WO2018171318A1 - Puce à réseau de nanofils de dioxyde de silicium destinée à collecter et à détecter des cellules tumorales circulantes dans le sang total et son procédé de préparation - Google Patents

Puce à réseau de nanofils de dioxyde de silicium destinée à collecter et à détecter des cellules tumorales circulantes dans le sang total et son procédé de préparation Download PDF

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WO2018171318A1
WO2018171318A1 PCT/CN2018/072966 CN2018072966W WO2018171318A1 WO 2018171318 A1 WO2018171318 A1 WO 2018171318A1 CN 2018072966 W CN2018072966 W CN 2018072966W WO 2018171318 A1 WO2018171318 A1 WO 2018171318A1
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circulating tumor
tumor cells
chip
substrate
concentration
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PCT/CN2018/072966
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English (en)
Chinese (zh)
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孟靖昕
李冠男
王树涛
江雷
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北京赛特超润界面科技有限公司
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Publication of WO2018171318A1 publication Critical patent/WO2018171318A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
    • C01B33/16—Preparation of silica xerogels
    • C01B33/163—Preparation of silica xerogels by hydrolysis of organosilicon compounds, e.g. ethyl orthosilicate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/10—Particle morphology extending in one dimension, e.g. needle-like
    • C01P2004/16—Nanowires or nanorods, i.e. solid nanofibres with two nearly equal dimensions between 1-100 nanometer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention relates to the field of biological materials and clinical detection, in particular to a silica nanowire array chip for whole blood capturing cancer cells and a preparation method thereof, in particular to a silica nanometer for enrichment and detection of circulating tumor cells in whole blood.
  • Line array chip and preparation method in particular to a silica nanometer for enrichment and detection of circulating tumor cells in whole blood.
  • Circulating tumor cells refer to tumor cells that enter the peripheral blood circulation from primary or metastatic lesions due to spontaneous or diagnostic procedures. Most of the tumor cells invading the circulatory system die in the short term due to the body's immune recognition, mechanical killing and auto-apoptosis, and only a very small number survive to form metastases in organs or tissues suitable for their own survival and proliferation. Therefore, the detection of circulating tumor cells (CTCs) has important application value for clinical diagnosis, prognosis judgment and monitoring efficacy of early tumor metastasis.
  • the current mature circulating tumor cell detection technology is mainly based on the physical properties (such as size) of circulating tumor cells (CTCs) and the biological properties based on their affinity and recognition based on antibodies, and both.
  • Comprehensive utilization including immunomagnetic separation technology, microfluidics technology, microfiltration technology, density gradient centrifugation technology, and the combination of several technologies.
  • these methods still have the disadvantages of low efficiency, low purity and long capture time.
  • the combination of two or three methods may become an ideal method, the research in the past decade has not made much progress.
  • Circulating tumor cells (CTCs) detection is a highly reproducible, minimally invasive diagnostic tool that is currently recognized clinically and has important clinical value. Therefore, the development of new functional biomedical materials, improve the capture efficiency of circulating tumor cells, and provide more information for the research and clinical application of circulating tumor cells (CTCs), which is an urgent problem to be solved in the field.
  • CTCs circulating tumor cells
  • the silica nanowire array chip for enrichment and detection of whole blood circulation tumor cells of the present invention characterized in that the silica nanowire array chip comprises: a polydimethylsiloxane (PDMS) substrate, a polydimethylsiloxane interface in situ grown silica (SiO 2 ) nanowire array and an antibody having a specific recognition of tumor cells on the surface of the silica (SiO 2 ) nanowire array (eg, Anti- EpCAM); wherein the antibody specifically recognizing the tumor cell is immobilized in an amount of not less than 0.1 ⁇ g/cm 2 .
  • PDMS polydimethylsiloxane
  • SiO 2 polydimethylsiloxane interface in situ grown silica
  • an antibody having a specific recognition of tumor cells on the surface of the silica (SiO 2 ) nanowire array eg, Anti- EpCAM
  • the silica nanowire array chip according to the present invention wherein the antibody that specifically recognizes circulating tumor cells includes, but is not limited to, a specific antibody that binds to a specific antigen such as EpCAM on the surface of a tumor cell (Anti-EpCAM) Specific antibodies that bind to the common antigen CD45 on the surface of leukocytes; markers for circulating tumor cells (CTCs) such as guanylate cyclase C, cytokeratin (CK18, CK19, CK20), epithelial cadherin ( E-cadherin An antibody that specifically recognizes an antibody that specifically recognizes tumor tissue markers such as carcinoembryonic antigen CEA, