014036951A1
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fractal structure
tumor cells
circulating tumor
substrate
fractal
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PCT/CN2013/082982
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French (fr)
Chinese (zh)
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王树涛
张鹏超
陈立
江雷
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中国科学院化学研究所
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    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for performing specific capture of circulating tumor cells using the surface of a fractal structure and a device for specifically capturing circulating tumor cells and uses thereof.
  • Background technique
  • Cancer is the second biggest killer of human death. 90% of deaths are caused by metastatic cancer. Metastatic cancer is mainly caused by cancer cells that are detached from the initial cancerous position. This part of the cancer cells spread to other tissues or organs of the body through blood vessels or lymphatic vessels, thereby forming metastatic cancer. The entry of cancer cells into the blood circulation is the initial stage of cancer metastasis, providing the possibility of eventually forming metastatic lesions of the tumor. The cancer cells that enter this part of human peripheral blood are Circulating Tumor Cells. By detecting circulating tumor cells in peripheral blood, it is possible to effectively predict the occurrence of cancer metastasis, conduct early diagnosis of cancer, predict the survival rate of cancer patients, and monitor the effects of treatment. Therefore, the detection of circulating tumor cells in the blood has important clinical application value. The detection of circulating tumor cells is of great significance for the early diagnosis, disease monitoring and treatment prognosis of cancer.
  • the prerequisite for the detection of circulating tumor cells is the capture and separation of circulating tumor cells in the blood. Due to its small amount in the blood (several to dozens per milliliter), the capture and separation of circulating tumor cells remains a huge challenge.
  • the methods of capturing and separating circulating tumor cells are mainly based on the specific morphology and biochemical characteristics of tumor cells, such as size, density, and proteins specifically expressed on the surface of tumor cells.
  • Existing capture and separation techniques mainly include ISET (Isolation by size of epithelial tumor cells) technology (Am J Pathol 2000, 156, 57-63), density gradient centrifugation (Cytometry 2002, 49, 150-158) and immunomagnetics. Separation technique (N Engl J Med 2004, 351, 781-91).
  • the ISET technique utilizes the difference between tumor cells and blood cell size for the separation of circulating tumor cells, but the separation purity of this technique is low and it is difficult to meet actual needs. Density gradient centrifugation is based on the difference in cell density in the blood. Cells with higher density such as red blood cells and neutrophils migrate to the bottom, and tumor cells are still at the top, thus achieving separation. However, the problem with this technique is also that the purity is low, and the circulating tumor cells are inevitably lost during the process of treatment and transfer, thereby affecting the capture efficiency. Immunomagnetic separation technology is one of the most widely studied and applied technologies.
  • the principle is to modify the specific antibody on the surface of magnetic particles and combine it with the surface antigen of circulating tumor cells to achieve the purpose of capturing and separating under the action of magnetic field. It can achieve higher capture efficiency, but its application is limited due to its complicated process and high cost.
  • the method for performing specific capture of circulating tumor cells by using the surface of the fractal structure of the present invention the basic principle is to prepare a surface of a desired fractal structure on the surface of the substrate, and immobilizing specific antibodies on the surface of the circulating tumor cells On the surface having the desired fractal structure, a sample of the circulating tumor cells to be captured is dropped onto the surface of the specific antibody on the surface of the circulating tumor cells, and the specific antigen on the surface of the circulating tumor cells in the sample is fixed thereto.
  • the method of the present invention for performing specific capture of circulating tumor cells using the surface of the fractal structure comprises the following steps:
  • the fractal structure is selected from a branched fractal structure, a broccoli fractal structure or a urchin fractal structure;
  • the material of the substrate is selected from one of a conductive metal, a conductive inorganic non-metal, a conductive inorganic non-metal compound, a non-conductive inorganic non-metal compound, and a non-conductive polymer.
  • the conductive metal is preferably selected from one of gold, silver, platinum, palladium, copper, iron, zinc, and aluminum.
  • the conductive inorganic nonmetal is preferably crystalline silicon.
  • the conductive inorganic non-metallic compound is preferably indium tin oxide (ITO) glass.
  • the non-conductive inorganic non-metallic compound is preferably quartz or ordinary glass.
  • the non-conductive polymer may be various non-conductive polymers capable of forming a block or a film, preferably selected from the group consisting of dimethyl siloxane, (meth) acrylate, urethane, carbonate, ethylene, propylene. a homopolymer and/or a copolymer formed of at least one of styrene, lactic acid, and vinyl alcohol as a monomer (eg, polydimethylsiloxane, poly(meth)acrylate, polyurethane, polycarbonate , polyethylene, polypropylene, polystyrene, polylactic acid, a copolymer of styrene and lactic acid, and at least one of polyvinyl alcohol).
  • a monomer eg, polydimethylsiloxane, poly(meth)acrylate, polyurethane, polycarbonate , polyethylene, polypropylene, polystyrene, polylactic acid, a copolymer of styren
  • the material of the surface of the fractal structure is selected from one of a metal, a metal compound, an inorganic non-metal, an inorganic non-metal compound, a conductive polymer, and a non-conductive polymer.
  • the metal is preferably selected from one of gold, silver, platinum, palladium, copper, iron, zinc, and aluminum.
  • the metal compound is preferably selected from one of copper oxide, iron oxide, zinc oxide and titanium dioxide.
  • the inorganic nonmetal is preferably crystalline silicon.
  • the inorganic non-metallic compound is preferably quartz.
  • the conductive polymer may be any of various conductive polymers capable of forming a block or a film, and is preferably one selected from the group consisting of polythiophene, polypyrrole, polyaniline, and polyphenylene.
  • the non-conductive polymer may be various non-conductive polymers capable of forming a block or a film, preferably selected from the group consisting of dimethyl siloxane, (meth) acrylate, urethane, carbonate, ethylene, propylene, a homopolymer and/or a copolymer formed of at least one of styrene, lactic acid, and vinyl alcohol as a monomer (eg, polydimethylsiloxane, poly(meth)acrylate, polyurethane, polycarbonate, Polyethylene, polypropylene, polystyrene, polylactic acid, a copolymer of styrene and lactic acid, and at least one of polyvinyl alcohol).
  • the material of the substrate and the material of the surface of the fractal structure may be the same or different.
  • the surface of the fractal structure is prepared as a metal material on the surface of the conductive metal substrate, the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate, it is preferably prepared by electrochemical deposition, that is, Using a conventional three-electrode system, using a platinum wire or a platinum plate as a counter electrode, a silver/silver chloride electrode as a reference electrode, the conductive metal substrate, the conductive inorganic non-metal substrate or the
  • the conductive inorganic non-metallic compound substrate is used as a working electrode, and is an aqueous solution of chloroauric acid, an aqueous solution of silver nitrate, an aqueous solution of chloroplatinic acid, an aqueous solution of chloropalladic acid, an aqueous solution of copper nitrate, an aqueous solution of ferrous chloride, an aqueous solution of zinc sulfate or an aqueous solution of aluminum
  • the electrolyte such as gold as the substrate can use chloroauric acid aqueous solution as the electrolyte, you can also choose other electrolytes
  • the working voltage -0.1 V ⁇ - 0.5 V
  • the concentration of ions (0.01 mol / L ⁇ 2 mol / L), electrochemical deposition at room temperature for 20 minutes ⁇ 180 minutes, can be in the conductive metal base , Inorganic non-metallic surface of said conductive substrate or said conductive non-metal inorganic compound can be prepared substrate surface dendritic structure formed on the surface of the sub-surface sea urchin-like or fractal structure cauliflower fractal structure, respectively.
  • the conductive polymer material is polythiophene as a fractal structure.
  • the method described in the literature (Yu Weili, Electrodeposition of Polythiophene Ordered Microstructures and Organic Solar Cells. Jilin University, 2009.) is preferably used in the conductive metal substrate.
  • the surface of the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate is prepared as a surface of a broccoli fractal structure or a surface of a urchin-shaped fractal structure; and the conductive polymer material is a polypyrrole as a fractal
  • the method described in the literature Wang Jie, Electrical Properties and Application of Electrochemical Synthesis of Polypyrrole Films. Xi'an Jiaotong University, 2008.
  • the conductive metal substrate, the conductive The surface of the inorganic non-metal substrate or the conductive inorganic non-metal compound substrate is prepared as a surface of a urchin-like fractal structure; for a surface having a conductive polymer material as polystyrene as a fractal structure, the literature is preferably used ( Scientific method, 2003, 48, 35-37), which can be prepared on the surface of the conductive metal substrate, the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate.
  • the surface of the urchin-like fractal structure for a surface having a conductive polymer material as polystyrene as a fractal structure, the literature is preferably used ( Scientific method, 2003, 48, 35-37), which can be prepared on the surface of the conductive metal substrate, the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate.
  • the metal compound is used as the copper oxide as a fractal
  • the method described in the literature Journal of Shaanxi Normal University (Natural Science Edition), 2009, 37, 60-62) is preferably employed, that is, in the conductive inorganic non-metal substrate,
  • the surface of the conductive inorganic non-metallic compound substrate or the non-conductive inorganic non-metal compound substrate is prepared as a surface of a broccoli-like fractal structure or a surface of a urchin-like fractal structure; for a surface having a metal compound as an iron oxide as a fractal structure
  • the method described in the literature Journal of Northeast Normal University (Natural Science Edition), 2012, 44, 155-157) is preferably employed, that is, in the conductive inorganic non-metal substrate.
  • the surface of the conductive inorganic non-metallic compound substrate or the non-conductive inorganic non-metal compound substrate is prepared as a surface of a broccoli-like fractal structure or
  • the conductive inorganic non-metal group a surface of the conductive inorganic non-metal compound substrate or the surface of the non-conductive inorganic non-metal compound substrate to prepare a surface of a dendritic fractal structure or a surface of a urchin-like fractal structure; for a metal compound as titanium dioxide as a fractal
  • the method described in the literature ACS Nano, 2009, 3, 1212-1218
  • the surface of the sheet or the non-conductive inorganic non-metallic compound substrate is prepared as a surface of a dendritic fractal structure or a surface of a urchin-like fractal structure.
  • the substrate described above is consistent with the chemical composition of the surface of the fractal structure
  • the surface of the conductive inorganic non-metal compound substrate is prepared as an inorganic non-metal compound material (the substrate described above is consistent with the chemical composition of the surface of the fractal structure)
  • the method described in the literature can be prepared on the surface of the conductive inorganic non-metal substrate or the non-conductive inorganic non-metal compound substrate. The surface of the cauliflower-like fractal structure.
  • the surface of the fractal structure is prepared on the surface of the non-conductive polymer substrate as a non-conductive polymer material (the substrate is in conformity with the chemical composition of the surface of the fractal structure), (Macromol. Rapid Commun. 2005, 26, 1805-1809), a surface of a broccoli-like fractal structure can be prepared on the surface of the non-conductive polymer substrate; using the literature (Angew. Chem. Int) Ed. 2002, 41, 1221-1223), the surface of the dendritic fractal structure can be prepared on the surface of the non-conductive polymer substrate.
  • the method of immobilizing the specific antibody on the surface of the circulating tumor cell on the surface of the substrate obtained in the step (1) having the desired fractal structure may be carried out by sequentially connecting the biotin on the surface of the fractal structure.
  • a method for immobilizing polyethylene glycol (having a functional group at one end), streptavidin, and specific antibodies on the surface of circulating tumor cells, and a preferred method of immobilization can be carried out as follows: (a) diluting a biotinylated polyethylene glycol having a functional group to one end with a deionized water to a 2 mM aqueous solution, and immersing the substrate having a surface having a desired fractal structure at room temperature at room temperature A biotinylated polyethylene glycol having a functional group attached at one end to a 2 mM aqueous solution (the surface having the surface having the desired fractal structure is rinsed with deionized water before use; preferably soaked) The time is more than 12 hours
  • step (b) diluting streptavidin with phosphate buffered saline (PBS) to a phosphate solution of streptavidin at 10 g/mL, and then drying the surface obtained in step (a) to have the desired
  • PBS phosphate buffered saline
  • the substrate of the surface of the fractal structure is immersed in the above 10 g/mL streptavidin phosphate solution, and left at room temperature (preferably left at room temperature for about 30 minutes); the surface is taken out to have a desired fractal structure. Substrate, washed with phosphate buffer;
  • step (c) Diluting specific antibodies (such as anti-EpCAM antibodies) on the surface of circulating tumor cells with phosphate buffered saline (PBS) to 10 g/mL, followed by dropwise addition to step (b) washing with phosphate buffered saline (PBS)
  • PBS phosphate buffered saline
  • the resulting surface is on the surface of the substrate having the surface of the desired fractal structure (e.g., 25 L is applied dropwise to the surface of the substrate having the surface having the desired fractal structure (area 1 cm 2 ) ), placed at room temperature (preferably at room temperature for 30 minutes).
  • the specific antibody that circulates the surface of the tumor cell is immobilized on the surface of the substrate obtained in the step (1) having a desired fractal structure, which is fixed on the surface of the circulating tumor cell on the surface having the desired fractal structure.
  • the immobilized amount of the specific antibody is preferably not less than 0.1 g/cm 2 (preferably from 0.1 to 5 g/cm 2 ).
  • the range of the characteristic parameter fractal dimension of the surface of the fractal structure is preferably: 2 ⁇ fractal dimension ⁇ 3.
  • the specific antibody on the surface of the circulating tumor cells may be an antibody against an antigen specifically expressed by various tumor cells, preferably an anti-EpCAM antibody.
  • biotinylated polyethylene glycol, streptavidin, and anti-EpCAM antibodies having functional groups at one end are commercially available products.
  • the functional group in the biotinylated polyethylene glycol to which the functional group is attached at one end is one of a thiol group, a carboxyl group and an amino group.
  • the sample of the circulating tumor cells to be captured is generally a peripheral blood sample.
  • the present invention also provides a device for specifically capturing circulating tumor cells, the device comprising a substrate having a fractal structure on a surface thereof, wherein a specific antibody having a surface of a circulating tumor cell is immobilized on a surface having a fractal structure on the substrate
  • the fractal structure is selected from the group consisting of a branched fractal structure, a broccoli fractal structure or a urchin fractal structure.
  • the present invention provides the use of the device of the present invention described above for early diagnosis, disease monitoring and prognosis of cancer.
  • the invention establishes a method for performing specific capture of circulating tumor cells by using the surface of the fractal structure, and the method (1) proves that the surface of the fractal structure can achieve efficient capture of circulating tumor cells; (2) the specificity is strong, Capture specific tumor cells and reduce adhesion of non-specific tumor cells.
  • the method and device of the present invention for performing specific capture of circulating tumor cells using the surface of the fractal structure can be used for the capture of cancer-associated cells.
  • the method can effectively improve the capturing efficiency of circulating tumor cells, and the cost is low. It is easy to operate and can be used for clinical diagnosis.
  • Fig. la Large-area scanning electron micrograph of the surface topography of a gold surface having a dendritic fractal structure prepared in Example 1 of the present invention
  • Fig. 1b is an enlarged photograph of a scanning electron microscope of a single particle having a gold-like surface of a dendritic fractal structure.
  • Fig. 2 is a schematic view showing the process of immobilizing an anti-EpCAM antibody on the surface of a circulating tumor cell of Example 1 of the present invention on a substrate having a gold-like surface of a dendritic fractal structure.
  • Figure 3 A histogram of the capture efficiency of Example 1 of the present invention. detailed description
  • MCF7 cells (ATCC® H B-22TM), Jurkat T cells (ATCC® CRL-2899TM), and Daudi cells (ATCC® CCL-213TM) were purchased from ATCC; anti-EpCAM antibodies were purchased from R&D.
  • the product number is BAF960; biotinylated polyethylene glycol with thiol, carboxyl or amino group at one end is purchased from Nanocs Co., Ltd., and the numbers are PG2-BNTH-5k, PG2-BNCA-5k and PG2-AMBN-5k;
  • the fixed amount of anti-EpCAM antibody on the cell surface was determined by means of an ELISA kit (purchased from Sangon Biotech, article number ESK5295). For details, see the kit instructions.
  • the fractal dimension of the gold surface having the dendritic fractal structure prepared in this example is 2.73; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a gold surface having a dendritic fractal structure is prepared on the surface of a crystalline silicon substrate by electrochemical deposition.
  • a crystal solution of a gold surface having a dendritic fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a).
  • the substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a crystalline silicon substrate having a gold surface having a dendritic fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was 0.1 g/cm 2 on a crystalline silicon substrate having a gold surface of a dendritic fractal structure of 1 cm 2 at room temperature for 30 minutes.
  • 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the gold surface having a dendritic fractal structure on the crystalline silicon substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter).
  • the surface of the anti-EpCAM antibody on the surface of the immobilized tumor cells (three 1 cm 2 crystal silicon substrates with a dendritic fractal gold surface were placed in each cell culture plate), and then placed in a cell culture incubator, Synergistic effect of anti-EpCAM antibody on the surface of tumor cells and dendritic fractal structure, specifically capturing anti-EpCAM on the surface of tumor cells immobilized on the gold surface with dendritic fractal structure added to the crystalline silicon substrate obtained in step (2)
  • the step (3) on the surface of the antibody was mixed to obtain a circulating tumor cell in a cell suspension obtained uniformly, and the capture time was 45 minutes.
  • the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
  • the crystalline silicon substrate of the gold surface having the dendritic fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • Each of the crystalline silicon substrates having a dendritic fractal-shaped gold surface captured by circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and a crystalline silicon substrate having a gold-like surface having a dendritic fractal structure captured by circulating tumor cells was photographed. The circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
  • the flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the gold surface having the cauliflower-like fractal structure prepared in this example is 2.54; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a gold surface having a broccoli fractal structure was prepared on the surface of an indium tin oxide (ITO) glass substrate by electrochemical deposition.
  • a platinum plate is used as a counter electrode
  • a silver/silver chloride electrode is used as a reference electrode
  • an indium tin oxide (yttrium) glass substrate is used as a working electrode
  • an aqueous solution of chloroauric acid (1 mg/mL) is used as an electrolyte.
  • the chloride ion concentration was 0.9 mo/L
  • a gold surface having a broccoli-like fractal structure was prepared on the surface of an indium tin oxide (ITO) glass substrate by electrochemical deposition for 90 minutes at an operating voltage of -0.15 V.
  • a biotinylated polyethylene glycol with a sulfhydryl group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and an indium tin oxide (ITO) glass substrate having a broccoli-like fractal gold surface at room temperature is used. Soaking in a 2 mM aqueous solution of biotinylated polyethylene glycol thiol-containing at one end is immersed for 12 hours (indium tin oxide (ITO) glass substrate with a broccoli-like fractal gold surface is used before use. Rinse with deionized water; remove the indium tin oxide (yttrium) glass substrate with a gold surface of broccoli fractal structure, wash with deionized water, and dry;
  • a phosphate solution having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a)
  • the tin (ITO) glass substrate was immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; an indium tin oxide (yttrium) glass substrate having a gold surface of a broccoli fractal structure was taken out, buffered with phosphate. Liquid washing;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • the immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was 0.5 g/cm 2 on an indium tin oxide (ITO) glass substrate having a cauliflower-like fractal gold structure of 1 cm 2 at room temperature for 30 minutes.
  • ITO indium tin oxide
  • MCF7 cell suspension 20 Take MCF7 cell suspension 20 and count it with a cell counter and calculate the concentration. Take a certain amount of the above cell suspension, dilute to 1 ⁇ 10 5 cells/mL with cell culture medium DMEM, mix well, and store at room temperature.
  • the Daudi cell suspension 20 was counted by a cell counter and the concentration thereof was calculated. A certain amount of the above cell suspension was taken, diluted with a cell culture medium 1640 to IX 10 5 cells/mL, uniformly mixed, and stored at room temperature.
  • the gold surface of the fractal structure is fixed on the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 of indium tin oxide ⁇ with a gold surface of broccoli-like fractal structure is placed on each cell culture plate)) And then placed in a cell culture incubator, the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the cauliflower-like fractal structure, specifically capturing the broccoli-like shape of the indium tin oxide ⁇ ) obtained on the glass substrate obtained by the step (2)
  • the gold surface of the fractal structure is fixed on the surface of the tumor cell surface of the anti-EpCAM antibody on the surface of the step (3) and the circulating tumor cells in the obtained cell suspension are uniformly mixed, and the capture time is 45 minutes.
  • the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
  • ITO indium tin oxide
  • PBS phosphate buffered saline
  • Triton-XlOO aqueous solution with a concentration of 0.4% was immersed for 10 minutes, and the 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • the flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the gold surface having the urchin-like fractal structure prepared in this embodiment is 2.23; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a gold surface having a urchin-like fractal structure is prepared on the surface of an iron substrate using a conventional three-electrode system, a platinum plate as a counter electrode and a silver/silver chloride electrode as a reference electrode.
  • Iron substrate as working electrode, chloroauric acid aqueous solution (l mg/mL) as electrolyte (chloride ion concentration 2 mo/L), electrochemical deposition for 180 minutes at working voltage of -0.1 V
  • the surface is prepared to prepare a gold surface having a urchin-like fractal structure.
  • step (1) (2) immobilizing an anti-EpCAM antibody on the surface of the tumor cell on the gold surface having the urchin-like fractal structure on the iron substrate obtained in the step (1) (a) diluting a biotinylated polyethylene glycol having a sulfhydryl group at one end with a deionized water to a 2 mM aqueous solution, and immersing an iron substrate having a gold surface having a urchin-like fractal structure at one end at room temperature
  • the thiol biotinylated polyethylene glycol is immersed in an aqueous solution of 2 mM for 12 hours (the iron substrate with the gold surface of the urchin-like fractal structure is rinsed with deionized water before use); the urchin-like fractal is taken out Structure of the gold surface of the iron substrate, washed with deionized water, dried;
  • an iron base having a gold surface having a urchin-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a) The sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; an iron substrate having a gold surface having a urchin-like fractal structure was taken out and washed with a phosphate buffer;
  • PBS phosphate buffered saline
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the iron substrate of a gold surface having a urchin-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 2 g/cm 2 .
  • 3 mL of the cell suspension obtained by uniformly mixing the step (3) was respectively added to the gold surface having the urchin-like fractal structure on the iron substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter).
  • Immobilized tumor cell surface on the surface of anti-EpCAM antibody (3 1 cm 2 gold substrate with gold surface of urchin-like fractal structure per cell culture plate), then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the urchin-like fractal structure, specifically capturing the surface of the anti-EpCAM antibody surface of the tumor cell surface fixed by the gold surface having the urchin-like fractal structure added to the iron substrate obtained in the step (2)
  • the circulating tumor cells in the cell suspension obtained after mixing were uniformly mixed, and the capture time was 45 minutes.
  • the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
  • the iron substrate of the gold surface having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • the iron substrate of each gold surface having a urchin-like fractal structure captured by circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the iron substrate of the gold surface having the urchin-like fractal structure of the circulating tumor cells was photographed. The captured circulating tumor cells were counted and the capture efficiency was calculated.
  • the flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the polypyrrole surface having the broccoli fractal structure prepared in this example is 2.51;
  • MCF7 and Daudi are examples of circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to a 2 mM aqueous solution, and an indium tin oxide (ITO) glass base having a cauliflower-like fractal structure on the surface of the polypyrrole at room temperature is used.
  • the sheet was immersed in an aqueous solution of biotinylated polyethylene glycol having a carboxyl group at one end and dissolved in a 2 mM aqueous solution for 12 hours (indium tin oxide ( ⁇ ) glass substrate having a cauliflower-like fractal structure on the polypyrrole surface was used. Rinse with deionized water before); remove the indium tin oxide ⁇ ) glass substrate with polycalyx surface with broccoli fractal structure, wash with deionized water, and dry;
  • step (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then oxidizing the surface of the polypyrrole having a cauliflower-like fractal structure obtained by drying the step (a)
  • the indium tin (ITO) glass substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the indium tin oxide ⁇ ) glass substrate having a cauliflower-like fractal structure is taken out, and the phosphate is used. Buffer washing;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • the immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 5 g/cm 2 on an indium tin oxide (ITO) glass substrate having a cauliflower-like fractal structure of 1 cm 2 and allowed to stand at room temperature for 30 minutes.
  • ITO indium tin oxide
  • the flat polypyrrole surface to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • the indium tin oxide ⁇ ) glass substrate of the polypyrrole surface having the broccoli-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked with a 4% by mass aqueous solution of paraformaldehyde. After 20 minutes, the Triton-XlOO aqueous solution with a concentration of 0.4% was immersed for 10 minutes, and the 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • Photographs of each indium tin oxide ⁇ ) glass substrate with a cauliflower-like fractal structure with broccoli-like fractal structure captured by Nikon inverted fluorescence microscope, and a polygalactose-like fractal structure captured by circulating tumor cells The circulating tumor cells captured on the glass substrate of the indium tin oxide on the surface of the pyrrole were counted, and the capture efficiency was calculated.
  • the flat polypyrrole surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the surface of polyphenylene having a urchin-like fractal structure prepared in this example was 2.41.
  • the capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a polyphenylene surface having a urchin-like fractal structure can be prepared on the surface of an indium tin oxide (ITO) glass substrate by the method described in the literature (Science Bulletin, 2003, 48, 35-37).
  • ITO indium tin oxide
  • a biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to an aqueous solution of 2 mM, and an indium tin oxide (ITO) glass base having a surface of a polystyrene having a urchin-like fractal structure is obtained at room temperature.
  • the sheet was immersed in an aqueous solution of biotinylated polyethylene glycol having a carboxyl group at one end and dissolved in a 2 mM aqueous solution for 12 hours (indium tin oxide with a surface of polystyrene having a urchin-like fractal structure).
  • the glass substrate was used before use. Rinse with deionized water); remove the indium tin oxide ⁇ ) glass substrate with a polystyrene surface having a urchin-like fractal structure, wash with deionized water, and dry;
  • step (b) oxidizing a streptavidin diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the polyphenylene surface having a urchin-like fractal structure obtained by drying the step (a) Indium tin (ITO) glass substrate is immersed in streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; indium tin oxide ⁇ ) glass substrate with urchin-like fractal structure is removed, using phosphate Buffer washing; (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the indium tin oxide (ITO) glass substrate having a surface of polystyrene having a urchin-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 0.5 g/cm 2 .
  • the step (3) on the surface of the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface of the polyphenylene having a urchin-like fractal structure is uniformly mixed to obtain a circulating tumor cell in the cell suspension, and the capture time is 45 minutes.
  • the flat polyphenyl surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same capture of circulating tumor cells in the test sample.
  • ITO indium tin oxide
  • PBS phosphate buffered saline
  • Photographs of indium tin oxide (ITO) glass substrates on the surface of polyphenylene having urchin-like fractal structures each capturing circulating tumor cells were photographed by Nikon inverted fluorescence microscope, and urchin-like fractal structures were captured for circulating tumor cells.
  • the circulating tumor cells captured on the polyphenylene surface of the indium tin oxide ⁇ ) glass substrate were counted, and the capture efficiency was calculated.
  • the smooth polystyrene surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the polyaniline having the urchin-like fractal structure prepared in this example was 2.41.
  • the capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps: (1) Indium tin oxide can be used in the method described in the literature (J. Phys. Chem. C 2010, 114, 8062-8067)
  • the surface of the glass substrate was prepared with a surface of a polyaniline having a urchin-like fractal structure.
  • a biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water into an aqueous solution of 2 mM, and an indium tin oxide ⁇ ) glass substrate having a surface of a polyaniline having a urchin-like fractal structure at room temperature Soaking in a 2 mM aqueous solution of biotinylated polyethylene glycol with carboxyl group at one end is immersed for 12 hours (indium tin oxide with a urchin-like fractal structure of indium tin oxide ⁇ ).
  • the glass substrate is used before use. Rinsing with deionized water); taking out an indium tin oxide ⁇ ) glass substrate having a surface of polyaniline having a urchin-like fractal structure, washing with deionized water, and drying;
  • step (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then oxidizing the surface of the polyaniline having a urchin-like fractal structure obtained by drying step (a) Indium tin ⁇ ) The glass substrate was immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the indium tin oxide ⁇ ) glass substrate having the surface of the polyaniline having a urchin-like fractal structure was taken out, buffered with phosphate Liquid washing;
  • PBS phosphate buffered saline
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the tablet is then placed in a cell culture incubator, and the anti-EpCAM antibody on the surface of the tumor cell synergizes with the urchin-like fractal structure to specifically capture the indium tin oxide (ITO) glass substrate obtained by the step (2).
  • the surface of the anti-EpCAM antibody on the surface of the tumor cell surface immobilized on the surface of the polyaniline having the urchin-like fractal structure (3) is uniformly mixed to obtain circulating tumor cells in the cell suspension, and the capture time is 45 minutes.
  • the flat polyaniline surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • ITO indium tin oxide
  • PBS phosphate buffered saline
  • Cyclic tumors were captured for each with a Nikon inverted fluorescence microscope Photographing the indium tin oxide ⁇ ) glass substrate of the polyaniline surface of the cell with a urchin-like fractal structure, and capturing it on a glass substrate of an indium tin oxide ⁇ ) surface of a polyaniline having a urchin-like fractal structure that captures circulating tumor cells The circulating tumor cells were counted and the capture efficiency was calculated.
  • the flat polyaniline surface that captured the circulating tumor cells was also subjected to the above procedure, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the polythiophene having the broccoli fractal structure prepared in this example was 2.51.
  • the capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a gold phosphate solution having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a).
  • PBS phosphate buffered saline
  • the substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the gold substrate having the surface of the polythiophene having a cauliflower-like fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the gold substrate of the polythiophene surface having a cauliflower-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 0.8 g/cm 2 .
  • 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the surface of the polythiophene having a cauliflower-like fractal structure on the gold substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter).
  • Fixed On the surface of the anti-EpCAM antibody on the surface of the tumor cells three 1 cm 2 gold substrate on the surface of the polythiophene with cauliflower-like fractal structure was placed in each cell culture dish), and then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the cauliflower-like fractal structure, specifically capturing the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface of the polythiophene having the cauliflower-like fractal structure added to the gold substrate obtained in the step (2) On the surface of the step (3), after mixing evenly, the circulating tumor cells in the cell suspension were obtained, and the capture time was 45 minutes.
  • the flat polythiophene surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • the gold substrate on the surface of the polythiophene having the cauliflower-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • the gold substrate of the polythiophene surface having the cauliflower-like fractal structure of each of the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the gold base of the polythiophene surface having the cauliflower-like fractal structure of the circulating tumor cells was captured.
  • the circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
  • the flat polythiophene surface that captured the circulating tumor cells was also subjected to the above procedure, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the zinc oxide having the dendritic fractal structure prepared in this example is 2.81; and the tumor cells MCF7 and Jurkat T are taken as the circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a zinc oxide surface having a dendritic fractal structure can be prepared on the surface of a conventional glass substrate by the method described in the literature (Adv. Funct. Mater., 2006, 16, 335-344).
  • step (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, Then, the ordinary glass substrate having the zinc oxide surface of the dendritic fractal structure obtained after the step (a) is dried is immersed in the streptavidin phosphate solution, and left at room temperature for 30 minutes; and the dendritic fractal structure is taken out. a common glass substrate on the surface of zinc oxide, washed with a phosphate buffer;
  • PBS phosphate buffered saline
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 2.8 g/cm 2 at room temperature for 30 minutes.
  • the capture time is 45 minutes.
  • the flat zinc oxide surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing circulating tumor cells in the sample to be tested.
  • the ordinary glass substrate of the zinc oxide surface having the dendritic fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass
  • PBS phosphate buffered saline
  • the Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing.
  • a common glass substrate of a zinc oxide surface having a dendritic fractal structure each capturing a circulating tumor cell was photographed by a Nikon inverted fluorescence microscope, and a common zinc oxide surface having a dendritic fractal structure was captured.
  • the circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
  • the flat zinc oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the copper oxide having the urchin-like fractal structure prepared in this embodiment is 2.21; MCF7 and JurkatT are examples of circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a copper oxide surface having a urchin-like fractal structure can be prepared on the surface of a common glass substrate by the method described in Journal of Shaanxi Normal University (Natural Science Edition, 2009, 37, 60-62).
  • a biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to a 2 mM aqueous solution, and a common glass substrate having a surface of a copper oxide having a urchin-like fractal structure is immersed at the above end at room temperature.
  • step (b) diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the surface of the copper oxide having a urchin-like fractal structure obtained by drying the step (a)
  • the glass substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a common glass substrate having a copper oxide surface having a urchin-like fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 4.1 g/cm 2 at room temperature for 30 minutes.
  • the surface of the flat copper oxide of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • a common glass substrate on which a copper oxide surface having a urchin-like fractal structure of circulating tumor cells was captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the aqueous solution of Triton-XlOO with a concentration of 0.4% was immersed for 10 minutes, and 2 ⁇ glmL of DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • a common glass substrate of a copper oxide surface having a urchin-like fractal structure in which a circulating tumor cell was captured was photographed by a Nikon inverted fluorescence microscope, and a common copper oxide surface having a urchin-like fractal structure in which a circulating tumor cell was captured was used.
  • the circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
  • the flat copper oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of MCF7 cells on the surface of copper oxide with urchin-like fractal structure is 35.3%, and the capture efficiency of Jurkat T cells is only 1.4%.
  • the capture efficiency of MCF7 cells on the surface of copper oxide in the control experiment is only At 2.4%, the capture efficiency of Jurkat T cells was only 1.3%.
  • the fractal dimension of the surface of the iron oxide having the urchin-like fractal structure prepared in this embodiment is 2.23.
  • the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • An iron oxide surface having a urchin-like fractal structure can be prepared on the surface of a common glass substrate by the method described in the literature (Journal of Northeast Normal University (Natural Science Edition), 2012, 44, 155-157).
  • step (b) a normal glass obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to 10 ⁇ g of a phosphate solution, and then drying the step (a) to obtain an iron oxide surface having a urchin-like fractal structure.
  • the substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a common glass substrate having an iron oxide surface having a urchin-like fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 5 g/cm 2 at room temperature for 30 minutes.
  • the anti-EpCAM antibody on the surface of the tumor cell and the urchin-like fractal structure specifically capturing the surface of the tumor cell fixed by the surface of the iron oxide having the urchin-like fractal structure on the ordinary glass substrate obtained in the step (2)
  • the anti-EpCAM antibody on the surface of the step (3) is mixed uniformly after the obtained cell suspension in the circulating tumor cells, the capture time is 45 minutes.
  • the surface of the flat iron oxide of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • the ordinary glass substrate of the iron oxide surface having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde, mass
  • PBS phosphate buffered saline
  • the Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing.
  • a common glass substrate for each surface of an iron oxide having a urchin-like fractal structure in which a circulating tumor cell was captured was photographed by a Nikon inverted fluorescence microscope, and an ordinary surface of an iron oxide having a urchin-like fractal structure in which a circulating tumor cell was captured was used.
  • the circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
  • the flat iron oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of MCF7 cells with iron oxide surface with urchin-like fractal structure is
  • the fractal dimension of the surface of the titanium dioxide having a dendritic fractal structure prepared in this example is 2.83.
  • the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a titanium oxide surface having a dendritic fractal structure can be prepared on the surface of a conventional glass substrate by the method described in the literature (ACS Nano, 2009, 3, 1212-1218).
  • step (b) a phosphate solution in which a streptavidin is diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then the step (a) is dried to obtain a titanium dioxide surface having a dendritic fractal structure.
  • the substrate is immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; a common glass substrate having a surface of titanium dioxide having a dendritic fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the amount of the anti-EpCAM antibody immobilized on the surface of the tumor cells was 0.1 g/cm 2 on a common glass substrate having a dendritic surface of 1 cm 2 of a dendritic structure at room temperature for 30 minutes.
  • 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the surface of the titanium oxide having a dendritic fractal structure on the ordinary glass substrate obtained in the step (2) placed on the cell culture plate (3.5 cm in diameter).
  • the anti-EpCAM antibody On the surface of the anti-EpCAM antibody on the surface of the immobilized tumor cells (three 1 cm 2 ordinary glass substrates with a dendritic fractal surface of titanium dioxide were placed in each cell culture plate), and then placed in a cell culture incubator, Synergistic effect of anti-EpCAM antibody on the surface of tumor cells and dendritic fractal structure, specifically capturing anti-EpCAM on the surface of tumor cells immobilized on the surface of titanium dioxide having a dendritic fractal structure added to the ordinary glass substrate obtained in step (2)
  • the step (3) on the surface of the antibody was mixed to obtain a circulating tumor cell in a cell suspension obtained uniformly, and the capture time was 45 minutes.
  • the flat titanium dioxide surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same capture of circulating tumor cells in the test sample.
  • a common glass substrate of a titanium oxide surface having a dendritic fractal structure in which circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • a common glass substrate of the surface of the titanium dioxide having a dendritic fractal structure each capturing a circulating tumor cell was photographed by a Nikon inverted fluorescence microscope, and a common glass base of the surface of the titanium dioxide having a dendritic fractal structure capturing the circulating tumor cells was respectively taken. The circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
  • the flat titanium dioxide surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of the MCF7 cells with the dendritic fractal structure is 69.3%, and the capture efficiency of Jurkat T cells is only 1.9%.
  • the capture efficiency of MCF7 cells on the surface of titanium dioxide is only 2.1%.
  • the capture efficiency of Jurkat T cells is only 0.6%.
  • the fractal dimension of the surface of the crystalline silicon having the broccoli fractal structure prepared in this example is 2.51.
  • the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a crystalline silicon surface having a broccoli-like fractal structure can be prepared on the surface of a crystalline silicon substrate by the method described in the literature (Macromol. Rapid Commun. 2005, 26, 1805-1809).
  • a crystal of a surface of a crystalline silicon having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a).
  • the silicon substrate is immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; a crystalline silicon substrate having a surface of crystalline silicon having a broccoli fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • the immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 0.8 g/cm 2 on a crystalline silicon substrate having a crystal silicon surface of 1 cm 2 having a cauliflower-like fractal structure at room temperature for 30 minutes.
  • the anti-EpCAM antibody on the surface of the step (3) is mixed uniformly after the obtained cell suspension in the circulating tumor cells, the capture time is 45 minutes.
  • the flat crystalline silicon surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to phase. The same method of capturing circulating tumor cells in the sample to be tested.
  • the crystalline silicon substrate of the crystalline silicon surface having the broccoli-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass
  • PBS phosphate buffered saline
  • the Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing.
  • the flat crystalline silicon surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of MCF7 cells with crystalline cauliflower-like fractal structure is 41.3%, and the capture efficiency of Daudi cells is only 1.5%.
  • the capture efficiency of MCF7 cells on the surface of gold in the control experiment is only 2.6%.
  • the capture efficiency of Daudi cells is only 0.7%.
  • the fractal dimension of the quartz surface having the broccoli fractal structure prepared in this example is 2.57; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a quartz surface having a broccoli-like fractal structure can be prepared on the surface of a quartz substrate by the method described in the literature (Macromol. Rapid Commun. 2005, 26, 1805-1809).
  • step (b) a quartz solution in which streptavidin is diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then the quartz surface having a broccoli-like fractal structure obtained by drying the step (a) is dried.
  • the sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; the quartz substrate having a quartz surface having a broccoli fractal structure was taken out and washed with a phosphate buffer;
  • PBS phosphate buffered saline
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the fixed amount of the EpCAM antibody was 1.0 g/cm 2 .
  • the flat quartz surface to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • the quartz substrate of the quartz surface having the broccoli fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • the quartz substrate with the broccoli-like fractal structure of each of the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the quartz substrate having the broccoli-like fractal structure of the circulating tumor cells was photographed on a quartz substrate. The captured circulating tumor cells were counted and the capture efficiency was calculated.
  • the flat quartz surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the polyvinyl alcohol having the dendritic fractal structure prepared in this example was 2.81.
  • the tumor cell MCF7 and Jurkat T were taken as the circulated tumor cells to be captured, and the capture system of the present invention was further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a polyvinyl alcohol surface having a dendritic fractal structure can be prepared on the surface of a polyvinyl alcohol substrate by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223).
  • step (1) (2) immobilizing an anti-EpCAM antibody on the surface of the tumor cell on the surface of the polyvinyl alcohol having a dendritic fractal structure on the polyvinyl alcohol substrate obtained in the step (1) (a) A biotinylated polyethylene glycol with an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and a polyvinyl alcohol substrate having a dendritic structure on the surface of the polyvinyl alcohol is immersed in a room temperature at room temperature.
  • biotinylated polyethylene glycol having an amino group at one end was immersed in an aqueous solution of 2 mM for 12 hours (the polyvinyl alcohol substrate having a dendritic structure on the surface of the polyvinyl alcohol was rinsed with deionized water before use). Clean); take out the polyvinyl alcohol substrate with the surface of the polyvinyl alcohol having a dendritic fractal structure, wash it with deionized water, and dry;
  • step (b) diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the surface of the polyvinyl alcohol having a dendritic fractal structure obtained by drying the step (a)
  • the polyvinyl alcohol substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the polyvinyl alcohol substrate having the surface of the polyvinyl alcohol having a dendritic fractal structure is taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 2.0 g/cm 2 on a polyvinyl alcohol substrate having a dendritic fractal structure of 1 cm 2 at room temperature for 30 minutes.
  • 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the polyglycolic structure on the polyvinyl alcohol substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter).
  • the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface of vinyl alcohol (three 1 cm 2 polyvinyl alcohol substrate with a dendritic fractal surface on each cell culture plate), and then placed In the cell culture incubator, the anti-EpCAM antibody on the surface of the tumor cell synergizes with the dendritic fractal structure to specifically capture the surface of the polyvinyl alcohol having a dendritic fractal structure added to the polyvinyl alcohol substrate obtained in the step (2).
  • the cells in the cell suspension obtained after the step (3) were uniformly mixed, and the capture time was 45 minutes.
  • the surface of the flat polyvinyl alcohol to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
  • the polyvinyl alcohol substrate on the surface of the polyvinyl alcohol having a dendritic fractal structure in which the circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde. Immerse for 10 minutes in a Triton-XlOO aqueous solution with a mass concentration of 0.4%, and soak for 2 minutes in a 2 ⁇ glmL DAPI aqueous solution to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • Each of the polyvinyl alcohol substrates having a dendritic fractal structure on which the circulating tumor cells were captured was photographed by a Nikon inverted fluorescence microscope, and a polyvinyl alcohol having a dendritic fractal structure in which circulating tumor cells were captured was taken.
  • the circulating tumor cells captured on the surface of the polyvinyl alcohol substrate were counted to calculate the capture efficiency.
  • the flat polyvinyl alcohol surface on which circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of MCF7 cells with a dendritic fractal structure on the surface of polyvinyl alcohol is 71.5%, and the capture efficiency of Jurkat T cells is only 1.5%.
  • the fractal dimension of the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure prepared in this example is 2.74; taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured as an example, the capture system of the present invention is further Explain and verify.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a styrene having a dendritic fractal structure can be prepared on the surface of a copolymer substrate of styrene and lactic acid by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223). Copolymer surface with lactic acid.
  • a biotinylated polyethylene glycol having an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and styrene and a surface of a copolymer having a dendritic fractal structure of styrene and lactic acid are allowed to be at room temperature.
  • a lactic acid copolymer substrate was immersed in an aqueous solution of a biotinylated polyethylene glycol having an amino group at one end and a concentration of 2 mM for 12 hours (benzene having a dendritic structure of a copolymer of styrene and lactic acid) a copolymer substrate of ethylene and lactic acid is rinsed with deionized water before use; a copolymer substrate of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure is taken out and washed with deionized water. Dry
  • step (b) a solution of streptavidin diluted to a phosphate solution of 10 g/mL with a phosphate buffered saline (PBS) solution, and then styrene and lactic acid having a dendritic fractal structure obtained by drying the step (a)
  • the copolymer substrate of styrene and lactic acid on the surface of the copolymer is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the styrene on the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure is taken out.
  • a copolymer substrate with lactic acid washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the copolymer of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the amount of anti-EpCAM antibody immobilized on the surface of the tumor cells was 1.5 g. /cm 2 .
  • the step (3) was obtained by uniformly mixing the circulating tumor cells in the cell suspension, and the capture time was 45 minutes.
  • the surface of the styrene and lactic acid copolymer of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
  • a copolymer substrate of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure in which circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then used at a mass concentration of 4%. Soaked in a solution of paraformaldehyde for 20 minutes, soaked in a 0.3% aqueous Triton-XlOO solution for 10 minutes, and soaked in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • Photographs of styrene and lactic acid copolymer substrates on the surface of each copolymer of styrene and lactic acid having a dendritic fractal structure, which captured the circulating tumor cells, were respectively photographed by Nikon inverted fluorescence microscope, and the cells with captured circulating tumor cells were captured.
  • the circulating tumor cells captured on the copolymer substrate of styrene and lactic acid on the surface of the copolymer of styrene and lactic acid of the dendritic fractal structure were counted, and the capture efficiency was calculated.
  • the surface of the copolymer of styrene and lactic acid which captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the fractal dimension of the surface of the polyurethane having a dendritic fractal structure prepared in this example is 2.74; and the tumor cells MCF7 and Jurkat T are taken as the example of the circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified.
  • a method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
  • a polyurethane surface having a dendritic fractal structure can be prepared on the surface of a polyurethane substrate by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223).
  • a biotinylated polyethylene glycol having an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and a polyurethane substrate having a dendritic fractal structure is immersed in the one end at room temperature at room temperature.
  • the amino group biotinylated polyethylene glycol was immersed in an aqueous solution of 2 mM for 12 hours (the polyurethane substrate having a dendritic fractal structure was rinsed with deionized water before use); the stripped fractal was taken out Structural polyurethane surface Polyurethane substrate, washed with deionized water, dried;
  • a phosphate-based solution of a polyurethane surface having a dendritic fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a).
  • PBS phosphate buffered saline
  • the sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; the polyurethane substrate having a polyurethane surface having a dendritic fractal structure was taken out and washed with a phosphate buffer;
  • step (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a polyurethane substrate having a dendritic fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the amount of anti-EpCAM antibody immobilized on the surface of the tumor cells was 2.0 g/cm 2 .
  • step (3) 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the polyurethane surface having a dendritic fractal structure on the polyurethane substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). Immobilized on the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 polyurethane substrates with a dendritic fractal surface were placed on each cell culture plate), and then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the dendritic fractal structure, specifically capturing the surface of the anti-EpCAM antibody surface of the tumor cell immobilized on the surface of the polyurethane having a dendritic fractal structure added to the polyurethane substrate obtained in the step (2)
  • the above step (3) circulates the tumor cells in the cell suspension obtained after mixing uniformly, and the capture time is 45 minutes.
  • the flat polyurethane surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same cycled tumor cell test in the sample to be tested.
  • the polyurethane substrate having the dendritic fractal-shaped polyurethane surface captured by circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing.
  • PBS phosphate buffered saline
  • Each of the polyurethane substrates having the dendritic fractal-shaped polyurethane surface captured by the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the polyurethane substrate having the dendritic fractal structure-captured polyurethane surface was captured. The captured circulating tumor cells were counted and the capture efficiency was calculated.
  • the flat polyurethane surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
  • the experimental results show that the capture efficiency of MCF7 cells with polyurethane surface with dendritic fractal structure is
  • the present invention utilizes the surface of the fractal structure for specific capture of circulating tumor cells to obtain higher capture efficiency and greater specificity.
  • Comparative Example 1 and Comparative Example 1 since the silicon nanowire array is not a fractal structure, its capturing efficiency is much lower than that of the device having a dendritic fractal structure used in the present invention (about 10-20% lower).

Abstract

The present invention provides a method and a device for specifically capturing a circulating tumor cell by utilizing a surface of a fractal structure, and an application thereof. According to the present invention, a surface of a substrate is formed as a surface with a fractal structure; before being put into a cell culture plate, a specific antibody on a circulating tumor cell surface is fixed on the surface with the fractal structure; before being put into a cell culture incubator, a sample containing the circulating tumor cell to be captured is dropwise added to the surface of the specific antibody on the circulating tumor cell surface. Due to the synergetic effect between the specific antibody on the circulating tumor cell surface and the fractal structure, the circulating tumor cell in the sample is specifically captured. The capture method based on the surface with the fractal structure according to the present invention can be used to capture cells associated with cancer.

Description

利用分形结构的表面进行循环肿瘤细胞的特异性捕获的方法和器件及其应用 技术领域  Method and device for utilizing surface of fractal structure for specific capture of circulating tumor cells and application thereof
本发明涉及生物技术领域， 具体地， 涉及利用分形结构的表面进行循环肿瘤细胞的 特异性捕获的方法以及用于特异性捕获循环肿瘤细胞的器件及其应用。 背景技术  The present invention relates to the field of biotechnology, and in particular to a method for performing specific capture of circulating tumor cells using the surface of a fractal structure and a device for specifically capturing circulating tumor cells and uses thereof. Background technique
癌症是导致人类死亡的第二大杀手。 其中 90%的死亡是由转移癌引起的。 转移癌主 要是由从初始癌变位置脱离的癌细胞引起的， 这部分癌细胞通过血管或者***扩散到 机体的其他组织或者器官， 从而形成转移癌。 癌细胞进入血液循环是实现癌症转移的最 初阶段， 为最终形成肿瘤的转移病灶提供了可能。 这部分进入人体外周血中的癌细胞就 是循环肿瘤细胞 （Circulating Tumor Cells )。 通过检测外周血中的循环肿瘤细胞可以有效 预测癌症转移的发生、 进行癌症的早期诊断、 预测癌症病人的生存几率以及监测治疗的 效果等。 因此， 检测血液中的循环肿瘤细胞具有重要的临床应用价值， 循环肿瘤细胞的 检测对于癌症的早期诊断、 病情监测、 治疗预后等具有重要意义。  Cancer is the second biggest killer of human death. 90% of deaths are caused by metastatic cancer. Metastatic cancer is mainly caused by cancer cells that are detached from the initial cancerous position. This part of the cancer cells spread to other tissues or organs of the body through blood vessels or lymphatic vessels, thereby forming metastatic cancer. The entry of cancer cells into the blood circulation is the initial stage of cancer metastasis, providing the possibility of eventually forming metastatic lesions of the tumor. The cancer cells that enter this part of human peripheral blood are Circulating Tumor Cells. By detecting circulating tumor cells in peripheral blood, it is possible to effectively predict the occurrence of cancer metastasis, conduct early diagnosis of cancer, predict the survival rate of cancer patients, and monitor the effects of treatment. Therefore, the detection of circulating tumor cells in the blood has important clinical application value. The detection of circulating tumor cells is of great significance for the early diagnosis, disease monitoring and treatment prognosis of cancer.
实现循环肿瘤细胞检测的前提是对血液中的循环肿瘤细胞进行捕获与分离。 由于其 在血液中的数量很少（每毫升几个到几十个）， 循环肿瘤细胞的捕获与分离仍是一个巨大 的挑战。 目前， 循环肿瘤细胞捕获与分离的方法主要是根据肿瘤细胞自身特殊的形貌与 生物化学特征进行的， 比如尺寸、 密度和肿瘤细胞表面特殊表达的蛋白质。 现有的捕获 与分离技术主要包括 ISET (Isolation by size of epithelial tumor cells)技术（Am J Pathol 2000, 156, 57-63)、密度梯度离心技术（Cytometry 2002， 49， 150-158)和免疫磁性分离技术 (N Engl J Med 2004， 351, 781-91)。 ISET技术利用肿瘤细胞与血细胞尺寸之间的差异进行 循环肿瘤细胞的分离， 但是该技术分离纯度低， 难以满足实际需求。 密度梯度离心技术 则是基于血液中各种细胞密度的差异， 红细胞、 中性白细胞等密度较大的细胞迁移至底 部， 肿瘤细胞则仍处于顶部， 从而达到分离的目的。 但是该技术存在的问题同样是纯度 较低， 而且在处理与转移的过程中会不可避免地损失循环肿瘤细胞， 进而影响捕获效率。 免疫磁性分离技术是目前研究和应用最广泛的技术之一， 其原理是将特异性抗体修饰在 磁性颗粒表面， 与循环肿瘤细胞表面抗原结合后在磁场作用下实现捕获与分离的目的， 该技术能够实现较高的捕获效率， 但是由于其过程复杂、 费用较高而限制了其应用。  The prerequisite for the detection of circulating tumor cells is the capture and separation of circulating tumor cells in the blood. Due to its small amount in the blood (several to dozens per milliliter), the capture and separation of circulating tumor cells remains a huge challenge. At present, the methods of capturing and separating circulating tumor cells are mainly based on the specific morphology and biochemical characteristics of tumor cells, such as size, density, and proteins specifically expressed on the surface of tumor cells. Existing capture and separation techniques mainly include ISET (Isolation by size of epithelial tumor cells) technology (Am J Pathol 2000, 156, 57-63), density gradient centrifugation (Cytometry 2002, 49, 150-158) and immunomagnetics. Separation technique (N Engl J Med 2004, 351, 781-91). The ISET technique utilizes the difference between tumor cells and blood cell size for the separation of circulating tumor cells, but the separation purity of this technique is low and it is difficult to meet actual needs. Density gradient centrifugation is based on the difference in cell density in the blood. Cells with higher density such as red blood cells and neutrophils migrate to the bottom, and tumor cells are still at the top, thus achieving separation. However, the problem with this technique is also that the purity is low, and the circulating tumor cells are inevitably lost during the process of treatment and transfer, thereby affecting the capture efficiency. Immunomagnetic separation technology is one of the most widely studied and applied technologies. The principle is to modify the specific antibody on the surface of magnetic particles and combine it with the surface antigen of circulating tumor cells to achieve the purpose of capturing and separating under the action of magnetic field. It can achieve higher capture efficiency, but its application is limited due to its complicated process and high cost.
在最新的生物学研究领域， 王树涛等（Angew. Chem. Int. Ed., 2009, 48， 8970 -8973)率 先将三维纳米结构基片应用于循环肿瘤细胞检测领域。 该技术通过增强细胞表面的纳米 组分 （例如， 微绒毛、 丝状伪足等） 与三维纳米结构之间的相互作用极大地提高了其捕 获效率。 随后， 导电聚合物（Adv. Mater. 2011, 23, 4788-4792)、 PDMS (Cancer 2012, 118, 1145-54)和 Ti02纳米纤维（Adv. Mater. 2012, 24, 2756-2760)等都用于三维纳米结构的构建 从而增强循环肿瘤细胞的捕获效率。 由于其捕获效率高、 灵敏度高， 这一技术越来越引 起科学家的关注。 但是该技术存在成本较高、 使用不便等缺点， 而且该技术背后的基本 设计原则尚不清楚。 因此， 迫切需求一种新的高效率、 高特异性的循环肿瘤细胞的捕获 技术。 发明内容 In the latest field of biological research, Wang Shutao et al. (Angew. Chem. Int. Ed., 2009, 48, 8970 -8973) pioneered the application of three-dimensional nanostructured substrates in the field of circulating tumor cell detection. This technique greatly enhances its capture efficiency by enhancing the interaction between nano-components on the cell surface (eg, microvilli, filopodia, etc.) and three-dimensional nanostructures. Subsequently, conductive polymers (Adv. Mater. 2011, 23, 4788-4792), PDMS (Cancer 2012, 118, 1145-54) and Ti0 2 nanofibers (Adv. Mater. 2012, 24, 2756-2760), etc. For the construction of 3D nanostructures Thereby enhancing the capture efficiency of circulating tumor cells. Due to its high capture efficiency and high sensitivity, this technology has attracted more and more attention from scientists. However, this technology has disadvantages such as high cost and inconvenient use, and the basic design principles behind the technology are still unclear. Therefore, there is an urgent need for a new high-efficiency, high-specific capture technique for circulating tumor cells. Summary of the invention
本发明的目的在于提供一种利用分形结构的表面进行循环肿瘤细胞的特异性捕获的 方法以及用于特异性捕获循环肿瘤细胞的器件及其应用。  It is an object of the present invention to provide a method for performing specific capture of circulating tumor cells using the surface of a fractal structure and a device for specifically capturing circulating tumor cells and uses thereof.
本发明的利用分形结构的表面进行循环肿瘤细胞的特异性捕获的方法， 其基本原理 是在基片的表面制备出所需的分形结构的表面， 将循环肿瘤细胞表面的特异性抗体固定 在该具有所需的分形结构的表面上， 将待捕获循环肿瘤细胞的样品滴加到该循环肿瘤细 胞表面的特异性抗体的表面上， 通过样品中的循环肿瘤细胞表面的特异性抗原与固定在 该具有所需的分形结构的表面上的循环肿瘤细胞表面的特异性抗体之间的特异性结合， 实现循环肿瘤细胞的特异性捕获， 通过样品中的肿瘤细胞表面的纳米组分 （例如： 微绒 毛、 丝状伪足等） 与所述基片表面的分形结构之间的相互作用， 提高循环肿瘤细胞的特 异性捕获效率， 从而达到高效、 高特异性循环肿瘤细胞捕获的目的。  The method for performing specific capture of circulating tumor cells by using the surface of the fractal structure of the present invention, the basic principle is to prepare a surface of a desired fractal structure on the surface of the substrate, and immobilizing specific antibodies on the surface of the circulating tumor cells On the surface having the desired fractal structure, a sample of the circulating tumor cells to be captured is dropped onto the surface of the specific antibody on the surface of the circulating tumor cells, and the specific antigen on the surface of the circulating tumor cells in the sample is fixed thereto. Specific binding between specific antibodies on the surface of circulating tumor cells on the surface of the desired fractal structure, achieving specific capture of circulating tumor cells, through the nanocomponents of the tumor cell surface in the sample (eg microvilli) The interaction between the filopodia and the fractal structure on the surface of the substrate enhances the specific capture efficiency of circulating tumor cells, thereby achieving the purpose of efficient and highly specific circulating tumor cell capture.
本发明的利用分形结构的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：  The method of the present invention for performing specific capture of circulating tumor cells using the surface of the fractal structure comprises the following steps:
( 1 ) 在基片的表面制备出具有所需的分形结构的表面；  (1) preparing a surface having a desired fractal structure on the surface of the substrate;
所述的分形结构选自枝状分形结构、 花椰菜状分形结构或海胆状分形结构； The fractal structure is selected from a branched fractal structure, a broccoli fractal structure or a urchin fractal structure;
(2) 将循环肿瘤细胞表面的特异性抗体固定在步骤 （1 ) 得到的基片上的具有所需 的分形结构的表面上， 然后放置在细胞培养板中； (2) immobilizing a specific antibody on the surface of the circulating tumor cell on the surface of the substrate obtained in the step (1) having a desired fractal structure, and then placing it in the cell culture plate;
(3 ) 将待捕获循环肿瘤细胞的样品滴加到步骤 （2) 得到的基片上的具有所需的分 形结构的表面所固定的循环肿瘤细胞表面的特异性抗体的表面上， 然后置于细胞培养箱 中， 由所述的循环肿瘤细胞表面的特异性抗体与所述的分形结构的协同作用， 特异性捕 获样品中的循环肿瘤细胞。  (3) dropping a sample of the circulating tumor cells to be captured onto the surface of the specific antibody on the surface of the circulating tumor cells immobilized on the surface of the substrate having the desired fractal structure obtained in the step (2), and then placing the cells on the surface In the incubator, the circulating tumor cells in the sample are specifically captured by the synergistic action of the specific antibody on the surface of the circulating tumor cells and the fractal structure.
在优选的实施方式中， 所述的基片的材料选自导电金属、 导电无机非金属、 导电无 机非金属化合物、 非导电无机非金属化合物和非导电聚合物中的一种。 所述的导电金属 优选选自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种。 所述的导电无机非金属优选是晶 体硅。 所述的导电无机非金属化合物优选是氧化铟锡（ITO)玻璃。 所述的非导电无机非 金属化合物优选是石英或普通玻璃。 所述的非导电聚合物可以为各种能够形成块状或者 薄膜的非导电聚合物， 优选选自以二甲基硅氧烷、 （甲基） 丙烯酸酯、 氨酯、 碳酸酯、 乙 烯、丙烯、苯乙烯、乳酸和乙烯醇中的至少一种作为单体而形成的均聚物和 /或共聚物（如 聚二甲基硅氧烷、 聚 （甲基） 丙烯酸酯、 聚氨酯、 聚碳酸酯、 聚乙烯、 聚丙烯、 聚苯乙 烯、 聚乳酸、 苯乙烯和乳酸的共聚物、 以及聚乙烯醇中的至少一种）。 在优选的实施方式中， 所述的分形结构的表面的材料选自金属、 金属化合物、 无机 非金属、 无机非金属化合物、 导电聚合物和非导电聚合物中的一种。 所述的金属优选选 自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种。 所述的金属化合物优选选自氧化铜、 氧 化铁、 氧化锌和二氧化钛中的一种。 所述的无机非金属优选是晶体硅。 所述的无机非金 属化合物优选是石英。 所述的导电聚合物可以为各种能够形成块状或者薄膜的导电聚合 物， 优选选自聚噻吩、 聚吡咯、 聚苯胺和聚苯中的一种。 所述的非导电聚合物可以各种 能够形成块状或者薄膜的非导电聚合物， 优选选自以二甲基硅氧烷、 （甲基） 丙烯酸酯、 氨酯、 碳酸酯、 乙烯、 丙烯、 苯乙烯、 乳酸和乙烯醇中的至少一种作为单体而形成的均 聚物和 /或共聚物 （如聚二甲基硅氧烷、 聚 （甲基） 丙烯酸酯、 聚氨酯、 聚碳酸酯、 聚乙 烯、聚丙烯、聚苯乙烯、聚乳酸、苯乙烯和乳酸的共聚物、 以及聚乙烯醇中的至少一种）。 In a preferred embodiment, the material of the substrate is selected from one of a conductive metal, a conductive inorganic non-metal, a conductive inorganic non-metal compound, a non-conductive inorganic non-metal compound, and a non-conductive polymer. The conductive metal is preferably selected from one of gold, silver, platinum, palladium, copper, iron, zinc, and aluminum. The conductive inorganic nonmetal is preferably crystalline silicon. The conductive inorganic non-metallic compound is preferably indium tin oxide (ITO) glass. The non-conductive inorganic non-metallic compound is preferably quartz or ordinary glass. The non-conductive polymer may be various non-conductive polymers capable of forming a block or a film, preferably selected from the group consisting of dimethyl siloxane, (meth) acrylate, urethane, carbonate, ethylene, propylene. a homopolymer and/or a copolymer formed of at least one of styrene, lactic acid, and vinyl alcohol as a monomer (eg, polydimethylsiloxane, poly(meth)acrylate, polyurethane, polycarbonate , polyethylene, polypropylene, polystyrene, polylactic acid, a copolymer of styrene and lactic acid, and at least one of polyvinyl alcohol). In a preferred embodiment, the material of the surface of the fractal structure is selected from one of a metal, a metal compound, an inorganic non-metal, an inorganic non-metal compound, a conductive polymer, and a non-conductive polymer. The metal is preferably selected from one of gold, silver, platinum, palladium, copper, iron, zinc, and aluminum. The metal compound is preferably selected from one of copper oxide, iron oxide, zinc oxide and titanium dioxide. The inorganic nonmetal is preferably crystalline silicon. The inorganic non-metallic compound is preferably quartz. The conductive polymer may be any of various conductive polymers capable of forming a block or a film, and is preferably one selected from the group consisting of polythiophene, polypyrrole, polyaniline, and polyphenylene. The non-conductive polymer may be various non-conductive polymers capable of forming a block or a film, preferably selected from the group consisting of dimethyl siloxane, (meth) acrylate, urethane, carbonate, ethylene, propylene, a homopolymer and/or a copolymer formed of at least one of styrene, lactic acid, and vinyl alcohol as a monomer (eg, polydimethylsiloxane, poly(meth)acrylate, polyurethane, polycarbonate, Polyethylene, polypropylene, polystyrene, polylactic acid, a copolymer of styrene and lactic acid, and at least one of polyvinyl alcohol).
其中， 基片的材料与分形结构的表面的材料可以相同或不同。  Wherein, the material of the substrate and the material of the surface of the fractal structure may be the same or different.
所述的在基片的表面制备出所需的分形结构的表面的方法有多种， 可以包括以下所 述的方法：  There are various methods for preparing the surface of the desired fractal structure on the surface of the substrate, and may include the methods described below:
在导电金属基片、 导电无机非金属基片或导电无机非金属化合物基片的表面制备出 所述的分形结构的表面为金属材料时， 较佳地是采用电化学沉积的方法进行制备， 即采 用传统的三电极体系， 以铂丝或铂片作为对电极， 以银 /氯化银电极作为参比电极， 以所 述的导电金属基片、 所述的导电无机非金属基片或所述的导电无机非金属化合物基片作 为工作电极， 以氯金酸水溶液、 硝酸银水溶液、 氯铂酸水溶液、 氯钯酸水溶液、 硝酸铜 水溶液、 氯化亚铁水溶液、 硫酸锌水溶液或硫酸铝水溶液等作为电解液 （如以金作为基 片可采用氯金酸水溶液作为电解液， 还可以选择其它所述的电解液）， 通过调整工作电压 (-0.1 V〜- 0.5 V) 以及电解液中相应酸根离子的浓度 （0.01 mol/L〜2 mol/L)， 室温下电 化学沉积 20分钟〜 180分钟， 即可在所述的导电金属基片、 所述的导电无机非金属基片 或所述的导电无机非金属化合物基片的表面可分别制备出枝状分形结构的表面、 花椰菜 状分形结构的表面或海胆状分形结构的表面。  When the surface of the fractal structure is prepared as a metal material on the surface of the conductive metal substrate, the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate, it is preferably prepared by electrochemical deposition, that is, Using a conventional three-electrode system, using a platinum wire or a platinum plate as a counter electrode, a silver/silver chloride electrode as a reference electrode, the conductive metal substrate, the conductive inorganic non-metal substrate or the The conductive inorganic non-metallic compound substrate is used as a working electrode, and is an aqueous solution of chloroauric acid, an aqueous solution of silver nitrate, an aqueous solution of chloroplatinic acid, an aqueous solution of chloropalladic acid, an aqueous solution of copper nitrate, an aqueous solution of ferrous chloride, an aqueous solution of zinc sulfate or an aqueous solution of aluminum sulfate. As the electrolyte (such as gold as the substrate can use chloroauric acid aqueous solution as the electrolyte, you can also choose other electrolytes), by adjusting the working voltage (-0.1 V ~ - 0.5 V) and the corresponding acid in the electrolyte The concentration of ions (0.01 mol / L ~ 2 mol / L), electrochemical deposition at room temperature for 20 minutes ~ 180 minutes, can be in the conductive metal base , Inorganic non-metallic surface of said conductive substrate or said conductive non-metal inorganic compound can be prepared substrate surface dendritic structure formed on the surface of the sub-surface sea urchin-like or fractal structure cauliflower fractal structure, respectively.
在导电金属基片、 导电无机非金属基片或导电无机非金属化合物基片的表面制备出 所述的分形结构的表面为导电聚合物材料时， 对于以导电聚合物材料为聚噻吩作为分形 结构的表面而言， 较佳地采用文献 （于伟利， 电沉积制备聚噻吩有序微结构及有机太阳 能电池. 吉林大学， 2009. ) 所述的方法， 即可在所述的导电金属基片、 所述的导电无机 非金属基片或所述的导电无机非金属化合物基片的表面制备出花椰菜状分形结构的表面 或海胆状分形结构的表面； 对于以导电聚合物材料为聚吡咯作为分形结构的表面而言， 较佳地采用文献（王杰， 电化学合成聚吡咯膜的电学性能与应用. 西安交通大学， 2008. ) 所述的方法， 即可在所述的导电金属基片、 所述的导电无机非金属基片或所述的导电无 机非金属化合物基片的表面制备出花椰菜状分形结构的表面或海胆状分形结构的表面； 对于以导电聚合物材料为聚苯胺作为分形结构的表面而言， 较佳地采用文献 （J. Phys. Chem. C 2010, 114, 8062-8067) 所述的方法， 即可在所述的导电金属基片、 所述的导电 无机非金属基片或所述的导电无机非金属化合物基片的表面制备出海胆状分形结构的表 面； 对于以导电聚合物材料为聚苯作为分形结构的表面而言， 较佳地采用文献 （科学通 报， 2003， 48， 35-37) 所述的方法， 即可在所述的导电金属基片、 所述的导电无机非金属 基片或所述的导电无机非金属化合物基片的表面制备出海胆状分形结构的表面。 When the surface of the fractal structure is prepared as a conductive polymer material on the surface of a conductive metal substrate, a conductive inorganic non-metal substrate or a conductive inorganic non-metal compound substrate, the conductive polymer material is polythiophene as a fractal structure. For the surface, the method described in the literature (Yu Weili, Electrodeposition of Polythiophene Ordered Microstructures and Organic Solar Cells. Jilin University, 2009.) is preferably used in the conductive metal substrate. The surface of the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate is prepared as a surface of a broccoli fractal structure or a surface of a urchin-shaped fractal structure; and the conductive polymer material is a polypyrrole as a fractal For the surface of the structure, the method described in the literature (Wang Jie, Electrical Properties and Application of Electrochemical Synthesis of Polypyrrole Films. Xi'an Jiaotong University, 2008.) is preferably used in the conductive metal substrate. a surface of the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate to prepare a surface of a broccoli fractal structure or The surface of the biliary fractal structure; for the surface having the conductive polymer material as the polyaniline as the fractal structure, the method described in the literature (J. Phys. Chem. C 2010, 114, 8062-8067) is preferably employed. The conductive metal substrate, the conductive The surface of the inorganic non-metal substrate or the conductive inorganic non-metal compound substrate is prepared as a surface of a urchin-like fractal structure; for a surface having a conductive polymer material as polystyrene as a fractal structure, the literature is preferably used ( Scientific method, 2003, 48, 35-37), which can be prepared on the surface of the conductive metal substrate, the conductive inorganic non-metal substrate or the conductive inorganic non-metal compound substrate. The surface of the urchin-like fractal structure.
在导电无机非金属基片、 导电无机非金属化合物基片或非导电无机非金属化合物基 片的表面制备出所述的分形结构的表面为金属化合物材料时， 对于以金属化合物为氧化 铜作为分形结构的表面而言，较佳地采用文献（陕西师范大学学报（自然科学版）， 2009, 37, 60-62) 所述的方法， 即可在所述的导电无机非金属基片、 所述的导电无机非金属化合物 基片或所述的非导电无机非金属化合物基片的表面制备出花椰菜状分形结构的表面或海 胆状分形结构的表面； 对于以金属化合物为氧化铁作为分形结构的表面而言， 较佳地采 用文献 （东北师大学报（自然科学版 )， 2012， 44， 155-157) 所述的方法， 即可在所述的导 电无机非金属基片、 所述的导电无机非金属化合物基片或所述的非导电无机非金属化合 物基片的表面制备出海胆状分形结构的表面； 对于以金属化合物为氧化锌作为分形结构 的表面而言， 较佳地采用文献（Adv. Funct. Mater., 2006， 16, 335-344)所述的方法， 即可 在所述的导电无机非金属基片、 所述的导电无机非金属化合物基片或所述的非导电无机 非金属化合物基片的表面制备出枝状分形结构的表面或海胆状分形结构的表面； 对于以 金属化合物为二氧化钛作为分形结构的表面而言， 较佳地采用文献 （ACS Nano, 2009， 3， 1212-1218 )所述的方法， 即可在所述的导电无机非金属基片、 所述的导电无机非金属化 合物基片或所述的非导电无机非金属化合物基片的表面制备出枝状分形结构的表面或海 胆状分形结构的表面。  When the surface of the fractal structure is prepared as a metal compound material on the surface of the conductive inorganic non-metal substrate, the conductive inorganic non-metal compound substrate or the non-conductive inorganic non-metal compound substrate, the metal compound is used as the copper oxide as a fractal In terms of the surface of the structure, the method described in the literature (Journal of Shaanxi Normal University (Natural Science Edition), 2009, 37, 60-62) is preferably employed, that is, in the conductive inorganic non-metal substrate, The surface of the conductive inorganic non-metallic compound substrate or the non-conductive inorganic non-metal compound substrate is prepared as a surface of a broccoli-like fractal structure or a surface of a urchin-like fractal structure; for a surface having a metal compound as an iron oxide as a fractal structure In other words, the method described in the literature (Journal of Northeast Normal University (Natural Science Edition), 2012, 44, 155-157) is preferably used in the conductive inorganic non-metal substrate, the conductive inorganic The surface of the non-metallic compound substrate or the non-conductive inorganic non-metal compound substrate is prepared as a surface of a urchin-like fractal structure; The metal compound is zinc oxide as the surface of the fractal structure, preferably by the method described in the literature (Adv. Funct. Mater., 2006, 16, 335-344), that is, the conductive inorganic non-metal group a surface of the conductive inorganic non-metal compound substrate or the surface of the non-conductive inorganic non-metal compound substrate to prepare a surface of a dendritic fractal structure or a surface of a urchin-like fractal structure; for a metal compound as titanium dioxide as a fractal For the surface of the structure, the method described in the literature (ACS Nano, 2009, 3, 1212-1218) is preferably employed, that is, the conductive inorganic non-metal substrate, the conductive inorganic non-metal compound group. The surface of the sheet or the non-conductive inorganic non-metallic compound substrate is prepared as a surface of a dendritic fractal structure or a surface of a urchin-like fractal structure.
在导电无机非金属基片的表面制备出所述的分形结构的表面为无机非金属材料时 (此时所述的基片是与所述的分形结构的表面的化学组成一致），或者在非导电无机非金 属化合物基片的表面制备出所述的分形结构的表面为无机非金属化合物材料时 （此时所 述的基片是与所述的分形结构的表面的化学组成一致）， 均较佳地采用文献 （Macromol. Rapid Commun. 2005， 26， 1805-1809)所述的方法， 即可在所述的导电无机非金属基片或 所述的非导电无机非金属化合物基片的表面制备出花椰菜状分形结构的表面。  When the surface of the fractal structure is prepared on the surface of the conductive inorganic non-metal substrate as an inorganic non-metal material (the substrate described above is consistent with the chemical composition of the surface of the fractal structure), or When the surface of the conductive inorganic non-metal compound substrate is prepared as an inorganic non-metal compound material (the substrate described above is consistent with the chemical composition of the surface of the fractal structure), Preferably, the method described in the literature (Macromol. Rapid Commun. 2005, 26, 1805-1809) can be prepared on the surface of the conductive inorganic non-metal substrate or the non-conductive inorganic non-metal compound substrate. The surface of the cauliflower-like fractal structure.
在非导电聚合物基片的表面制备出所述的分形结构的表面为非导电聚合物材料时 (此时所述的基片是与所述的分形结构的表面的化学组成一致）， 采用文献 （Macromol. Rapid Commun. 2005, 26, 1805-1809)所述的方法， 即可在所述的非导电聚合物基片的表 面制备出花椰菜状分形结构的表面；采用文献 (Angew. Chem. Int. Ed. 2002, 41, 1221-1223) 所述的方法， 即可在所述的非导电聚合物基片的表面制备出枝状分形结构的表面。  When the surface of the fractal structure is prepared on the surface of the non-conductive polymer substrate as a non-conductive polymer material (the substrate is in conformity with the chemical composition of the surface of the fractal structure), (Macromol. Rapid Commun. 2005, 26, 1805-1809), a surface of a broccoli-like fractal structure can be prepared on the surface of the non-conductive polymer substrate; using the literature (Angew. Chem. Int) Ed. 2002, 41, 1221-1223), the surface of the dendritic fractal structure can be prepared on the surface of the non-conductive polymer substrate.
所述的将循环肿瘤细胞表面的特异性抗体固定在步骤（1 )得到的基片上的具有所需 的分形结构的表面上的方法有多种， 可以采用在分形结构的表面上依次连接生物素化的 聚乙二醇 （一端接有功能基团）、 链霉亲和素和循环肿瘤细胞表面的特异性抗体的方法， 较佳的固定方法可按照如下步骤进行固定： (a)将一端接有功能基团的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室温下， 将表面为具有所需的分形结构的表面的基片浸泡在上述一端接有功能基团的生 物素化的聚乙二醇的浓度为 2 mM的水溶液中 （表面为具有所需的分形结构的表面的基 片在使用前用去离子水冲洗干净； 优选浸泡的时间为 12小时以上）； 取出表面为具有所 需的分形结构的表面的基片， 用去离子水洗涤， 干燥； The method of immobilizing the specific antibody on the surface of the circulating tumor cell on the surface of the substrate obtained in the step (1) having the desired fractal structure may be carried out by sequentially connecting the biotin on the surface of the fractal structure. A method for immobilizing polyethylene glycol (having a functional group at one end), streptavidin, and specific antibodies on the surface of circulating tumor cells, and a preferred method of immobilization can be carried out as follows: (a) diluting a biotinylated polyethylene glycol having a functional group to one end with a deionized water to a 2 mM aqueous solution, and immersing the substrate having a surface having a desired fractal structure at room temperature at room temperature A biotinylated polyethylene glycol having a functional group attached at one end to a 2 mM aqueous solution (the surface having the surface having the desired fractal structure is rinsed with deionized water before use; preferably soaked) The time is more than 12 hours); the substrate having the surface having the desired fractal structure is taken out, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)稀释为 10 g/mL的链霉亲和素的磷酸盐 溶液， 然后将步骤（a)干燥后得到的表面为具有所需的分形结构的表面的基片浸泡在上 述 10 g/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）； 取出表面为具有所需的分形结构的表面的基片， 用磷酸盐缓冲液洗涤；  (b) diluting streptavidin with phosphate buffered saline (PBS) to a phosphate solution of streptavidin at 10 g/mL, and then drying the surface obtained in step (a) to have the desired The substrate of the surface of the fractal structure is immersed in the above 10 g/mL streptavidin phosphate solution, and left at room temperature (preferably left at room temperature for about 30 minutes); the surface is taken out to have a desired fractal structure. Substrate, washed with phosphate buffer;
(c)将循环肿瘤细胞表面的特异性抗体（如抗 EpCAM抗体）用磷酸盐缓冲液（PBS) 稀释至 10 g/mL， 然后滴加到步骤 （b)用磷酸盐缓冲液（PBS)洗涤后得到的表面为具 有所需的分形结构的表面的基片的表面上（如取 25 L滴加到表面为具有所需的分形结构 的表面的基片的表面上 （面积为 1 cm2)), 室温放置 （优选室温下放置 30分钟）。 (c) Diluting specific antibodies (such as anti-EpCAM antibodies) on the surface of circulating tumor cells with phosphate buffered saline (PBS) to 10 g/mL, followed by dropwise addition to step (b) washing with phosphate buffered saline (PBS) The resulting surface is on the surface of the substrate having the surface of the desired fractal structure (e.g., 25 L is applied dropwise to the surface of the substrate having the surface having the desired fractal structure (area 1 cm 2 ) ), placed at room temperature (preferably at room temperature for 30 minutes).
所述的将循环肿瘤细胞表面的特异性抗体固定在步骤（1 )得到的基片上的具有所需 的分形结构的表面上， 其固定在具有所需的分形结构的表面上的循环肿瘤细胞表面的特 异性抗体的固定量优选不少于 0.1 g/cm2 (优选为 0.1-5 g/cm2)。 The specific antibody that circulates the surface of the tumor cell is immobilized on the surface of the substrate obtained in the step (1) having a desired fractal structure, which is fixed on the surface of the circulating tumor cell on the surface having the desired fractal structure. The immobilized amount of the specific antibody is preferably not less than 0.1 g/cm 2 (preferably from 0.1 to 5 g/cm 2 ).
所述的分形结构的表面的表征参数分维的范围优选是： 2<分维 <3。  The range of the characteristic parameter fractal dimension of the surface of the fractal structure is preferably: 2 < fractal dimension <3.
所述的循环肿瘤细胞表面的特异性抗体可以为抗各种肿瘤细胞特异性表达的抗原的 抗体， 优选为抗 EpCAM抗体。  The specific antibody on the surface of the circulating tumor cells may be an antibody against an antigen specifically expressed by various tumor cells, preferably an anti-EpCAM antibody.
所述的一端接有功能基团的生物素化的聚乙二醇、 链霉亲和素以及抗 EpCAM抗体 均为市售产品。  The biotinylated polyethylene glycol, streptavidin, and anti-EpCAM antibodies having functional groups at one end are commercially available products.
所述的一端接有功能基团的生物素化的聚乙二醇中的功能基团为巯基、 羧基和氨基 中的一种。  The functional group in the biotinylated polyethylene glycol to which the functional group is attached at one end is one of a thiol group, a carboxyl group and an amino group.
本发明中， 所述的待捕获循环肿瘤细胞的样品一般为外周血样品。  In the present invention, the sample of the circulating tumor cells to be captured is generally a peripheral blood sample.
本发明还提供了一种用于特异性捕获循环肿瘤细胞的器件， 该器件包括表面具有分 形结构的基片， 其中， 基片上的具有分形结构的表面上固定有循环肿瘤细胞表面的特异 性抗体， 所述的分形结构选自枝状分形结构、 花椰菜状分形结构或海胆状分形结构。 根 据本发明优选实施方式的器件与上述方法对应相同， 因而不再赘述。  The present invention also provides a device for specifically capturing circulating tumor cells, the device comprising a substrate having a fractal structure on a surface thereof, wherein a specific antibody having a surface of a circulating tumor cell is immobilized on a surface having a fractal structure on the substrate The fractal structure is selected from the group consisting of a branched fractal structure, a broccoli fractal structure or a urchin fractal structure. The device according to the preferred embodiment of the present invention is the same as the above method, and thus will not be described again.
此外， 本发明还提供了上述本发明的器件在癌症的早期诊断、 病情监测和治疗预后 中的应用。  Furthermore, the present invention provides the use of the device of the present invention described above for early diagnosis, disease monitoring and prognosis of cancer.
本发明建立了一种利用分形结构的表面进行循环肿瘤细胞的特异性捕获的方法， 此 方法 （1 )证明了分形结构的表面可以实现循环肿瘤细胞的高效捕获； （2)特异性强， 可 以捕获特异性肿瘤细胞， 减少非特异性肿瘤细胞的粘附。  The invention establishes a method for performing specific capture of circulating tumor cells by using the surface of the fractal structure, and the method (1) proves that the surface of the fractal structure can achieve efficient capture of circulating tumor cells; (2) the specificity is strong, Capture specific tumor cells and reduce adhesion of non-specific tumor cells.
本发明的利用分形结构的表面进行循环肿瘤细胞的特异性捕获的方法和器件可用于 癌症相关的细胞的捕获。所述的方法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， 可用于临床诊断。 附图说明 The method and device of the present invention for performing specific capture of circulating tumor cells using the surface of the fractal structure can be used for the capture of cancer-associated cells. The method can effectively improve the capturing efficiency of circulating tumor cells, and the cost is low. It is easy to operate and can be used for clinical diagnosis. DRAWINGS
图 la.本发明实施例 1制备的具有枝状分形结构的金表面的表面形貌的大面积扫描电 镜照片； 图 lb是具有枝状分形结构的金表面的单个颗粒的扫描电镜的放大照片。  Fig. la. Large-area scanning electron micrograph of the surface topography of a gold surface having a dendritic fractal structure prepared in Example 1 of the present invention; Fig. 1b is an enlarged photograph of a scanning electron microscope of a single particle having a gold-like surface of a dendritic fractal structure.
图 2.本发明实施例 1的循环肿瘤细胞表面的抗 EpCAM抗体固定在具有枝状分形结 构的金表面的基片上的过程示意图。  Fig. 2 is a schematic view showing the process of immobilizing an anti-EpCAM antibody on the surface of a circulating tumor cell of Example 1 of the present invention on a substrate having a gold-like surface of a dendritic fractal structure.
图 3.本发明实施例 1的捕获效率的柱状图。 具体实施方式  Figure 3. A histogram of the capture efficiency of Example 1 of the present invention. detailed description
下面结合实施例， 对本发明作进一步阐述。 应理解的是， 这些实施例仅用于说明本 发明而不是用于限制本发明的保护范围。  The present invention will be further described below in conjunction with the embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
以下实施例中， MCF7细胞 （ATCC® H B-22™)、 Jurkat T细胞 （ATCC® CRL-2899™) 和 Daudi细胞（ATCC®CCL-213™)均购自 ATCC;抗 EpCAM抗体购自 R&D，货号为 BAF960; 一端接有巯基、 羧基或氨基的生物素化的聚乙二醇购自 Nanocs有限公司， 货号分别为 PG2-BNTH-5k、 PG2-BNCA-5k和 PG2-AMBN-5k; 肿瘤细胞表面的抗 EpCAM抗体的固 定量的测定借助 ELISA试剂盒 （购自 Sangon Biotech, 货号为 ESK5295 ) 完成， 具体操 作参见试剂盒说明书； 对照实验中， 平整的表面利用真空蒸镀法制得； 捕获效率 （％) = 单位面积基片上所捕获的循环肿瘤细胞数 /单位面积总的循环肿瘤细胞数 X 100%。 实施例 1  In the following examples, MCF7 cells (ATCC® H B-22TM), Jurkat T cells (ATCC® CRL-2899TM), and Daudi cells (ATCC® CCL-213TM) were purchased from ATCC; anti-EpCAM antibodies were purchased from R&D. , the product number is BAF960; biotinylated polyethylene glycol with thiol, carboxyl or amino group at one end is purchased from Nanocs Co., Ltd., and the numbers are PG2-BNTH-5k, PG2-BNCA-5k and PG2-AMBN-5k; The fixed amount of anti-EpCAM antibody on the cell surface was determined by means of an ELISA kit (purchased from Sangon Biotech, article number ESK5295). For details, see the kit instructions. In the control experiment, the flat surface was prepared by vacuum evaporation; (%) = number of circulating tumor cells per unit area of the substrate/unit area Total circulating tumor cells X 100%. Example 1
本实施例所制备的具有枝状分形结构的金表面的分维为 2.73; 以肿瘤细胞 MCF7和 Jurkat T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 分形结构的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：  The fractal dimension of the gold surface having the dendritic fractal structure prepared in this example is 2.73; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用电化学沉积的方法， 在晶体硅基片的表面制备出具有枝状分形结构的金表 面  (1) A gold surface having a dendritic fractal structure is prepared on the surface of a crystalline silicon substrate by electrochemical deposition.
采用传统的三电极体系， 铂丝作为对电极， 银 /氯化银电极作为参比电极， 晶体硅基 片作为工作电极， 氯金酸水溶液（I mg/mL)作为电解液（氯离子浓度为 0.01 mO/L)， 在 工作电压为 -0.5V下电化学沉积 20分钟即在晶体硅基片的表面制备出具有枝状分形结构 的金表面。 表面形貌的大面积扫描电镜照片如图 la所示， 单个颗粒的扫描电镜的放大照 片如图 lb所示。 Using a traditional three-electrode system, platinum wire is used as the counter electrode, silver/silver chloride electrode is used as the reference electrode, crystalline silicon substrate is used as the working electrode, and aqueous solution of chloroauric acid (I mg/mL) is used as the electrolyte (the chloride ion concentration is 0.01 m O / L), a gold surface having a dendritic fractal structure was prepared on the surface of a crystalline silicon substrate by electrochemical deposition for 20 minutes at an operating voltage of -0.5V. A large-area scanning electron micrograph of the surface topography is shown in Figure la, and a magnified photograph of a single particle scanning electron microscope is shown in Figure lb.
(2) 固定过程如图 2所示， 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得 到的晶体硅基片上的具有枝状分形结构的金表面上  (2) Fixation process As shown in Fig. 2, the anti-EpCAM antibody on the surface of the tumor cell is immobilized on the gold surface having a dendritic fractal structure on the crystalline silicon substrate obtained in the step (1).
(a)将一端接有巯基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的金表面的晶体硅基片浸泡在上述一端接有巯基的生物素化 的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有枝状分形结构的金表面的晶体 硅基片在使用前用去离子水冲洗干净）； 取出具有枝状分形结构的金表面的晶体硅基片， 用去离子水洗涤， 干燥； (a) diluting a biotinylated polyethylene glycol having a sulfhydryl group at one end with a deionized water to a 2 mM aqueous solution, and immersing a crystalline silicon substrate having a gold-like surface having a dendritic fractal structure at one end at room temperature Thiol-based biotinylation The polyethylene glycol was immersed in an aqueous solution of 2 mM for 12 hours (the crystalline silicon substrate with the gold surface of the dendritic structure was rinsed with deionized water before use); the gold surface with the dendritic fractal structure was taken out. Crystalline silicon substrate, washed with deionized water, dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的金表面的晶体硅基片浸泡在链霉亲和素 的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝状分形结构的金表面的晶体硅基片， 用磷酸盐缓冲液洗涤；  (b) a crystal solution of a gold surface having a dendritic fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a). The substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a crystalline silicon substrate having a gold surface having a dendritic fractal structure is taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 枝状分形结构的金表面的晶体硅基片上， 室温放置 30 分钟， 测得肿瘤细胞表面的抗 EpCAM抗体的固定量为 0.1 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was 0.1 g/cm 2 on a crystalline silicon substrate having a gold surface of a dendritic fractal structure of 1 cm 2 at room temperature for 30 minutes.
(3 ) 准备待测样品  (3) Preparing the sample to be tested
取 MCF7细胞悬浮液 20 μL, 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， 用细胞培养基 DMEM稀释至 1 X 105个细胞 /mL， 混合均匀， 室温下保存。 Take 20 μL of MCF7 cell suspension, count with a cell counter and calculate the concentration. Take a certain amount of the above cell suspension, dilute to 1×10 5 cells/mL with cell culture medium DMEM, mix well, and store at room temperature.
取 Jurkat T细胞悬浮液 20 μL,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， 用细胞培养基 1640稀释至 I X 105个细胞 /mL， 混合均匀， 室温下保存。 Take 20 μL of Jurkat T cell suspension, count with a cell counter and calculate the concentration. Take a certain amount of the above cell suspension, dilute to IX 10 5 cells/mL with cell culture medium 1640, mix well, and store at room temperature.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的晶体硅基片上的具有枝状分形结构的金表面所固定的肿 瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有枝状分形 结构的金表面的晶体硅基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗 体与枝状分形结构的协同作用， 特异性捕获滴加到步骤（2)得到的晶体硅基片上的具有 枝状分形结构的金表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混 合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the gold surface having a dendritic fractal structure on the crystalline silicon substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). The surface of the anti-EpCAM antibody on the surface of the immobilized tumor cells (three 1 cm 2 crystal silicon substrates with a dendritic fractal gold surface were placed in each cell culture plate), and then placed in a cell culture incubator, Synergistic effect of anti-EpCAM antibody on the surface of tumor cells and dendritic fractal structure, specifically capturing anti-EpCAM on the surface of tumor cells immobilized on the gold surface with dendritic fractal structure added to the crystalline silicon substrate obtained in step (2) The step (3) on the surface of the antibody was mixed to obtain a circulating tumor cell in a cell suspension obtained uniformly, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整金表面也进行相同的 捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的金表面的晶体硅基片用磷酸盐缓冲液 (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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到 染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有枝状分形 结构的金表面的晶体硅基片拍照， 并对捕获了循环肿瘤细胞的具有枝状分形结构的金表 面的晶体硅基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The crystalline silicon substrate of the gold surface having the dendritic fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing. Each of the crystalline silicon substrates having a dendritic fractal-shaped gold surface captured by circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and a crystalline silicon substrate having a gold-like surface having a dendritic fractal structure captured by circulating tumor cells was photographed. The circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整金表面也按上述步骤进行， 并计算捕获效率。  The flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有枝状分形结构的金表面的 MCF7细胞的捕获效率为 63.3%， Jurkat T细胞的捕获效率仅为 1.2%; 对照实验中平整金表面的 MCF7细胞的捕获效率仅 为 2.8%， Jurkat T细胞的捕获效率仅为 0.3%， 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。 如图 3所示的捕获效率柱状图。 实施例 2 The experimental results show that the capture efficiency of the gold-surfaced MCF7 cells with a dendritic fractal structure is 63.3%. The capture efficiency of Jurkat T cells was only 1.2%; in the control experiment, the capture efficiency of MCF7 cells on the surface of gold was only 2.8%, and the capture efficiency of Jurkat T cells was only 0.3%. These data indicate that the method can achieve circulating tumor cells. Efficient and specific capture. A capture efficiency histogram as shown in Figure 3. Example 2
本实施例所制备的具有花椰菜状分形结构的金表面的分维为 2.54;以肿瘤细胞 MCF7 和 Daudi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 用分形结构的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：  The fractal dimension of the gold surface having the cauliflower-like fractal structure prepared in this example is 2.54; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用电化学沉积的方法， 在氧化铟锡 （ITO) 玻璃基片的表面制备出具有花椰 菜状分形结构的金表面  (1) A gold surface having a broccoli fractal structure was prepared on the surface of an indium tin oxide (ITO) glass substrate by electrochemical deposition.
采用传统的三电极体系， 铂片作为对电极， 银 /氯化银电极作为参比电极， 氧化铟锡 (ΙΤΟ) 玻璃基片作为工作电极， 氯金酸水溶液 （l mg/mL) 作为电解液 （氯离子浓度为 0.9 mo/L), 在工作电压为 -0.15 V下电化学沉积 90分钟即可在氧化铟锡（ITO)玻璃基片 的表面制备出具有花椰菜状分形结构的金表面。  Using a traditional three-electrode system, a platinum plate is used as a counter electrode, a silver/silver chloride electrode is used as a reference electrode, an indium tin oxide (yttrium) glass substrate is used as a working electrode, and an aqueous solution of chloroauric acid (1 mg/mL) is used as an electrolyte. (The chloride ion concentration was 0.9 mo/L), and a gold surface having a broccoli-like fractal structure was prepared on the surface of an indium tin oxide (ITO) glass substrate by electrochemical deposition for 90 minutes at an operating voltage of -0.15 V.
(2)将肿瘤细胞表面的抗 EpCAM抗体固定在步骤（1 )得到的氧化铟锡 （ITO)玻 璃基片上的具有花椰菜状分形结构的金表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the gold surface having the broccoli-like fractal structure on the indium tin oxide (ITO) glass substrate obtained in the step (1).
(a)将一端接有巯基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有花椰菜状分形结构的金表面的氧化铟锡（ITO)玻璃基片浸泡在上述一端接 有巯基的生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有花椰菜状分 形结构的金表面的氧化铟锡 （ITO) 玻璃基片在使用前用去离子水冲洗干净）； 取出具有 花椰菜状分形结构的金表面的氧化铟锡 （ΙΤΟ) 玻璃基片， 用去离子水洗涤， 干燥； (a) A biotinylated polyethylene glycol with a sulfhydryl group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and an indium tin oxide (ITO) glass substrate having a broccoli-like fractal gold surface at room temperature is used. Soaking in a 2 mM aqueous solution of biotinylated polyethylene glycol thiol-containing at one end is immersed for 12 hours (indium tin oxide (ITO) glass substrate with a broccoli-like fractal gold surface is used before use. Rinse with deionized water; remove the indium tin oxide (yttrium) glass substrate with a gold surface of broccoli fractal structure, wash with deionized water, and dry;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤 （a) 干燥后得到的具有花椰菜状分形结构的金表面的氧化铟锡 （ITO) 玻璃基 片浸泡在链霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有花椰菜状分形结构 的金表面的氧化铟锡 （ΙΤΟ) 玻璃基片， 用磷酸盐缓冲液洗涤； (b) a phosphate solution having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a) The tin (ITO) glass substrate was immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; an indium tin oxide (yttrium) glass substrate having a gold surface of a broccoli fractal structure was taken out, buffered with phosphate. Liquid washing;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 花椰菜状分形结构的金表面的氧化铟锡 （ITO) 玻璃基片上， 室温放置 30分钟， 测得肿 瘤细胞表面的抗 EpCAM抗体的固定量为 0.5 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was 0.5 g/cm 2 on an indium tin oxide (ITO) glass substrate having a cauliflower-like fractal gold structure of 1 cm 2 at room temperature for 30 minutes.
( 3 ) 准备待测样品  (3) Preparing the sample to be tested
取 MCF7细胞悬浮液 20 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， 用细胞培养基 DMEM稀释至 1 X 105个细胞 /mL， 混合均匀， 室温下保存。 Take MCF7 cell suspension 20 and count it with a cell counter and calculate the concentration. Take a certain amount of the above cell suspension, dilute to 1 × 10 5 cells/mL with cell culture medium DMEM, mix well, and store at room temperature.
取 Daudi细胞悬浮液 20 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， 用细胞培养基 1640稀释至 I X 105个细胞 /mL， 混合均匀， 室温下保存。 The Daudi cell suspension 20 was counted by a cell counter and the concentration thereof was calculated. A certain amount of the above cell suspension was taken, diluted with a cell culture medium 1640 to IX 10 5 cells/mL, uniformly mixed, and stored at room temperature.
(4) 捕获待测样品中的循环肿瘤细胞 分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有花椰菜状分形结构 的金表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有花椰菜状状分形结构的金表面的氧化铟锡 ατο) 玻璃基片）， 然后置于细胞 培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与花椰菜状分形结构的协同作用， 特异性 捕获滴加到步骤 （2) 得到的氧化铟锡 ατο) 玻璃基片上的具有花椰菜状分形结构的金 表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混合均匀后得到的细 胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 (4) Capture circulating tumor cells in the sample to be tested 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the indium tin oxide (ITO) glass substrate obtained in the step (2) placed on the cell culture plate (diameter: 3.5 cm) with cauliflower-like shape. The gold surface of the fractal structure is fixed on the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 of indium tin oxide ατο with a gold surface of broccoli-like fractal structure is placed on each cell culture plate)) And then placed in a cell culture incubator, the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the cauliflower-like fractal structure, specifically capturing the broccoli-like shape of the indium tin oxide ατο) obtained on the glass substrate obtained by the step (2) The gold surface of the fractal structure is fixed on the surface of the tumor cell surface of the anti-EpCAM antibody on the surface of the step (3) and the circulating tumor cells in the obtained cell suspension are uniformly mixed, and the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整金表面也进行相同的 捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有花椰菜状分形结构的金表面的氧化铟锡（ITO)玻璃基 片上用磷酸盐缓冲液（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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细 胞的具有花椰菜状分形结构的金表面的氧化铟锡 ατο)玻璃基片拍照， 并对捕获了循环 肿瘤细胞的具有花椰菜状分形结构的金表面的氧化铟锡 ατο)玻璃基片上所捕获的循环 肿瘤细胞进行计数， 计算捕获效率。  The indium tin oxide (ITO) glass substrate of the gold surface having the broccoli-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked with a 4% by mass aqueous solution of paraformaldehyde. After 20 minutes, the Triton-XlOO aqueous solution with a concentration of 0.4% was immersed for 10 minutes, and the 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographing each of the indium tin oxide ατο) glass substrates with broccoli-like fractal structures on the circulated tumor cells by Nikon inverted fluorescence microscope, and gold surface with broccoli fractal structure captured by circulating tumor cells Circulating tumor cells captured on the indium tin oxide ατο) glass substrate were counted and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整金表面也按上述步骤进行， 并计算捕获效率。  The flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有花椰菜状分形结构的金表面的 MCF7 细胞的捕获效率为 The experimental results show that the capture efficiency of the MCF7 cells with the broccoli fractal structure on the gold surface is
35.3%, Daudi细胞的捕获效率仅为 0.9%;对照实验中平整金表面的 MCF7细胞的捕获效 率仅为 2.1%， Daudi细胞的捕获效率仅为 0.2%， 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。 实施例 3 At 35.3%, the capture efficiency of Daudi cells was only 0.9%; in the control experiment, the capture efficiency of MCF7 cells on the surface of gold was only 2.1%, and the capture efficiency of Daudi cells was only 0.2%. These data indicate that the method can achieve circulating tumor cells. Efficient and specific capture. Example 3
本实施例所制备的具有海胆状分形结构的金表面的分维为 2.23; 以肿瘤细胞 MCF7 和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the gold surface having the urchin-like fractal structure prepared in this embodiment is 2.23; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用电化学沉积的方法， 在铁基片的表面制备出具有海胆状分形结构的金表面 采用传统的三电极体系， 铂片作为对电极， 银 /氯化银电极作为参比电极， 铁基片作 为工作电极， 氯金酸水溶液 （l mg/mL) 作为电解液 （氯离子浓度为 2 mo/L)， 在工作电 压为 -0.1 V下电化学沉积 180分钟即可在铁基片的表面制备出具有海胆状分形结构的金 表面的制备。  (1) Using a method of electrochemical deposition, a gold surface having a urchin-like fractal structure is prepared on the surface of an iron substrate using a conventional three-electrode system, a platinum plate as a counter electrode and a silver/silver chloride electrode as a reference electrode. Iron substrate as working electrode, chloroauric acid aqueous solution (l mg/mL) as electrolyte (chloride ion concentration 2 mo/L), electrochemical deposition for 180 minutes at working voltage of -0.1 V The surface is prepared to prepare a gold surface having a urchin-like fractal structure.
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的铁基片上的具有海 胆状分形结构的金表面上 (a)将一端接有巯基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有海胆状分形结构的金表面的铁基片浸泡在上述一端接有巯基的生物素化的 聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有海胆状分形结构的金表面的铁基 片在使用前用去离子水冲洗干净）； 取出具有海胆状分形结构的金表面的铁基片， 用去离 子水洗涤， 干燥； (2) immobilizing an anti-EpCAM antibody on the surface of the tumor cell on the gold surface having the urchin-like fractal structure on the iron substrate obtained in the step (1) (a) diluting a biotinylated polyethylene glycol having a sulfhydryl group at one end with a deionized water to a 2 mM aqueous solution, and immersing an iron substrate having a gold surface having a urchin-like fractal structure at one end at room temperature The thiol biotinylated polyethylene glycol is immersed in an aqueous solution of 2 mM for 12 hours (the iron substrate with the gold surface of the urchin-like fractal structure is rinsed with deionized water before use); the urchin-like fractal is taken out Structure of the gold surface of the iron substrate, washed with deionized water, dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有海胆状分形结构的金表面的铁基片浸泡在链霉亲和素的 磷酸盐溶液中， 室温下放置 30分钟； 取出具有海胆状分形结构的金表面的铁基片， 用磷 酸盐缓冲液洗涤；  (b) an iron base having a gold surface having a urchin-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a) The sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; an iron substrate having a gold surface having a urchin-like fractal structure was taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 海胆状分形结构的金表面的铁基片上，室温放置 30分钟，测得肿瘤细胞表面的抗 EpCAM 抗体的固定量为 2 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The iron substrate of a gold surface having a urchin-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 2 g/cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的铁基片上的具有海胆状分形结构的金表面所固定的肿瘤 细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有海胆状分形 结构的金表面的金基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与 海胆状分形结构的协同作用， 特异性捕获滴加到步骤（2)得到的铁基片上的具有海胆状 分形结构的金表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混合均 匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was respectively added to the gold surface having the urchin-like fractal structure on the iron substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). Immobilized tumor cell surface on the surface of anti-EpCAM antibody (3 1 cm 2 gold substrate with gold surface of urchin-like fractal structure per cell culture plate), then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the urchin-like fractal structure, specifically capturing the surface of the anti-EpCAM antibody surface of the tumor cell surface fixed by the gold surface having the urchin-like fractal structure added to the iron substrate obtained in the step (2) On the above step (3), the circulating tumor cells in the cell suspension obtained after mixing were uniformly mixed, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整金表面也进行相同的 捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat gold surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same experiment of capturing circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有海胆状分形结构的金表面的铁基片用磷酸盐缓冲液 (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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到 染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有海胆状分 形结构的金表面的铁基片拍照， 并对捕获了循环肿瘤细胞的具有海胆状分形结构的金表 面的铁基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The iron substrate of the gold surface having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. The iron substrate of each gold surface having a urchin-like fractal structure captured by circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the iron substrate of the gold surface having the urchin-like fractal structure of the circulating tumor cells was photographed. The captured circulating tumor cells were counted and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整金表面也按上述步骤进行， 并计算捕获效率。  The flat gold surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有枝状分形结构的金表面的 MCF7细胞的捕获效率为 30.9%， Jurkat T细胞的捕获效率仅为 0.7%; 对照实验中平整金表面的 MCF7细胞的捕获效率仅 为 2.5%， Jurkat T细胞的捕获效率仅为 0.2%， 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。 实施例 4 The experimental results show that the capture efficiency of MCF7 cells with gold-like fractal structure is 30.9%, and the capture efficiency of Jurkat T cells is only 0.7%. The capture efficiency of MCF7 cells on the surface of gold in the control experiment is only At 2.5%, the capture efficiency of Jurkat T cells is only 0.2%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 4
本实施例所制备的具有花椰菜状分形结构的聚吡咯表面的分维为 2.51 ; 以肿瘤细胞 The fractal dimension of the polypyrrole surface having the broccoli fractal structure prepared in this example is 2.51;
MCF7和 Daudi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。 利用分形结构的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： MCF7 and Daudi are examples of circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用文献（王杰，电化学合成聚吡咯膜的电学性能与应用. 西安交通大学，2008.) 所述的方法即可在氧化铟锡 ατο)玻璃基片的表面制备出具有花椰菜状分形结构的聚吡 咯表面。  (1) The method described in the literature (Wang Jie, Electrical Properties and Application of Electrochemical Synthetic Polypyrrole Films. Xi'an Jiaotong University, 2008.) can be used to prepare broccoli on the surface of indium tin oxide (ατο) glass substrate. The polypyrrole surface of the fractal structure.
(2)将肿瘤细胞表面的抗 EpCAM抗体固定在步骤（1 )得到的氧化铟锡 （ITO)玻 璃基片上的具有花椰菜状分形结构的聚吡咯表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the polypyrrole having a cauliflower-like fractal structure on the indium tin oxide (ITO) glass substrate obtained in the step (1).
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有花椰菜状分形结构的聚吡咯表面的氧化铟锡（ITO)玻璃基片浸泡在上述一 端接有羧基的生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有花椰菜 状分形结构的聚吡咯表面的氧化铟锡 （ιτο) 玻璃基片在使用前用去离子水冲洗干净）； 取出具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 ατο)玻璃基片，用去离子水洗涤， 干燥；  (a) A biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to a 2 mM aqueous solution, and an indium tin oxide (ITO) glass base having a cauliflower-like fractal structure on the surface of the polypyrrole at room temperature is used. The sheet was immersed in an aqueous solution of biotinylated polyethylene glycol having a carboxyl group at one end and dissolved in a 2 mM aqueous solution for 12 hours (indium tin oxide (ιτο) glass substrate having a cauliflower-like fractal structure on the polypyrrole surface was used. Rinse with deionized water before); remove the indium tin oxide ατο) glass substrate with polycalyx surface with broccoli fractal structure, wash with deionized water, and dry;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤 （a) 干燥后得到的具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 （ITO) 玻 璃基片浸泡在链霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有花椰菜状分形 结构的聚吡咯表面的氧化铟锡 ατο) 玻璃基片， 用磷酸盐缓冲液洗涤；  (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then oxidizing the surface of the polypyrrole having a cauliflower-like fractal structure obtained by drying the step (a) The indium tin (ITO) glass substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the indium tin oxide ατο) glass substrate having a cauliflower-like fractal structure is taken out, and the phosphate is used. Buffer washing;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 花椰菜状分形结构的聚吡咯表面的氧化铟锡 （ITO) 玻璃基片上， 室温放置 30分钟， 测 得肿瘤细胞表面的抗 EpCAM抗体的固定量为 5 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 5 g/cm 2 on an indium tin oxide (ITO) glass substrate having a cauliflower-like fractal structure of 1 cm 2 and allowed to stand at room temperature for 30 minutes.
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有花椰菜状分形结构 的聚吡咯表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养皿放置 3 个 1 cm2的具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 （ITO) 玻璃基片）， 然后置 于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与花椰菜状分形结构的协同作用， 特异性捕获滴加到步骤 （2) 得到的氧化铟锡 ατο) 玻璃基片上的具有花椰菜状分形结 构的聚吡咯表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混合均匀 后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the indium tin oxide (ITO) glass substrate obtained in the step (2) placed on the cell culture plate (diameter: 3.5 cm) with cauliflower-like shape. the upper surface of the polypyrrole surface fractal structure of the fixed surface of tumor cells anti-EpCAM antibody (each cell culture dish placed indium tin oxide having a polypyrrole surface cauliflower fractal structure of three 1 cm 2 of (ITO) glass substrate And then placed in a cell culture incubator, the synergistic effect of the anti-EpCAM antibody on the surface of the tumor cells and the cauliflower-like fractal structure, specifically capturing the indium tin oxide ατο) obtained on the glass substrate obtained by the step (2) Cauliflower-like fractal knot The polypyrrole surface is fixed on the surface of the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface of the cell (3) and the circulating tumor cells in the obtained cell suspension are uniformly obtained, and the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚吡咯表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat polypyrrole surface to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 ατο)玻 璃基片用磷酸盐缓冲液 （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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸 泡 15分钟， 从而达到染色的目的。用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤 细胞的具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 ατο)玻璃基片拍照， 并对捕获 了循环肿瘤细胞的具有花椰菜状分形结构的聚吡咯表面的氧化铟锡 ατο)玻璃基片上所 捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The indium tin oxide ατο) glass substrate of the polypyrrole surface having the broccoli-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked with a 4% by mass aqueous solution of paraformaldehyde. After 20 minutes, the Triton-XlOO aqueous solution with a concentration of 0.4% was immersed for 10 minutes, and the 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Photographs of each indium tin oxide ατο) glass substrate with a cauliflower-like fractal structure with broccoli-like fractal structure captured by Nikon inverted fluorescence microscope, and a polygalactose-like fractal structure captured by circulating tumor cells The circulating tumor cells captured on the glass substrate of the indium tin oxide on the surface of the pyrrole were counted, and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整聚吡咯表面也按上述步骤进行， 并计算捕获效率。  The flat polypyrrole surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有花椰菜状分形结构的聚吡咯表面的 MCF7细胞的捕获效率为 38.8%, Daudi细胞的捕获效率仅为 0.7%;对照实验中平整聚吡咯表面的 MCF7细胞的捕 获效率仅为 2.1%， Daudi细胞的捕获效率仅为 0.2%， 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。 实施例 5  The experimental results showed that the capture efficiency of MCF7 cells with polycalyx surface with cauliflower-like fractal structure was 38.8%, and the capture efficiency of Daudi cells was only 0.7%. The capture efficiency of MCF7 cells with flat polypyrrole surface was only 2.1 in the control experiment. %, Daudi cells have a capture efficiency of only 0.2%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 5
本实施例所制备的具有海胆状分形结构的聚苯表面的分维为 2.41 ;以肿瘤细胞 MCF7 和 Daudi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 用分形结构的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：  The fractal dimension of the surface of polyphenylene having a urchin-like fractal structure prepared in this example was 2.41. The capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 （科学通报， 2003， 48， 35-37) 所述的方法即可在氧化铟锡 （ITO) 玻 璃基片的表面制备出具有海胆状分形结构的聚苯表面。  (1) A polyphenylene surface having a urchin-like fractal structure can be prepared on the surface of an indium tin oxide (ITO) glass substrate by the method described in the literature (Science Bulletin, 2003, 48, 35-37).
(2)将肿瘤细胞表面的抗 EpCAM抗体固定在步骤（1 )得到的氧化铟锡 （ITO)玻 璃基片上的具有海胆状分形结构的聚苯表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the polyphenylene having a urchin-like fractal structure on the indium tin oxide (ITO) glass substrate obtained in the step (1).
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有海胆状分形结构的聚苯表面的氧化铟锡（ITO)玻璃基片浸泡在上述一端接 有羧基的生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有海胆状分形 结构的聚苯表面的氧化铟锡 ατο) 玻璃基片在使用前用去离子水冲洗干净）； 取出具有 海胆状分形结构的聚苯表面的氧化铟锡 ατο) 玻璃基片， 用去离子水洗涤， 干燥； (a) A biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to an aqueous solution of 2 mM, and an indium tin oxide (ITO) glass base having a surface of a polystyrene having a urchin-like fractal structure is obtained at room temperature. The sheet was immersed in an aqueous solution of biotinylated polyethylene glycol having a carboxyl group at one end and dissolved in a 2 mM aqueous solution for 12 hours (indium tin oxide with a surface of polystyrene having a urchin-like fractal structure). The glass substrate was used before use. Rinse with deionized water); remove the indium tin oxide ατο) glass substrate with a polystyrene surface having a urchin-like fractal structure, wash with deionized water, and dry;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤 （a) 干燥后得到的具有海胆状分形结构的聚苯表面的氧化铟锡 （ITO) 玻璃基 片浸泡在链霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有海胆状分形结构的 聚苯表面的氧化铟锡 ατο) 玻璃基片， 用磷酸盐缓冲液洗涤； (c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 海胆状分形结构的聚苯表面的氧化铟锡 （ITO) 玻璃基片上， 室温放置 30分钟， 测得肿 瘤细胞表面的抗 EpCAM抗体的固定量为 0.5 g/cm2。 (b) oxidizing a streptavidin diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the polyphenylene surface having a urchin-like fractal structure obtained by drying the step (a) Indium tin (ITO) glass substrate is immersed in streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; indium tin oxide ατο) glass substrate with urchin-like fractal structure is removed, using phosphate Buffer washing; (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The indium tin oxide (ITO) glass substrate having a surface of polystyrene having a urchin-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 0.5 g/cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有海胆状分形结构的 聚苯表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养皿放置 3个 1 cm2的具有海胆状分形结构的聚苯表面的氧化铟锡 （ITO) 玻璃基片）， 然后置于细胞培 养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与海胆状分形结构的协同作用， 特异性捕获 滴加到步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有海胆状分形结构的聚苯表面 所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混合均匀后得到的细胞悬 浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the indium tin oxide (ITO) glass substrate obtained in the step (2) placed on the cell culture plate (diameter: 3.5 cm) with a urchin shape. The surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface of the polyphenylene of the fractal structure (three ingots of indium tin oxide (ITO) glass base having a cholerular fractal structure of 3 cm 2 per cell culture dish The tablet is then placed in a cell culture incubator, and the anti-EpCAM antibody on the surface of the tumor cell synergizes with the urchin-like fractal structure to specifically capture the indium tin oxide (ITO) glass substrate obtained by the step (2). The step (3) on the surface of the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface of the polyphenylene having a urchin-like fractal structure is uniformly mixed to obtain a circulating tumor cell in the cell suspension, and the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚苯表面也进行相同 的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat polyphenyl surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same capture of circulating tumor cells in the test sample.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有海胆状分形结构的聚苯表面的氧化铟锡（ITO)玻璃基 片上用磷酸盐缓冲液（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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细 胞的具有海胆状分形结构的聚苯表面的氧化铟锡（ITO)玻璃基片拍照， 并对捕获了循环 肿瘤细胞的具有海胆状分形结构的聚苯表面的氧化铟锡 ατο)玻璃基片上所捕获的循环 肿瘤细胞进行计数， 计算捕获效率。  The indium tin oxide (ITO) glass substrate on the polyphenylene surface having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then a 4% by mass aqueous solution of polyformaldehyde was used. Soak for 20 minutes, soak in a Triton-XlOO aqueous solution with a concentration of 0.4% for 10 minutes, and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing. Photographs of indium tin oxide (ITO) glass substrates on the surface of polyphenylene having urchin-like fractal structures each capturing circulating tumor cells were photographed by Nikon inverted fluorescence microscope, and urchin-like fractal structures were captured for circulating tumor cells. The circulating tumor cells captured on the polyphenylene surface of the indium tin oxide ατο) glass substrate were counted, and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整聚苯表面也按上述步骤进行， 并计算捕获效率。  The smooth polystyrene surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有海胆状分形结构的聚苯表面的 MCF7 细胞的捕获效率为 37.8%, Daudi细胞的捕获效率仅为 0.5%;对照实验中平整聚苯表面的 MCF7细胞的捕获 效率仅为 2.3%， Daudi细胞的捕获效率仅为 0.3%， 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。 实施例 6  The experimental results show that the capture efficiency of MCF7 cells with urchin-like fractal structure is 37.8%, and the capture efficiency of Daudi cells is only 0.5%. The capture efficiency of MCF7 cells on the surface of polyphenylene in control experiments is only 2.3. The capture efficiency of %, Daudi cells is only 0.3%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 6
本实施例所制备的具有海胆状分形结构的聚苯胺表面的分维为 2.41 ; 以肿瘤细胞 MCF7和 Daudi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。 利用分形结构的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 )采用文献（J. Phys. Chem. C 2010， 114, 8062-8067)所述的方法即可在氧化铟锡The fractal dimension of the surface of the polyaniline having the urchin-like fractal structure prepared in this example was 2.41. The capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps: (1) Indium tin oxide can be used in the method described in the literature (J. Phys. Chem. C 2010, 114, 8062-8067)
( ιτο ) 玻璃基片的表面制备出具有海胆状分形结构的聚苯胺表面。 ( ιτο ) The surface of the glass substrate was prepared with a surface of a polyaniline having a urchin-like fractal structure.
(2)将肿瘤细胞表面的抗 EpCAM抗体固定在步骤（1 )得到的氧化铟锡 （ITO)玻 璃基片上的具有海胆状分形结构的聚苯胺表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the polyaniline having a urchin-like fractal structure on the indium tin oxide (ITO) glass substrate obtained in the step (1)
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有海胆状分形结构的聚苯胺表面的氧化铟锡 ατο)玻璃基片浸泡在上述一端 接有羧基的生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有海胆状分 形结构的聚苯胺表面的氧化铟锡 ατο) 玻璃基片在使用前用去离子水冲洗干净）； 取出 具有海胆状分形结构的聚苯胺表面的氧化铟锡 ατο)玻璃基片，用去离子水洗涤，干燥;  (a) A biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water into an aqueous solution of 2 mM, and an indium tin oxide ατο) glass substrate having a surface of a polyaniline having a urchin-like fractal structure at room temperature Soaking in a 2 mM aqueous solution of biotinylated polyethylene glycol with carboxyl group at one end is immersed for 12 hours (indium tin oxide with a urchin-like fractal structure of indium tin oxide ατο). The glass substrate is used before use. Rinsing with deionized water); taking out an indium tin oxide ατο) glass substrate having a surface of polyaniline having a urchin-like fractal structure, washing with deionized water, and drying;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤 （a) 干燥后得到的具有海胆状分形结构的聚苯胺表面的氧化铟锡 ατο) 玻璃 基片浸泡在链霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有海胆状分形结构 的聚苯胺表面的氧化铟锡 ατο) 玻璃基片， 用磷酸盐缓冲液洗涤；  (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then oxidizing the surface of the polyaniline having a urchin-like fractal structure obtained by drying step (a) Indium tin ατο) The glass substrate was immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the indium tin oxide ατο) glass substrate having the surface of the polyaniline having a urchin-like fractal structure was taken out, buffered with phosphate Liquid washing;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 海胆状分形结构的聚苯胺表面的氧化铟锡 ατο) 玻璃基片上， 室温放置 30分钟， 测得 肿瘤细胞表面的抗 EpCAM抗体的固定量为 0.1 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). On a glass substrate of 1 cm 2 of a polyaniline surface having a urchin-like fractal structure, it was allowed to stand at room temperature for 30 minutes, and the amount of anti-EpCAM antibody immobilized on the surface of the tumor cells was 0.1 g/cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有海胆状分形结构的 聚苯胺表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上 （每个细胞培养皿放置 3 个 1 cm2的具有海胆状分形结构的聚苯胺表面的氧化铟锡 （ITO) 玻璃基片）， 然后置于 细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与海胆状分形结构的协同作用， 特异 性捕获滴加到步骤 （2) 得到的氧化铟锡 （ITO) 玻璃基片上的具有海胆状分形结构的聚 苯胺表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤（3 )混合均匀后得到 的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the indium tin oxide (ITO) glass substrate obtained in the step (2) placed on the cell culture plate (diameter: 3.5 cm) with a urchin shape. The surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface of the polyaniline of the fractal structure (three ingots of 1 cm 2 of indium tin oxide (ITO) glass based on the surface of the polyaniline having a urchin-like fractal structure were placed in each cell culture dish. The tablet is then placed in a cell culture incubator, and the anti-EpCAM antibody on the surface of the tumor cell synergizes with the urchin-like fractal structure to specifically capture the indium tin oxide (ITO) glass substrate obtained by the step (2). The surface of the anti-EpCAM antibody on the surface of the tumor cell surface immobilized on the surface of the polyaniline having the urchin-like fractal structure (3) is uniformly mixed to obtain circulating tumor cells in the cell suspension, and the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚苯胺表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat polyaniline surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有海胆状分形结构的聚苯胺表面的氧化铟锡（ITO)玻璃 基片上用磷酸盐缓冲液 （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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸 泡 15分钟， 从而达到染色的目的。用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤 细胞的具有海胆状分形结构的聚苯胺表面的氧化铟锡 ατο)玻璃基片拍照， 并对捕获了 循环肿瘤细胞的具有海胆状分形结构的聚苯胺表面的氧化铟锡 ατο)玻璃基片上所捕获 的循环肿瘤细胞进行计数， 计算捕获效率。 The indium tin oxide (ITO) glass substrate on the surface of the polyaniline having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then a 4% by mass aqueous solution of polyformaldehyde was used. Soak for 20 minutes, soak in a Triton-XlOO aqueous solution with a concentration of 0.4% for 10 minutes, and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing. Cyclic tumors were captured for each with a Nikon inverted fluorescence microscope Photographing the indium tin oxide ατο) glass substrate of the polyaniline surface of the cell with a urchin-like fractal structure, and capturing it on a glass substrate of an indium tin oxide ατο) surface of a polyaniline having a urchin-like fractal structure that captures circulating tumor cells The circulating tumor cells were counted and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整聚苯胺表面也按上述步骤进行， 并计算捕获效率。  The flat polyaniline surface that captured the circulating tumor cells was also subjected to the above procedure, and the capture efficiency was calculated.
实验结果表明， 该具有海胆状分形结构的聚苯胺表面的 MCF7 细胞的捕获效率为 The experimental results show that the capture efficiency of MCF7 cells with polyamine surface with urchin-like fractal structure is
40.1%, Daudi细胞的捕获效率仅为 0.9%;对照实验中平整聚苯胺表面的 MCF7细胞的捕 获效率仅为 1.8%， Daudi细胞的捕获效率仅为 1.2%， 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。 实施例 Ί At 40.1%, the capture efficiency of Daudi cells was only 0.9%; in the control experiment, the capture efficiency of MCF7 cells on the surface of polyaniline was only 1.8%, and the capture efficiency of Daudi cells was only 1.2%. These data indicate that the method can achieve circulating tumors. Efficient and specific capture of cells. Example Ί
本实施例所制备的具有花椰菜状分形结构的聚噻吩表面的分维为 2.51 ; 以肿瘤细胞 MCF7和 Daudi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。 利用分形结构的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：  The fractal dimension of the surface of the polythiophene having the broccoli fractal structure prepared in this example was 2.51. The capture system of the present invention was further elaborated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 （于伟利， 电沉积制备聚噻吩有序微结构及有机太阳能电池. 吉林大 学， 2009. ) 所述的方法即可在金基片的表面制备出具有花椰菜状分形结构的聚噻吩表面。  (1) Using the method described in the literature (Yu Weili, Electrodeposition of Polythiophene Ordered Microstructures and Organic Solar Cells. Jilin University, 2009.), a broccoli-like fractal structure can be prepared on the surface of a gold substrate. Polythiophene surface.
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的金基片上的具有花 椰菜状分形结构的聚噻吩表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the polythiophene having a broccoli-like fractal structure on the gold substrate obtained in the step (1).
( a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有花椰菜状分形结构的聚噻吩表面的金基片浸泡在上述一端接有羧基的生物 素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有花椰菜状分形结构的聚噻 吩表面的金基片在使用前用去离子水冲洗干净）；取出具有花椰菜状分形结构的聚噻吩表 面的金基片， 用去离子水洗涤， 干燥；  (a) diluting a biotinylated polyethylene glycol having a carboxyl group at one end with a deionized water to a 2 mM aqueous solution, and immersing a gold substrate having a broccoli-like fractal structure on the surface of the polythiophene at one end at room temperature Soaking in a 2 mM aqueous solution of a carboxylated biotinylated polyethylene glycol for 12 hours (a gold substrate with a cauliflower-like fractal structure on the surface of the polythiophene was rinsed with deionized water before use); a gold substrate on the surface of a polythiophene having a fractal structure, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS )溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有花椰菜状分形结构的聚噻吩表面的金基片浸泡在链霉亲 和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有花椰菜状分形结构的聚噻吩表面的 金基片， 用磷酸盐缓冲液洗涤；  (b) a gold phosphate solution having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a). The substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the gold substrate having the surface of the polythiophene having a cauliflower-like fractal structure is taken out and washed with a phosphate buffer;
( c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS ) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS )洗涤后得到的面积为 1 cm2的具有 花椰菜状分形结构的聚噻吩表面的金基片上， 室温放置 30分钟， 测得肿瘤细胞表面的抗 EpCAM抗体的固定量为 0.8 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The gold substrate of the polythiophene surface having a cauliflower-like fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 0.8 g/cm 2 .
( 3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的金基片上的具有花椰菜状分形结构的聚噻吩表面所固定 的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养皿放置 3个 1 cm2的具有花椰 菜状分形结构的聚噻吩表面的金基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与花椰菜状状分形结构的协同作用， 特异性捕获滴加到步骤 （2) 得到的金 基片上的具有花椰菜状分形结构的聚噻吩表面所固定的肿瘤细胞表面的抗 EpCAM抗体 的表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45 分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the surface of the polythiophene having a cauliflower-like fractal structure on the gold substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). Fixed On the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 gold substrate on the surface of the polythiophene with cauliflower-like fractal structure was placed in each cell culture dish), and then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the cauliflower-like fractal structure, specifically capturing the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface of the polythiophene having the cauliflower-like fractal structure added to the gold substrate obtained in the step (2) On the surface of the step (3), after mixing evenly, the circulating tumor cells in the cell suspension were obtained, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚噻吩表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat polythiophene surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有花椰菜状分形结构的聚噻吩表面的金基片上用磷酸盐 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从 而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有花 椰菜状分形结构的聚噻吩表面的金基片拍照， 并对捕获了循环肿瘤细胞的具有花椰菜状 分形结构的聚噻吩表面的金基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The gold substrate on the surface of the polythiophene having the cauliflower-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing. The gold substrate of the polythiophene surface having the cauliflower-like fractal structure of each of the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the gold base of the polythiophene surface having the cauliflower-like fractal structure of the circulating tumor cells was captured. The circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整聚噻吩表面也按上述步骤进行， 并计算捕获效率。  The flat polythiophene surface that captured the circulating tumor cells was also subjected to the above procedure, and the capture efficiency was calculated.
实验结果表明， 该具有花椰菜状分形结构的聚噻吩表面的 MCF7细胞的捕获效率为 39.8%, Daudi细胞的捕获效率仅为 0.9%;对照实验中平整聚噻吩表面的 MCF7细胞的捕 获效率仅为 2.3%， Daudi细胞的捕获效率仅为 0.8%， 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。 实施例 8  The experimental results showed that the capture efficiency of MCF7 cells on the surface of polythiophene with cauliflower-like fractal structure was 39.8%, and the capture efficiency of Daudi cells was only 0.9%. The capture efficiency of MCF7 cells on the surface of polythiophene was only 2.3 in the control experiment. %, Daudi cells have a capture efficiency of only 0.8%, and these data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 8
本实施例所制备的具有枝状分形结构的氧化锌表面的分维为 2.81 ;以肿瘤细胞 MCF7 和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the zinc oxide having the dendritic fractal structure prepared in this example is 2.81; and the tumor cells MCF7 and Jurkat T are taken as the circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 （Adv. Funct. Mater., 2006， 16, 335-344) 所述的方法即可在普通玻璃 基片的表面制备出具有枝状分形结构的氧化锌表面。  (1) A zinc oxide surface having a dendritic fractal structure can be prepared on the surface of a conventional glass substrate by the method described in the literature (Adv. Funct. Mater., 2006, 16, 335-344).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的普通玻璃基片上的 具有枝状分形结构的氧化锌表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the zinc oxide having a dendritic fractal structure on the ordinary glass substrate obtained in the step (1)
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的氧化锌表面的普通玻璃基片浸泡在上述一端接有羧基的生 物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有枝状分形结构的氧化锌 表面的普通玻璃基片在使用前用去离子水冲洗干净）；取出具有枝状分形结构的氧化锌表 面的普通玻璃基片， 用去离子水洗涤， 干燥；  (a) diluting a biotinylated polyethylene glycol having a carboxyl group to one end with a deionized water to a 2 mM aqueous solution, and immersing a common glass substrate having a dendritic fractal structure on the zinc oxide surface at the above end at room temperature Soaking in a 2 mM aqueous solution of a carboxylated biotinylated polyethylene glycol for 12 hours (a common glass substrate having a zinc oxide surface with a dendritic structure is rinsed with deionized water before use); a common glass substrate having a zinc oxide surface having a dendritic fractal structure, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的氧化锌表面的普通玻璃基片浸泡在链霉 亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝状分形结构的氧化锌表面的普 通玻璃基片， 用磷酸盐缓冲液洗涤； (b) Diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, Then, the ordinary glass substrate having the zinc oxide surface of the dendritic fractal structure obtained after the step (a) is dried is immersed in the streptavidin phosphate solution, and left at room temperature for 30 minutes; and the dendritic fractal structure is taken out. a common glass substrate on the surface of zinc oxide, washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 枝状分形结构的氧化锌表面的普通玻璃基片上， 室温放置 30分钟， 测得肿瘤细胞表面的 抗 EpCAM抗体的固定量为 2.8 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). On a common glass substrate of a 1 cm 2 zinc oxide surface having a dendritic fractal structure, the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 2.8 g/cm 2 at room temperature for 30 minutes.
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的普通玻璃基片上的具有枝状分形结构的氧化锌表面所固 定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有枝 状分形结构的氧化锌表面的普通玻璃基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的 抗 EpCAM抗体与枝状分形结构的协同作用， 特异性捕获滴加到步骤 （2)得到的普通玻 璃基片上的具有枝状分形结构的氧化锌表面所固定的肿瘤细胞表面的抗 EpCAM抗体的 表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分 钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the zinc oxide having a dendritic fractal structure on the ordinary glass substrate obtained in the step (2) placed on the cell culture plate (3.5 cm in diameter). On the surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface (three 1 cm 2 ordinary glass substrates with a zinc oxide surface with a dendritic fractal structure were placed in each cell culture plate), and then placed in a cell culture incubator. , by the synergistic action of the anti-EpCAM antibody on the surface of the tumor cell and the dendritic fractal structure, specifically capturing the surface of the tumor cell fixed by the surface of the zinc oxide having a dendritic fractal structure added to the ordinary glass substrate obtained in the step (2) The anti-EpCAM antibody on the surface of the step (3) is mixed uniformly after the obtained cell suspension in the circulating tumor cells, the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整氧化锌表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat zinc oxide surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的氧化锌表面的普通玻璃基片用磷酸盐 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从 而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有枝 状分形结构的氧化锌表面的普通玻璃基片拍照， 并对捕获了循环肿瘤细胞的具有枝状分 形结构的氧化锌表面的普通玻璃基片上所捕获的循环肿瘤细胞进行计数，计算捕获效率。  The ordinary glass substrate of the zinc oxide surface having the dendritic fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass The Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. A common glass substrate of a zinc oxide surface having a dendritic fractal structure each capturing a circulating tumor cell was photographed by a Nikon inverted fluorescence microscope, and a common zinc oxide surface having a dendritic fractal structure was captured. The circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整氧化锌表面也按上述步骤进行， 并计算捕获效率。  The flat zinc oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有枝状分形结构的氧化锌表面的 MCF7 细胞的捕获效率为 65.3%, Jurkat T细胞的捕获效率仅为 1.1%; 对照实验中平整氧化锌表面的 MCF7细胞的 捕获效率仅为 2.4%， Jurkat T细胞的捕获效率仅为 1.3%， 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。 实施例 9  The experimental results show that the capture efficiency of MCF7 cells with zincated surface of the dendritic fractal structure is 65.3%, and the capture efficiency of Jurkat T cells is only 1.1%. The capture efficiency of MCF7 cells on the surface of zinc oxide in the control experiment is only At 2.4%, the capture efficiency of Jurkat T cells was only 1.3%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 9
本实施例所制备的具有海胆状分形结构的氧化铜表面的分维为 2.21 ; 以肿瘤细胞 MCF7和 JurkatT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。 利用分形结构的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： The fractal dimension of the surface of the copper oxide having the urchin-like fractal structure prepared in this embodiment is 2.21; MCF7 and JurkatT are examples of circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
(1) 采用文献 （陕西师范大学学报（自然科学版 )，2009，37，60-62) 所述的方法即可 在普通玻璃基片的表面制备出具有海胆状分形结构的氧化铜表面。  (1) A copper oxide surface having a urchin-like fractal structure can be prepared on the surface of a common glass substrate by the method described in Journal of Shaanxi Normal University (Natural Science Edition, 2009, 37, 60-62).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1) 得到的普通玻璃基片上的 具有海胆状分形结构的氧化铜表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the copper oxide having a urchin-like fractal structure on the ordinary glass substrate obtained in the step (1)
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有海胆状分形结构的氧化铜表面的普通玻璃基片浸泡在上述一端接有羧基的 生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有海胆状分形结构的氧 化铜表面的普通玻璃基片在使用前用去离子水冲洗干净）；取出具有海胆状分形结构的氧 化铜表面的普通玻璃基片， 用去离子水洗涤， 干燥；  (a) A biotinylated polyethylene glycol having a carboxyl group attached to one end thereof is diluted with deionized water to a 2 mM aqueous solution, and a common glass substrate having a surface of a copper oxide having a urchin-like fractal structure is immersed at the above end at room temperature. Soaking in a 2 mM aqueous solution of a carboxylated biotinylated polyethylene glycol for 12 hours (a common glass substrate having a copper oxide surface having a urchin-like fractal structure is rinsed with deionized water before use); a common glass substrate having a copper oxide surface having a urchin-like fractal structure, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有海胆状分形结构的氧化铜表面的普通玻璃基片浸泡在链 霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有海胆状分形结构的氧化铜表面 的普通玻璃基片， 用磷酸盐缓冲液洗涤；  (b) diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the surface of the copper oxide having a urchin-like fractal structure obtained by drying the step (a) The glass substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a common glass substrate having a copper oxide surface having a urchin-like fractal structure is taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 海胆状分形结构的氧化铜表面的普通玻璃基片上， 室温放置 30分钟， 测得肿瘤细胞表面 的抗 EpCAM抗体的固定量为 4.1 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). On a common glass substrate of a 1 cm 2 copper oxide surface having a urchin-like fractal structure, the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 4.1 g/cm 2 at room temperature for 30 minutes.
(3) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的普通玻璃基片上的具有海胆状分形结构的氧化铜表面所 固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有 海胆状分形结构的氧化铜表面的普通玻璃基片）， 然后置于细胞培养箱中， 由肿瘤细胞表 面的抗 EpCAM抗体与海胆状分形结构的协同作用， 特异性捕获滴加到步骤 （2)得到的 普通玻璃基片上的具有海胆状分形结构的氧化铜表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤 （3) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕 获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the copper oxide having a urchin-like fractal structure on the ordinary glass substrate obtained in the step (2) of the cell culture plate (3.5 cm in diameter). On the surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface (three 1 cm 2 ordinary glass substrates with a copper oxide surface with a urchin-like fractal structure were placed in each cell culture plate), and then placed in a cell culture incubator. Synergistic action of the anti-EpCAM antibody on the surface of the tumor cell and the urchin-like fractal structure, specifically capturing the surface of the tumor cell fixed by the surface of the copper oxide having the urchin-like fractal structure added to the ordinary glass substrate obtained in the step (2) Steps on the surface of the anti-EpCAM antibody (3) After mixing evenly, the circulating tumor cells in the cell suspension were obtained, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整氧化铜表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the surface of the flat copper oxide of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
(5) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有海胆状分形结构的氧化铜表面的普通玻璃基片用磷酸 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-XlOO水溶液浸泡 10分钟， 2 ^glmL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有 海胆状分形结构的氧化铜表面的普通玻璃基片拍照， 并对捕获了循环肿瘤细胞的具有海 胆状分形结构的氧化铜表面的普通玻璃基片上所捕获的循环肿瘤细胞进行计数， 计算捕 获效率。 A common glass substrate on which a copper oxide surface having a urchin-like fractal structure of circulating tumor cells was captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde. The aqueous solution of Triton-XlOO with a concentration of 0.4% was immersed for 10 minutes, and 2 ^ glmL of DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. A common glass substrate of a copper oxide surface having a urchin-like fractal structure in which a circulating tumor cell was captured was photographed by a Nikon inverted fluorescence microscope, and a common copper oxide surface having a urchin-like fractal structure in which a circulating tumor cell was captured was used. The circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整氧化铜表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有海胆状分形结构的氧化铜表面的 MCF7 细胞的捕获效率为 35.3%, Jurkat T细胞的捕获效率仅为 1.4%; 对照实验中平整氧化铜表面的 MCF7细胞的 捕获效率仅为 2.4%， Jurkat T细胞的捕获效率仅为 1.3%，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。 实施例 10  The flat copper oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of MCF7 cells on the surface of copper oxide with urchin-like fractal structure is 35.3%, and the capture efficiency of Jurkat T cells is only 1.4%. The capture efficiency of MCF7 cells on the surface of copper oxide in the control experiment is only At 2.4%, the capture efficiency of Jurkat T cells was only 1.3%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 10
本实施例所制备的具有海胆状分形结构的氧化铁表面的分维为 2.23 ; 以肿瘤细胞 MCF7和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the iron oxide having the urchin-like fractal structure prepared in this embodiment is 2.23. The capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 （东北师大学报（自然科学版 )，2012， 44， 155-157) 所述的方法即可在 普通玻璃基片的表面制备出具有海胆状分形结构的氧化铁表面。  (1) An iron oxide surface having a urchin-like fractal structure can be prepared on the surface of a common glass substrate by the method described in the literature (Journal of Northeast Normal University (Natural Science Edition), 2012, 44, 155-157).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的普通玻璃基片上的 具有海胆状分形结构的氧化铁表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the iron oxide having a urchin-like fractal structure on the ordinary glass substrate obtained in the step (1)
( a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有海胆状分形结构的氧化铁表面的普通玻璃基片浸泡在上述一端接有羧基的 生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有海胆状分形结构的氧 化铁表面的普通玻璃基片在使用前用去离子水冲洗干净）；取出具有海胆状分形结构的氧 化铁表面的普通玻璃基片， 用去离子水洗涤， 干燥；  (a) diluting a biotinylated polyethylene glycol having a carboxyl group at one end with a deionized water to a 2 mM aqueous solution, and immersing a common glass substrate having an iron oxide surface having a urchin-like fractal structure at the one end at room temperature Soaking in a 2 mM aqueous solution of a carboxylated biotinylated polyethylene glycol for 12 hours (a common glass substrate having an iron oxide surface having a urchin-like fractal structure is rinsed with deionized water before use); a common glass substrate having an iron oxide surface having a urchin-like fractal structure, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS )溶液稀释为 10 ^glmL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有海胆状分形结构的氧化铁表面的普通玻璃基片浸泡在链 霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有海胆状分形结构的氧化铁表面 的普通玻璃基片， 用磷酸盐缓冲液洗涤；  (b) a normal glass obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to 10 μg of a phosphate solution, and then drying the step (a) to obtain an iron oxide surface having a urchin-like fractal structure. The substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; a common glass substrate having an iron oxide surface having a urchin-like fractal structure is taken out and washed with a phosphate buffer;
( c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS ) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS )洗涤后得到的面积为 1 cm2的具有 海胆状分形结构的氧化铁表面的普通玻璃基片上， 室温放置 30分钟， 测得肿瘤细胞表面 的抗 EpCAM抗体的固定量为 5 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). On a common glass substrate of 1 cm 2 of an iron oxide surface having a urchin-like fractal structure, the immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was measured to be 5 g/cm 2 at room temperature for 30 minutes.
( 3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞 分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的普通玻璃基片上的具有海胆状分形结构的氧化铁表面所 固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有 海胆状分形结构的氧化铁表面的普通玻璃基片）， 然后置于细胞培养箱中， 由肿瘤细胞表 面的抗 EpCAM抗体与海胆状分形结构的协同作用， 特异性捕获滴加到步骤 （2)得到的 普通玻璃基片上的具有海胆状分形结构的氧化铁表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕 获时间是 45分钟。 (4) Capture circulating tumor cells in the sample to be tested 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the iron oxide having a urchin-like fractal structure on the ordinary glass substrate obtained in the step (2) of the cell culture plate (3.5 cm in diameter). The surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface (three 1 cm 2 ordinary glass substrates with an iron oxide surface having a urchin-like fractal structure were placed in each cell culture plate), and then placed in a cell culture incubator. , by the synergistic action of the anti-EpCAM antibody on the surface of the tumor cell and the urchin-like fractal structure, specifically capturing the surface of the tumor cell fixed by the surface of the iron oxide having the urchin-like fractal structure on the ordinary glass substrate obtained in the step (2) The anti-EpCAM antibody on the surface of the step (3) is mixed uniformly after the obtained cell suspension in the circulating tumor cells, the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整氧化铁表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the surface of the flat iron oxide of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有海胆状分形结构的氧化铁表面的普通玻璃基片用磷酸 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有 海胆状分形结构的氧化铁表面的普通玻璃基片拍照， 并对捕获了循环肿瘤细胞的具有海 胆状分形结构的氧化铁表面的普通玻璃基片上所捕获的循环肿瘤细胞进行计数， 计算捕 获效率。  The ordinary glass substrate of the iron oxide surface having the urchin-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde, mass The Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. A common glass substrate for each surface of an iron oxide having a urchin-like fractal structure in which a circulating tumor cell was captured was photographed by a Nikon inverted fluorescence microscope, and an ordinary surface of an iron oxide having a urchin-like fractal structure in which a circulating tumor cell was captured was used. The circulating tumor cells captured on the glass substrate were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整氧化铁表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有海胆状分形结构的氧化铁表面的 MCF7 细胞的捕获效率为 The flat iron oxide surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of MCF7 cells with iron oxide surface with urchin-like fractal structure is
29.3%, Jurkat T细胞的捕获效率仅为 1.5%; 对照实验中平整氧化铁表面的 MCF7细胞的 捕获效率仅为 2.1%， Jurkat T细胞的捕获效率仅为 0.3%， 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。 实施例 11 29.3%, the capture efficiency of Jurkat T cells was only 1.5%; in the control experiment, the capture efficiency of MCF7 cells on the surface of iron oxide was only 2.1%, and the capture efficiency of Jurkat T cells was only 0.3%. These data indicate that the method can be achieved. Efficient and specific capture of circulating tumor cells. Example 11
本实施例所制备的具有枝状分形结构的二氧化钛表面的分维为 2.83 ; 以肿瘤细胞 MCF7和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the titanium dioxide having a dendritic fractal structure prepared in this example is 2.83. The capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用文献 （ACS Nano，2009，3， 1212-1218)所述的方法即可在普通玻璃基片的 表面制备出具有枝状分形结构的二氧化钛表面。  (1) A titanium oxide surface having a dendritic fractal structure can be prepared on the surface of a conventional glass substrate by the method described in the literature (ACS Nano, 2009, 3, 1212-1218).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的普通玻璃基片上的 具有枝状分形结构的二氧化钛表面的基片上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the substrate of the titanium oxide surface having a dendritic fractal structure on the ordinary glass substrate obtained in the step (1).
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的二氧化钛表面的普通玻璃基片浸泡在上述一端接有羧基的 生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有枝状分形结构的二氧 化钛表面的普通玻璃基片在使用前用去离子水冲洗干净）；取出具有枝状分形结构的二氧 化钛表面的普通玻璃基片， 用去离子水洗涤， 干燥； (a) diluting a biotinylated polyethylene glycol having a carboxyl group to one end with a deionized water to a 2 mM aqueous solution, and immersing a common glass substrate having a dendritic fractal structure on the surface of the titanium oxide at one end at room temperature Soaking in a 2 mM aqueous solution with carboxylated biotinylated polyethylene glycol for 12 hours (dioxygen with dendritic fractal structure) A common glass substrate on the surface of titanium is rinsed with deionized water before use; a common glass substrate having a surface of titanium dioxide having a dendritic fractal structure is taken out, washed with deionized water, and dried;
(b )将链霉亲和素用磷酸盐缓冲液（PBS )溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的二氧化钛表面的普通玻璃基片浸泡在链 霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝状分形结构的二氧化钛表面 的普通玻璃基片， 用磷酸盐缓冲液洗涤；  (b) a phosphate solution in which a streptavidin is diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then the step (a) is dried to obtain a titanium dioxide surface having a dendritic fractal structure. The substrate is immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; a common glass substrate having a surface of titanium dioxide having a dendritic fractal structure is taken out and washed with a phosphate buffer;
( c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS ) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b )用磷酸盐缓冲液（PBS )洗涤后得到的面积为 1 cm2的具有 枝状分形结构的二氧化钛表面的普通玻璃基片上， 室温放置 30分钟， 测得肿瘤细胞表面 的抗 EpCAM抗体的固定量为 0.1 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The amount of the anti-EpCAM antibody immobilized on the surface of the tumor cells was 0.1 g/cm 2 on a common glass substrate having a dendritic surface of 1 cm 2 of a dendritic structure at room temperature for 30 minutes.
( 3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
( 4 ) 捕获待测样品中的循环肿瘤细胞  (4) capturing circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2 )得到的普通玻璃基片上的具有枝状分形结构的二氧化钛表面所 固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有 枝状分形结构的二氧化钛表面的普通玻璃基片）， 然后置于细胞培养箱中， 由肿瘤细胞表 面的抗 EpCAM抗体与枝状分形结构的协同作用， 特异性捕获滴加到步骤 （2 )得到的普 通玻璃基片上的具有枝状分形结构的二氧化钛表面所固定的肿瘤细胞表面的抗 EpCAM 抗体的表面上的步骤（3 )混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the surface of the titanium oxide having a dendritic fractal structure on the ordinary glass substrate obtained in the step (2) placed on the cell culture plate (3.5 cm in diameter). On the surface of the anti-EpCAM antibody on the surface of the immobilized tumor cells (three 1 cm 2 ordinary glass substrates with a dendritic fractal surface of titanium dioxide were placed in each cell culture plate), and then placed in a cell culture incubator, Synergistic effect of anti-EpCAM antibody on the surface of tumor cells and dendritic fractal structure, specifically capturing anti-EpCAM on the surface of tumor cells immobilized on the surface of titanium dioxide having a dendritic fractal structure added to the ordinary glass substrate obtained in step (2) The step (3) on the surface of the antibody was mixed to obtain a circulating tumor cell in a cell suspension obtained uniformly, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整二氧化钛表面也进行 相同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat titanium dioxide surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same capture of circulating tumor cells in the test sample.
( 5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的二氧化钛表面的普通玻璃基片用磷酸 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有 枝状分形结构的二氧化钛表面的普通玻璃基片拍照， 并对捕获了循环肿瘤细胞的具有枝 状分形结构的二氧化钛表面的普通玻璃基片上所捕获的循环肿瘤细胞进行计数， 计算捕 获效率。  A common glass substrate of a titanium oxide surface having a dendritic fractal structure in which circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass concentration Soak for 10 minutes in a 0.4% Triton-XlOO aqueous solution and soak for 2 minutes in a 2 g/mL DAPI aqueous solution to achieve the purpose of dyeing. A common glass substrate of the surface of the titanium dioxide having a dendritic fractal structure each capturing a circulating tumor cell was photographed by a Nikon inverted fluorescence microscope, and a common glass base of the surface of the titanium dioxide having a dendritic fractal structure capturing the circulating tumor cells was respectively taken. The circulating tumor cells captured on the sheet were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整二氧化钛表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有枝状分形结构的二氧化钛表面的 MCF7 细胞的捕获效率为 69.3%, Jurkat T细胞的捕获效率仅为 1.9%; 对照实验中平整二氧化钛表面的 MCF7细胞 的捕获效率仅为 2.1%， Jurkat T细胞的捕获效率仅为 0.6%， 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。 实施例 12 The flat titanium dioxide surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of the MCF7 cells with the dendritic fractal structure is 69.3%, and the capture efficiency of Jurkat T cells is only 1.9%. In the control experiment, the capture efficiency of MCF7 cells on the surface of titanium dioxide is only 2.1%. The capture efficiency of Jurkat T cells is only 0.6%. These data indicate that the method can be implemented. Efficient and specific capture of circulating tumor cells. Example 12
本实施例所制备的具有花椰菜状分形结构的晶体硅表面的分维为 2.51 ; 以肿瘤细胞 MCF7和 Daudi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。 利用分形结构的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：  The fractal dimension of the surface of the crystalline silicon having the broccoli fractal structure prepared in this example is 2.51. The capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用文献（Macromol. Rapid Commun. 2005, 26， 1805-1809)所述的方法即可在 晶体硅基片的表面制备出具有花椰菜状分形结构的晶体硅表面。  (1) A crystalline silicon surface having a broccoli-like fractal structure can be prepared on the surface of a crystalline silicon substrate by the method described in the literature (Macromol. Rapid Commun. 2005, 26, 1805-1809).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的晶体硅基片上的具 有花椰菜状分形结构的晶体硅表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the crystalline silicon having a broccoli-like fractal structure on the crystalline silicon substrate obtained in the step (1)
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有花椰菜状分形结构的晶体硅表面的晶体硅基片浸泡在上述一端接有羧基的 生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有花椰菜状分形结构的 晶体硅表面的晶体硅基片在使用前用去离子水冲洗干净）；取出具有花椰菜状分形结构的 晶体硅表面的晶体硅基片， 用去离子水洗涤， 干燥；  (a) diluting a biotinylated polyethylene glycol having a carboxyl group to one end with a deionized water to a 2 mM aqueous solution, and immersing a crystalline silicon substrate having a broccoli-like fractal crystal surface at the one end at room temperature Soaking in a 2 mM aqueous solution of carboxylated biotinylated polyethylene glycol for 12 hours (the crystalline silicon substrate with the crystalline silicon surface of the broccoli fractal structure was rinsed with deionized water before use); a crystalline silicon substrate having a crystalline silicon surface of a cauliflower-like fractal structure, washed with deionized water, and dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有花椰菜状分形结构的晶体硅表面的晶体硅基片浸泡在链 霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有花椰菜状分形结构的晶体硅表 面的晶体硅基片， 用磷酸盐缓冲液洗涤；  (b) a crystal of a surface of a crystalline silicon having a broccoli-like fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a). The silicon substrate is immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; a crystalline silicon substrate having a surface of crystalline silicon having a broccoli fractal structure is taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 花椰菜状分形结构的晶体硅表面的晶体硅基片上， 室温放置 30分钟， 测得肿瘤细胞表面 的抗 EpCAM抗体的固定量为 0.8 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The immobilized amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 0.8 g/cm 2 on a crystalline silicon substrate having a crystal silicon surface of 1 cm 2 having a cauliflower-like fractal structure at room temperature for 30 minutes.
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的晶体硅基片上的具有花椰菜状分形结构的晶体硅表面所 固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有 花椰菜状分形结构的晶体硅表面的晶体硅基片）， 然后置于细胞培养箱中， 由肿瘤细胞表 面的抗 EpCAM抗体与花椰菜状分形结构的协同作用， 特异性捕获滴加到步骤 （2)得到 的晶体硅基片上的具有花椰菜状分形结构的晶体硅表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕 获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the crystalline silicon having a broccoli-like fractal structure on the crystalline silicon substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). On the surface of the anti-EpCAM antibody on the surface of the tumor cell immobilized on the surface (three 1 cm 2 crystalline silicon substrate with a crystalline silicon surface with a cauliflower-like fractal structure was placed in each cell culture plate), and then placed in a cell culture incubator. Synergistic action of the anti-EpCAM antibody on the surface of the tumor cell and the cauliflower-like fractal structure, specifically capturing the surface of the tumor cell immobilized on the surface of the crystalline silicon having a cauliflower-like fractal structure added to the crystalline silicon substrate obtained in the step (2) The anti-EpCAM antibody on the surface of the step (3) is mixed uniformly after the obtained cell suspension in the circulating tumor cells, the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整晶体硅表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。 As a control experiment, the flat crystalline silicon surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to phase. The same method of capturing circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有花椰菜状分形结构的晶体硅表面的晶体硅基片用磷酸 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有 花椰菜状分形结构的晶体硅表面的晶体硅基片拍照， 并对捕获了循环肿瘤细胞的具有花 椰菜状分形结构的晶体硅表面的晶体硅基片上所捕获的循环肿瘤细胞进行计数， 计算捕 获效率。  The crystalline silicon substrate of the crystalline silicon surface having the broccoli-like fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde, mass The Triton-XlOO aqueous solution was immersed for 10 minutes in a concentration of 0.4%, and immersed in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographing the crystalline silicon substrate of the crystalline silicon surface with broccoli-like fractal structure of each of the circulating tumor cells by Nikon inverted fluorescence microscope, and crystallizing the surface of the crystalline silicon having the broccoli fractal structure of the circulating tumor cells The circulating tumor cells captured on the silicon substrate were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整晶体硅表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有花椰菜状分形结构的晶体硅表面的 MCF7细胞的捕获效率为 41.3%, Daudi细胞的捕获效率仅为 1.5%;对照实验中平整金表面的 MCF7细胞的捕获效 率仅为 2.6%， Daudi细胞的捕获效率仅为 0.7%， 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。 实施例 13  The flat crystalline silicon surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of MCF7 cells with crystalline cauliflower-like fractal structure is 41.3%, and the capture efficiency of Daudi cells is only 1.5%. The capture efficiency of MCF7 cells on the surface of gold in the control experiment is only 2.6%. The capture efficiency of Daudi cells is only 0.7%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 13
本实施例所制备的具有花椰菜状分形结构的石英表面的分维为 2.57; 以肿瘤细胞 MCF7和 Daudi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。 利用分形结构的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：  The fractal dimension of the quartz surface having the broccoli fractal structure prepared in this example is 2.57; the capture system of the present invention is further illustrated and verified by taking the tumor cells MCF7 and Daudi as the circulating tumor cells to be captured. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 )采用文献（Macromol. Rapid Commun. 2005, 26, 1805-1809)所述的方法即可在 石英基片的表面制备出具有花椰菜状分形结构的石英表面。  (1) A quartz surface having a broccoli-like fractal structure can be prepared on the surface of a quartz substrate by the method described in the literature (Macromol. Rapid Commun. 2005, 26, 1805-1809).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的石英基片上的具有 花椰菜状分形结构的石英表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the quartz surface having a cauliflower-like fractal structure on the quartz substrate obtained in the step (1)
(a)将一端接有羧基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有花椰菜状分形结构的石英表面的石英基片浸泡在上述一端接有羧基的生物 素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有花椰菜状分形结构的石英 表面的石英基片在使用前用去离子水冲洗干净）；取出具有花椰菜状分形结构的石英表面 的石英基片， 用去离子水洗涤， 干燥；  (a) diluting a biotinylated polyethylene glycol having a carboxyl group to one end with a deionized water to a 2 mM aqueous solution, and immersing a quartz substrate having a broccoli-like fractal structure on the one end at room temperature The carboxylated biotinylated polyethylene glycol was soaked in an aqueous solution of 2 mM for 12 hours (the quartz substrate with the broccoli-like fractal quartz surface was rinsed with deionized water before use); the broccoli-like fractal was taken out. a quartz substrate of a structured quartz surface, washed with deionized water, dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有花椰菜状分形结构的石英表面的石英基片浸泡在链霉亲 和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有花椰菜状分形结构的石英表面的石 英基片， 用磷酸盐缓冲液洗涤；  (b) a quartz solution in which streptavidin is diluted with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then the quartz surface having a broccoli-like fractal structure obtained by drying the step (a) is dried. The sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; the quartz substrate having a quartz surface having a broccoli fractal structure was taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 花椰菜状分形结构的石英表面的石英基片上， 室温放置 30分钟， 测得肿瘤细胞表面的抗 EpCAM抗体的固定量为 1.0 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). On a quartz substrate of 1 cm 2 of quartz surface having a broccoli-like fractal structure, it was allowed to stand at room temperature for 30 minutes to measure the surface resistance of the tumor cells. The fixed amount of the EpCAM antibody was 1.0 g/cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 2。  Same as Embodiment 2.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的石英基片上的具有花椰菜状分形结构的石英表面所固定 的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养皿放置 3个 1 cm2的具有花椰 菜状状分形结构的金表面的石英基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与花椰菜状分形结构的协同作用， 特异性捕获滴加到步骤 （2) 得到的石英 基片上的具有花椰菜状分形结构的石英表面所固定的肿瘤细胞表面的抗 EpCAM抗体的 表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分 钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the quartz surface having a broccoli-like fractal structure on the quartz substrate obtained in the step (2) placed on the cell culture plate (3.5 cm in diameter). Immobilized on the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 quartz substrates with a gold surface of broccoli-like fractal structure were placed in each cell culture dish), and then placed in a cell culture incubator, by tumor Synergistic action of the anti-EpCAM antibody on the cell surface and the cauliflower-like fractal structure, specifically capturing the anti-EpCAM antibody on the surface of the tumor cell immobilized on the quartz surface having the cauliflower-like fractal structure on the quartz substrate obtained in the step (2) Step (3) on the surface of the circulating tumor cells in the cell suspension obtained after mixing uniformly, the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整石英表面也进行相同 的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat quartz surface to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有花椰菜状分形结构的石英表面的石英基片上用磷酸盐 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从 而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有花 椰菜状分形结构的石英表面的石英基片拍照， 并对捕获了循环肿瘤细胞的具有花椰菜状 分形结构的石英表面的石英基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The quartz substrate of the quartz surface having the broccoli fractal structure of the circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% by mass aqueous solution of paraformaldehyde for 20 minutes at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. The quartz substrate with the broccoli-like fractal structure of each of the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the quartz substrate having the broccoli-like fractal structure of the circulating tumor cells was photographed on a quartz substrate. The captured circulating tumor cells were counted and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整石英表面也按上述步骤进行， 并计算捕获效率。  The flat quartz surface on which the circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有花椰菜状分形结构的石英表面的 MCF7 细胞的捕获效率为 51.7%， Daudi细胞的捕获效率仅为 1.9%;对照实验中平整石英表面的 MCF7细胞的捕获 效率仅为 2.4%， Daudi细胞的捕获效率仅为 1.2%， 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。 实施例 14  The experimental results showed that the capture efficiency of MCF7 cells with broccoli fractal structure was 51.7%, and the capture efficiency of Daudi cells was only 1.9%. The capture efficiency of MCF7 cells on the surface of quartz in the control experiment was only 2.4%. The capture efficiency of Daudi cells is only 1.2%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 14
本实施例所制备的具有枝状分形结构的聚乙烯醇表面的分维为 2.81 ; 以肿瘤细胞 MCF7和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the polyvinyl alcohol having the dendritic fractal structure prepared in this example was 2.81. The tumor cell MCF7 and Jurkat T were taken as the circulated tumor cells to be captured, and the capture system of the present invention was further elaborated and verified. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 (Angew. Chem. Int. Ed. 2002, 41, 1221-1223)所述的方法即可在聚乙烯 醇基片的表面制备出具有枝状分形结构的聚乙烯醇表面。  (1) A polyvinyl alcohol surface having a dendritic fractal structure can be prepared on the surface of a polyvinyl alcohol substrate by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的聚乙烯醇基片上的 具有枝状分形结构的聚乙烯醇表面上 ( a)将一端接有氨基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的聚乙烯醇表面的聚乙烯醇基片浸泡在上述一端接有氨基的 生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有枝状分形结构的聚乙 烯醇表面的聚乙烯醇基片在使用前用去离子水冲洗干净）；取出具有枝状分形结构的聚乙 烯醇表面的聚乙烯醇基片， 用去离子水洗涤， 干燥； (2) immobilizing an anti-EpCAM antibody on the surface of the tumor cell on the surface of the polyvinyl alcohol having a dendritic fractal structure on the polyvinyl alcohol substrate obtained in the step (1) (a) A biotinylated polyethylene glycol with an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and a polyvinyl alcohol substrate having a dendritic structure on the surface of the polyvinyl alcohol is immersed in a room temperature at room temperature. The above-mentioned biotinylated polyethylene glycol having an amino group at one end was immersed in an aqueous solution of 2 mM for 12 hours (the polyvinyl alcohol substrate having a dendritic structure on the surface of the polyvinyl alcohol was rinsed with deionized water before use). Clean); take out the polyvinyl alcohol substrate with the surface of the polyvinyl alcohol having a dendritic fractal structure, wash it with deionized water, and dry;
(b)将链霉亲和素用磷酸盐缓冲液（PBS )溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的聚乙烯醇表面的聚乙烯醇基片浸泡在链 霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝状分形结构的聚乙烯醇表面 的聚乙烯醇基片， 用磷酸盐缓冲液洗涤；  (b) diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the surface of the polyvinyl alcohol having a dendritic fractal structure obtained by drying the step (a) The polyvinyl alcohol substrate is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the polyvinyl alcohol substrate having the surface of the polyvinyl alcohol having a dendritic fractal structure is taken out and washed with a phosphate buffer;
( c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS ) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS )洗涤后得到的面积为 1 cm2的具有 枝状分形结构的聚乙烯醇表面的聚乙烯醇基片上， 室温放置 30分钟， 测得肿瘤细胞表面 的抗 EpCAM抗体的固定量为 2.0 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The immobilization amount of the anti-EpCAM antibody on the surface of the tumor cells was determined to be 2.0 g/cm 2 on a polyvinyl alcohol substrate having a dendritic fractal structure of 1 cm 2 at room temperature for 30 minutes.
( 3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的聚乙烯醇基片上的具有枝状分形结构的聚乙烯醇表面所 固定的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有 枝状分形结构的聚乙烯醇表面的聚乙烯醇基片）， 然后置于细胞培养箱中， 由肿瘤细胞表 面的抗 EpCAM抗体与枝状分形结构的协同作用， 特异性捕获滴加到步骤 （2)得到的聚 乙烯醇基片上的具有枝状分形结构的聚乙烯醇表面所固定的肿瘤细胞表面的抗 EpCAM 抗体的表面上的步骤（3 )混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the polyglycolic structure on the polyvinyl alcohol substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). On the surface of the anti-EpCAM antibody on the surface of the tumor cells immobilized on the surface of vinyl alcohol (three 1 cm 2 polyvinyl alcohol substrate with a dendritic fractal surface on each cell culture plate), and then placed In the cell culture incubator, the anti-EpCAM antibody on the surface of the tumor cell synergizes with the dendritic fractal structure to specifically capture the surface of the polyvinyl alcohol having a dendritic fractal structure added to the polyvinyl alcohol substrate obtained in the step (2). On the surface of the anti-EpCAM antibody immobilized on the surface of the tumor cells, the cells in the cell suspension obtained after the step (3) were uniformly mixed, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚乙烯醇表面也进行 相同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the surface of the flat polyvinyl alcohol to which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same method of capturing the circulating tumor cells in the sample to be tested.
( 5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的聚乙烯醇表面的聚乙烯醇基片用磷酸 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-XlOO水溶液浸泡 10分钟， 2 ^glmL DAPI水溶液浸泡 15分钟， 从而达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有 枝状分形结构的聚乙烯醇表面的聚乙烯醇基片拍照， 并对捕获了循环肿瘤细胞的具有枝 状分形结构的聚乙烯醇表面的聚乙烯醇基片上所捕获的循环肿瘤细胞进行计数， 计算捕 获效率。  The polyvinyl alcohol substrate on the surface of the polyvinyl alcohol having a dendritic fractal structure in which the circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde. Immerse for 10 minutes in a Triton-XlOO aqueous solution with a mass concentration of 0.4%, and soak for 2 minutes in a 2 ^ glmL DAPI aqueous solution to achieve the purpose of dyeing. Each of the polyvinyl alcohol substrates having a dendritic fractal structure on which the circulating tumor cells were captured was photographed by a Nikon inverted fluorescence microscope, and a polyvinyl alcohol having a dendritic fractal structure in which circulating tumor cells were captured was taken. The circulating tumor cells captured on the surface of the polyvinyl alcohol substrate were counted to calculate the capture efficiency.
捕获了循环肿瘤细胞的平整聚乙烯醇表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有枝状分形结构的聚乙烯醇表面的 MCF7 细胞的捕获效率为 71.5%, Jurkat T细胞的捕获效率仅为 1.5%; 对照实验中平整聚乙烯醇表面的 MCF7细胞 的捕获效率仅为 2.5%， Jurkat T细胞的捕获效率仅为 0.8%， 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。 实施例 15 The flat polyvinyl alcohol surface on which circulating tumor cells were captured was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of MCF7 cells with a dendritic fractal structure on the surface of polyvinyl alcohol is 71.5%, and the capture efficiency of Jurkat T cells is only 1.5%. The capture efficiency of MCF7 cells on the surface of polyvinyl alcohol in a control experiment. At only 2.5%, the capture efficiency of Jurkat T cells is only 0.8%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 15
本实施例所制备的具有枝状分形结构的苯乙烯和乳酸的共聚物表面的分维为 2.74; 以肿瘤细胞 MCF7和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure prepared in this example is 2.74; taking the tumor cells MCF7 and Jurkat T as the circulating tumor cells to be captured as an example, the capture system of the present invention is further Explain and verify. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 （Angew. Chem. Int. Ed. 2002, 41， 1221-1223 ) 所述的方法即可在苯乙 烯和乳酸的共聚物基片的表面制备出具有枝状分形结构的苯乙烯和乳酸的共聚物表面。  (1) A styrene having a dendritic fractal structure can be prepared on the surface of a copolymer substrate of styrene and lactic acid by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223). Copolymer surface with lactic acid.
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的苯乙烯和乳酸的共 聚物基片上的具有枝状分形结构的苯乙烯和乳酸的共聚物表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure on the copolymer substrate of styrene and lactic acid obtained in the step (1)
(a)将一端接有氨基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸的共聚物基片 浸泡在上述一端接有氨基的生物素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时 (具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸的共聚物基片在使用 前用去离子水冲洗干净）；取出具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯 和乳酸的共聚物基片， 用去离子水洗涤， 干燥；  (a) A biotinylated polyethylene glycol having an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and styrene and a surface of a copolymer having a dendritic fractal structure of styrene and lactic acid are allowed to be at room temperature. A lactic acid copolymer substrate was immersed in an aqueous solution of a biotinylated polyethylene glycol having an amino group at one end and a concentration of 2 mM for 12 hours (benzene having a dendritic structure of a copolymer of styrene and lactic acid) a copolymer substrate of ethylene and lactic acid is rinsed with deionized water before use; a copolymer substrate of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure is taken out and washed with deionized water. Dry
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和 乳酸的共聚物基片浸泡在链霉亲和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝 状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸的共聚物基片， 用磷酸盐缓冲 液洗涤；  (b) a solution of streptavidin diluted to a phosphate solution of 10 g/mL with a phosphate buffered saline (PBS) solution, and then styrene and lactic acid having a dendritic fractal structure obtained by drying the step (a) The copolymer substrate of styrene and lactic acid on the surface of the copolymer is immersed in a streptavidin phosphate solution and allowed to stand at room temperature for 30 minutes; the styrene on the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure is taken out. a copolymer substrate with lactic acid, washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸的共聚物基片上， 室温放置 30分钟， 测得肿瘤细胞表面的抗 EpCAM抗体的固定量为 1.5 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). The copolymer of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the amount of anti-EpCAM antibody immobilized on the surface of the tumor cells was 1.5 g. /cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的苯乙烯和乳酸的共聚物基片上的具有枝状分形结构的苯 乙烯和乳酸的共聚物表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面上 （每个细胞 培养板放置 3个 1 cm2的具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸 的共聚物基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与枝状分形 结构的协同作用， 特异性捕获滴加到步骤（2)得到的苯乙烯和乳酸的共聚物基片上的具 有枝状分形结构的苯乙烯和乳酸的共聚物表面所固定的肿瘤细胞表面的抗 EpCAM抗体 的表面上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45 分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the copolymer substrate of styrene and lactic acid obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). The fractal structure of the copolymer of styrene and lactic acid is immobilized on the surface of the anti-EpCAM antibody on the surface of the tumor cells (per cell The culture plate was placed with three 1 cm 2 copolymer substrates of styrene and lactic acid on the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure, and then placed in a cell culture incubator, and the anti-EpCAM on the surface of the tumor cells. Synergistic action of the antibody and the dendritic fractal structure, specifically capturing the tumor immobilized on the surface of the copolymer of styrene and lactic acid having a dendritic fractal structure on the copolymer substrate of styrene and lactic acid obtained in the step (2) On the surface of the cell surface of the anti-EpCAM antibody, the step (3) was obtained by uniformly mixing the circulating tumor cells in the cell suspension, and the capture time was 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整苯乙烯和乳酸的共聚 物表面也进行相同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the surface of the styrene and lactic acid copolymer of the anti-EpCAM antibody immobilized on the surface of the tumor cells was also subjected to the same process of capturing the circulating tumor cells in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯 和乳酸的共聚物基片用磷酸盐缓冲液 （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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI 水溶液浸泡 15分钟， 从而达到染色的目的。用 Nikon倒置荧光显微镜分别对每个捕获了 循环肿瘤细胞的具有枝状分形结构的苯乙烯和乳酸的共聚物表面的苯乙烯和乳酸的共聚 物基片拍照， 并对捕获了循环肿瘤细胞的具有枝状分形结构的苯乙烯和乳酸的共聚物表 面的苯乙烯和乳酸的共聚物基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  A copolymer substrate of styrene and lactic acid on the surface of a copolymer of styrene and lactic acid having a dendritic fractal structure in which circulating tumor cells were captured was washed three times with phosphate buffered saline (PBS), and then used at a mass concentration of 4%. Soaked in a solution of paraformaldehyde for 20 minutes, soaked in a 0.3% aqueous Triton-XlOO solution for 10 minutes, and soaked in a 2 g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs of styrene and lactic acid copolymer substrates on the surface of each copolymer of styrene and lactic acid having a dendritic fractal structure, which captured the circulating tumor cells, were respectively photographed by Nikon inverted fluorescence microscope, and the cells with captured circulating tumor cells were captured. The circulating tumor cells captured on the copolymer substrate of styrene and lactic acid on the surface of the copolymer of styrene and lactic acid of the dendritic fractal structure were counted, and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整苯乙烯和乳酸的共聚物表面也按上述步骤进行， 并计算 捕获效率。  The surface of the copolymer of styrene and lactic acid which captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated.
实验结果表明， 该具有枝状分形结构的苯乙烯和乳酸的共聚物表面的 MCF7细胞的 捕获效率为 67.4%， Jurkat T细胞的捕获效率仅为 2.1%;对照实验中平整苯乙烯和乳酸的 共聚物表面的 MCF7细胞的捕获效率仅为 2.3%， Jurkat T细胞的捕获效率仅为 1.8%， 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。 实施例 16  The experimental results show that the capture efficiency of MCF7 cells on the surface of the copolymer of styrene and lactic acid with branched fractal structure is 67.4%, and the capture efficiency of Jurkat T cells is only 2.1%. The copolymerization of styrene and lactic acid in the control experiment The capture efficiency of MCF7 cells on the surface was only 2.3%, and the capture efficiency of Jurkat T cells was only 1.8%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells. Example 16
本实施例所制备的具有枝状分形结构的聚氨酯表面的分维为 2.74;以肿瘤细胞 MCF7 和 Jurkat T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。 利用分形结构的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：  The fractal dimension of the surface of the polyurethane having a dendritic fractal structure prepared in this example is 2.74; and the tumor cells MCF7 and Jurkat T are taken as the example of the circulating tumor cells to be captured, and the capture system of the present invention is further elaborated and verified. A method for performing specific capture of circulating tumor cells using the surface of a fractal structure includes the following steps:
( 1 ) 采用文献 (Angew. Chem. Int. Ed. 2002, 41, 1221-1223)所述的方法即可在聚氨酯 基片的表面制备出具有枝状分形结构的聚氨酯表面。  (1) A polyurethane surface having a dendritic fractal structure can be prepared on the surface of a polyurethane substrate by the method described in the literature (Angew. Chem. Int. Ed. 2002, 41, 1221-1223).
(2) 将肿瘤细胞表面的抗 EpCAM抗体固定在步骤 （1 ) 得到的聚氨酯基片上的具 有枝状分形结构的聚氨酯表面上  (2) immobilizing the anti-EpCAM antibody on the surface of the tumor cell on the surface of the polyurethane having a dendritic fractal structure on the polyurethane substrate obtained in the step (1)
(a)将一端接有氨基的生物素化的聚乙二醇用去离子水稀释为 2 mM的水溶液， 室 温下， 将具有枝状分形结构的聚氨酯表面的聚氨酯基片浸泡在上述一端接有氨基的生物 素化的聚乙二醇的浓度为 2 mM的水溶液中浸泡 12小时（具有枝状分形结构的聚氨酯表 面的聚氨酯基片在使用前用去离子水冲洗干净）；取出具有枝状分形结构的聚氨酯表面的 聚氨酯基片， 用去离子水洗涤， 干燥； (a) A biotinylated polyethylene glycol having an amino group attached to one end is diluted with deionized water to a 2 mM aqueous solution, and a polyurethane substrate having a dendritic fractal structure is immersed in the one end at room temperature at room temperature. The amino group biotinylated polyethylene glycol was immersed in an aqueous solution of 2 mM for 12 hours (the polyurethane substrate having a dendritic fractal structure was rinsed with deionized water before use); the stripped fractal was taken out Structural polyurethane surface Polyurethane substrate, washed with deionized water, dried;
(b)将链霉亲和素用磷酸盐缓冲液（PBS)溶液稀释为 10 g/mL的磷酸盐溶液， 然 后将步骤（a)干燥后得到的具有枝状分形结构的聚氨酯表面的聚氨酯基片浸泡在链霉亲 和素的磷酸盐溶液中， 室温下放置 30分钟； 取出具有枝状分形结构的聚氨酯表面的聚氨 酯基片， 用磷酸盐缓冲液洗涤；  (b) a phosphate-based solution of a polyurethane surface having a dendritic fractal structure obtained by diluting streptavidin with a phosphate buffered saline (PBS) solution to a phosphate solution of 10 g/mL, and then drying the step (a). The sheet was immersed in a phosphate solution of streptavidin and allowed to stand at room temperature for 30 minutes; the polyurethane substrate having a polyurethane surface having a dendritic fractal structure was taken out and washed with a phosphate buffer;
(c) 将肿瘤细胞表面的抗 EpCAM抗体用磷酸盐缓冲液 （PBS) 稀释至 10 g/mL， 然后取 25 L滴加到步骤（b)用磷酸盐缓冲液（PBS)洗涤后得到的面积为 1 cm2的具有 枝状分形结构的聚氨酯表面的聚氨酯基片上， 室温放置 30分钟， 测得肿瘤细胞表面的抗 EpCAM抗体的固定量为 2.0 g/cm2。 (c) Dilute the anti-EpCAM antibody on the surface of the tumor cells to 10 g/mL with phosphate buffered saline (PBS), and then add 25 L to the area obtained in step (b) after washing with phosphate buffered saline (PBS). A polyurethane substrate having a dendritic fractal structure of 1 cm 2 was allowed to stand at room temperature for 30 minutes, and the amount of anti-EpCAM antibody immobilized on the surface of the tumor cells was 2.0 g/cm 2 .
(3 ) 准备待测样品  (3) Preparing the sample to be tested
同实施例 1。  Same as Embodiment 1.
(4) 捕获待测样品中的循环肿瘤细胞  (4) Capture circulating tumor cells in the sample to be tested
分别将步骤 （3 ) 混合均匀后得到的细胞悬浮液 3 mL滴加到放置在细胞培养板 （直 径 3.5 cm) 中的步骤（2)得到的聚氨酯基片上的具有枝状分形结构的聚氨酯表面所固定 的肿瘤细胞表面的抗 EpCAM抗体的表面上（每个细胞培养板放置 3个 1 cm2的具有枝状 分形结构的聚氨酯表面的聚氨酯基片）， 然后置于细胞培养箱中， 由肿瘤细胞表面的抗 EpCAM抗体与枝状分形结构的协同作用， 特异性捕获滴加到步骤 （2) 得到的聚氨酯基 片上的具有枝状分形结构的聚氨酯表面所固定的肿瘤细胞表面的抗 EpCAM抗体的表面 上的步骤 （3 ) 混合均匀后得到的细胞悬浮液中的循环肿瘤细胞， 捕获时间是 45分钟。 3 mL of the cell suspension obtained by uniformly mixing the step (3) was added dropwise to the polyurethane surface having a dendritic fractal structure on the polyurethane substrate obtained in the step (2) placed in the cell culture plate (3.5 cm in diameter). Immobilized on the surface of the anti-EpCAM antibody on the surface of the tumor cells (three 1 cm 2 polyurethane substrates with a dendritic fractal surface were placed on each cell culture plate), and then placed in a cell culture incubator, by tumor cells Synergistic action of the surface anti-EpCAM antibody and the dendritic fractal structure, specifically capturing the surface of the anti-EpCAM antibody surface of the tumor cell immobilized on the surface of the polyurethane having a dendritic fractal structure added to the polyurethane substrate obtained in the step (2) The above step (3) circulates the tumor cells in the cell suspension obtained after mixing uniformly, and the capture time is 45 minutes.
作为对照实验， 固定了肿瘤细胞表面的抗 EpCAM抗体的平整聚氨酯表面也进行相 同的捕获待测样品中的循环肿瘤细胞实验。  As a control experiment, the flat polyurethane surface on which the anti-EpCAM antibody on the surface of the tumor cells was immobilized was also subjected to the same cycled tumor cell test in the sample to be tested.
(5 ) 捕获效果的评价  (5) Evaluation of capture effect
将捕获了循环肿瘤细胞的具有枝状分形结构的聚氨酯表面的聚氨酯基片用磷酸盐缓 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-XlOO水溶液浸泡 10分钟， 2 g/mL DAPI水溶液浸泡 15分钟， 从而 达到染色的目的。 用 Nikon倒置荧光显微镜分别对每个捕获了循环肿瘤细胞的具有枝状 分形结构的聚氨酯表面的聚氨酯基片拍照， 并对捕获了循环肿瘤细胞的具有枝状分形结 构的聚氨酯表面的聚氨酯基片上所捕获的循环肿瘤细胞进行计数， 计算捕获效率。  The polyurethane substrate having the dendritic fractal-shaped polyurethane surface captured by circulating tumor cells was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% by mass aqueous solution of paraformaldehyde at a mass concentration of 0.4% Triton-XlOO aqueous solution was immersed for 10 minutes, and 2 g/mL DAPI aqueous solution was immersed for 15 minutes to achieve the purpose of dyeing. Each of the polyurethane substrates having the dendritic fractal-shaped polyurethane surface captured by the circulating tumor cells was photographed by a Nikon inverted fluorescence microscope, and the polyurethane substrate having the dendritic fractal structure-captured polyurethane surface was captured. The captured circulating tumor cells were counted and the capture efficiency was calculated.
捕获了循环肿瘤细胞的平整聚氨酯表面也按上述步骤进行， 并计算捕获效率。 实验结果表明， 该具有枝状分形结构的聚氨酯表面的 MCF7 细胞的捕获效率为 The flat polyurethane surface that captured the circulating tumor cells was also carried out as described above, and the capture efficiency was calculated. The experimental results show that the capture efficiency of MCF7 cells with polyurethane surface with dendritic fractal structure is
77.4%, Jurkat T细胞的捕获效率仅为 2.1%; 对照实验中平整聚聚氨酯表面的 MCF7细胞 的捕获效率仅为 1.3%， Jurkat T细胞的捕获效率仅为 2.8%， 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。 对比例 1 利用文献 "Angew. Chem. Int. Ed., 2009, 48， 8970-8973 " 中的硅纳米线阵列进行循环 肿瘤细胞的捕获 （具体操作与实施例 1的步骤 （4) 相同）， 其对 MCF7细胞的捕获效率 约为 40%， 对 Daudi细胞的捕获效率约为 4%。 从以上实施例可以看出， 本发明利用分形结构的表面进行循环肿瘤细胞的特异性捕 获能够获得较高的捕获效率， 且特异性较强。 比较实施例 1和对比例 1可以看出， 由于 硅纳米线阵列不是分形结构， 其捕获效率远低于本发明中所用具有枝状分形结构的器件 的捕获效率 （低约 10-20%)。 At 77.4%, the capture efficiency of Jurkat T cells was only 2.1%; in the control experiment, the capture efficiency of MCF7 cells on the surface of polyurethane was only 1.3%, and the capture efficiency of Jurkat T cells was only 2.8%. These data indicate that the method can be achieved. Efficient and specific capture of circulating tumor cells. Comparative example 1 Capture of circulating tumor cells using a silicon nanowire array in the literature "Angew. Chem. Int. Ed., 2009, 48, 8970-8973" (specifically the same as step (4) of Example 1), which is for MCF7 The cell capture efficiency is about 40%, and the capture efficiency for Daudi cells is about 4%. As can be seen from the above examples, the present invention utilizes the surface of the fractal structure for specific capture of circulating tumor cells to obtain higher capture efficiency and greater specificity. As can be seen from Comparative Example 1 and Comparative Example 1, since the silicon nanowire array is not a fractal structure, its capturing efficiency is much lower than that of the device having a dendritic fractal structure used in the present invention (about 10-20% lower).

Claims

权利要求 Rights request
1. 一种利用分形结构的表面进行循环肿瘤细胞的特异性捕获的方法， 其特征是， 所 述的方法包括以下步骤： 1. A method for specifically capturing circulating tumor cells using the surface of a fractal structure, characterized in that the method includes the following steps:
( 1 ) 在基片的表面制备出具有分形结构的表面； (1) Prepare a surface with a fractal structure on the surface of the substrate;
(2) 将循环肿瘤细胞表面的特异性抗体固定在步骤 （1 ) 得到的基片上的具有分形 结构的表面上， 然后放置在细胞培养板中； (2) Immobilize specific antibodies on the surface of circulating tumor cells on the surface with a fractal structure on the substrate obtained in step (1), and then place it in a cell culture plate;
(3 ) 将待捕获循环肿瘤细胞的样品滴加到步骤 （2) 得到的基片上的具有分形结构 的表面所固定的循环肿瘤细胞表面的特异性抗体的表面上， 然后置于细胞培养箱中， 由 所述的循环肿瘤细胞表面的特异性抗体与所述的分形结构的协同作用， 特异性捕获样品 中的循环肿瘤细胞； (3) Drop the sample of circulating tumor cells to be captured onto the surface of the specific antibodies immobilized on the surface of circulating tumor cells on the surface with a fractal structure on the substrate obtained in step (2), and then place it in a cell culture incubator. , due to the synergistic effect of the specific antibodies on the surface of the circulating tumor cells and the fractal structure, the circulating tumor cells in the sample are specifically captured;
所述的分形结构选自枝状分形结构、 花椰菜状分形结构或海胆状分形结构。 The fractal structure is selected from a branch-like fractal structure, a cauliflower-like fractal structure or a sea urchin-like fractal structure.
2. 根据权利要求 1所述的方法， 其特征是： 所述的将循环肿瘤细胞表面的特异性抗 体固定在步骤（1 )得到的基片上的具有分形结构的表面上， 其固定在具有分形结构的表 面上的循环肿瘤细胞表面的特异性抗体的固定量不少于 0.1 μ§/οηι2,优选为 0.1-5 g/cm2。 2. The method according to claim 1, characterized in that: the specific antibodies on the surface of circulating tumor cells are fixed on the surface with a fractal structure on the substrate obtained in step (1), which is fixed on the surface with a fractal structure. The fixed amount of specific antibodies on the surface of circulating tumor cells on the surface of the structure is not less than 0.1μ§ / οηι2 , preferably 0.1-5 g/ cm2 .
3. 根据权利要求 1或 2所述的方法， 其特征是： 所述的分形结构的表面的表征参数 分维的范围是： 2<分维 <3。 3. The method according to claim 1 or 2, characterized in that: the range of the fractal dimension of the characterization parameter of the surface of the fractal structure is: 2<fractal dimension<3.
4. 根据权利要求 1或 2所述的方法， 其特征是： 所述的循环肿瘤细胞表面的特异性 抗体为抗 EpCAM抗体。 4. The method according to claim 1 or 2, characterized in that: the specific antibody on the surface of circulating tumor cells is an anti-EpCAM antibody.
5. 根据权利要求 1或 2所述的方法， 其特征是： 所述的基片的材料选自导电金属、 导电无机非金属、 导电无机非金属化合物、 非导电无机非金属化合物和非导电聚合物中 的一种。 5. The method according to claim 1 or 2, characterized in that: the material of the substrate is selected from the group consisting of conductive metals, conductive inorganic non-metals, conductive inorganic non-metal compounds, non-conductive inorganic non-metal compounds and non-conductive polymers One of the things.
6. 根据权利要求 5所述的方法， 其特征是： 所述的导电金属选自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种； 6. The method according to claim 5, characterized in that: the conductive metal is selected from one of gold, silver, platinum, palladium, copper, iron, zinc and aluminum;
所述的导电无机非金属是晶体硅； The conductive inorganic non-metal is crystalline silicon;
所述的导电无机非金属化合物是氧化铟锡玻璃； The conductive inorganic non-metallic compound is indium tin oxide glass;
所述的非导电无机非金属化合物是石英或普通玻璃； The non-conductive inorganic non-metallic compound is quartz or ordinary glass;
所述的非导电聚合物选自以二甲基硅氧烷、 （甲基）丙烯酸酯、氨酯、碳酸酯、 乙烯、 丙烯、 苯乙烯、 乳酸和乙烯醇中的至少一种作为单体而形成的均聚物和 /或共聚物。 The non-conductive polymer is selected from the group consisting of at least one of dimethylsiloxane, (meth)acrylate, urethane, carbonate, ethylene, propylene, styrene, lactic acid and vinyl alcohol as a monomer. Homopolymers and/or copolymers formed.
7. 根据权利要求 1或 2所述的方法， 其特征是： 所述的分形结构的表面的材料选自 金属、 金属化合物、 无机非金属、 无机非金属化合物、 导电聚合物和非导电聚合物中的 一种。 7. The method according to claim 1 or 2, characterized in that: the material of the surface of the fractal structure is selected from the group consisting of: One of metals, metal compounds, inorganic non-metals, inorganic non-metal compounds, conductive polymers and non-conductive polymers.
8. 根据权利要求 7所述的方法， 其特征是： 所述的金属选自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种； 8. The method according to claim 7, characterized in that: the metal is selected from one of gold, silver, platinum, palladium, copper, iron, zinc and aluminum;
所述的金属化合物选自氧化铜、 氧化铁、 氧化锌和二氧化钛中的一种； The metal compound is selected from one of copper oxide, iron oxide, zinc oxide and titanium dioxide;
所述的无机非金属是晶体硅； The inorganic non-metal is crystalline silicon;
所述的无机非金属化合物是石英； The inorganic non-metallic compound is quartz;
所述的导电聚合物选自聚噻吩、 聚吡咯、 聚苯胺和聚苯中的一种； The conductive polymer is selected from one of polythiophene, polypyrrole, polyaniline and polyphenylene;
所述的非导电聚合物选自以二甲基硅氧烷、 （甲基）丙烯酸酯、氨酯、碳酸酯、 乙烯、 丙烯、 苯乙烯、 乳酸和乙烯醇中的至少一种作为单体而形成的均聚物和 /或共聚物。 The non-conductive polymer is selected from the group consisting of at least one of dimethylsiloxane, (meth)acrylate, urethane, carbonate, ethylene, propylene, styrene, lactic acid and vinyl alcohol as a monomer. Homopolymers and/or copolymers formed.
9. 一种用于特异性捕获循环肿瘤细胞的器件， 其特征在于， 该器件包括表面具有分 形结构的基片， 其中， 基片上的具有分形结构的表面上固定有循环肿瘤细胞表面的特异 性抗体， 所述的分形结构选自枝状分形结构、 花椰菜状分形结构或海胆状分形结构。 9. A device for specifically capturing circulating tumor cells, characterized in that the device includes a substrate with a fractal structure on the surface, wherein the surface of the substrate with a fractal structure is fixed with specificity on the surface of circulating tumor cells. Antibody, the fractal structure is selected from a branch-like fractal structure, a cauliflower-like fractal structure or a sea urchin-like fractal structure.
10. 根据权利要求 9所述的器件，其特征是： 固定在具有分形结构的表面上的循环肿 瘤细胞表面的特异性抗体的固定量不少于 0.1 ug/cm2, 优选为 0.1-5.0 ug/cm2。 10. The device according to claim 9, characterized in that: the fixed amount of specific antibodies fixed on the surface of circulating tumor cells on the surface with a fractal structure is not less than 0.1 ug/cm 2 , preferably 0.1-5.0 ug. /cm 2 .
11. 根据权利要求 9或 10所述的器件， 其特征是： 所述的分形结构的表面的表征参 数分维的范围是： 2<分维 <3。 11. The device according to claim 9 or 10, characterized in that: the range of the fractal dimension, the characterization parameter of the surface of the fractal structure, is: 2<fractal dimension<3.
12. 根据权利要求 9或 10所述的器件， 其特征是： 所述的循环肿瘤细胞表面的特异 性抗体为抗 EpCAM抗体。 12. The device according to claim 9 or 10, characterized in that: the specific antibody on the surface of circulating tumor cells is an anti-EpCAM antibody.
13. 根据权利要求 9或 10所述的器件，其特征是：所述的基片的材料选自导电金属、 导电无机非金属、 导电无机非金属化合物、 非导电无机非金属化合物和非导电聚合物中 的一种。 13. The device according to claim 9 or 10, characterized in that: the material of the substrate is selected from the group consisting of conductive metals, conductive inorganic non-metals, conductive inorganic non-metal compounds, non-conductive inorganic non-metal compounds and non-conductive polymers One of the things.
14. 根据权利要求 13所述的器件， 其特征是： 所述的导电金属选自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种； 14. The device according to claim 13, characterized in that: the conductive metal is selected from one of gold, silver, platinum, palladium, copper, iron, zinc and aluminum;
所述的导电无机非金属是晶体硅； The conductive inorganic non-metal is crystalline silicon;
所述的导电无机非金属化合物是氧化铟锡玻璃； The conductive inorganic non-metallic compound is indium tin oxide glass;
所述的非导电无机非金属化合物是石英或普通玻璃； The non-conductive inorganic non-metallic compound is quartz or ordinary glass;
所述的非导电聚合物选自以二甲基硅氧烷、 （甲基）丙烯酸酯、氨酯、碳酸酯、 乙烯、 丙烯、 苯乙烯、 乳酸和乙烯醇中的至少一种作为单体而形成的均聚物和 /或共聚物。 The non-conductive polymer is selected from the group consisting of at least one of dimethylsiloxane, (meth)acrylate, urethane, carbonate, ethylene, propylene, styrene, lactic acid and vinyl alcohol as a monomer. Homopolymers and/or copolymers formed.
15. 根据权利要求 9或 10所述的器件， 其特征是： 所述的分形结构的表面的材料选 自金属、 金属化合物、 无机非金属、 无机非金属化合物、 导电聚合物和非导电聚合物中 的一种。 15. The device according to claim 9 or 10, characterized in that: the material of the surface of the fractal structure is selected from metals, metal compounds, inorganic non-metals, inorganic non-metal compounds, conductive polymers and non-conductive polymers one of them.
16. 根据权利要求 15所述的器件， 其特征是： 所述的金属选自金、 银、 铂、 钯、 铜、 铁、 锌和铝中的一种； 16. The device according to claim 15, characterized in that: the metal is selected from one of gold, silver, platinum, palladium, copper, iron, zinc and aluminum;
所述的金属化合物选自氧化铜、 氧化铁、 氧化锌和二氧化钛中的一种； The metal compound is selected from one of copper oxide, iron oxide, zinc oxide and titanium dioxide;
所述的无机非金属是晶体硅； The inorganic non-metal is crystalline silicon;
所述的无机非金属化合物是石英； The inorganic non-metallic compound is quartz;
所述的导电聚合物选自聚噻吩、 聚吡咯、 聚苯胺和聚苯中的一种； The conductive polymer is selected from one of polythiophene, polypyrrole, polyaniline and polyphenylene;
所述的非导电聚合物选自以二甲基硅氧烷、 （甲基）丙烯酸酯、氨酯、碳酸酯、 乙烯、 丙烯、 苯乙烯、 乳酸和乙烯醇中的至少一种作为单体而形成的均聚物和 /或共聚物。 The non-conductive polymer is selected from the group consisting of at least one of dimethylsiloxane, (meth)acrylate, urethane, carbonate, ethylene, propylene, styrene, lactic acid and vinyl alcohol as a monomer. Homopolymers and/or copolymers formed.
17. 权利要求 9-16中任意一项所述的器件在癌症的早期诊断、 病情监测和治疗预后 中的应用。 17. Application of the device according to any one of claims 9 to 16 in early diagnosis, condition monitoring and treatment prognosis of cancer.
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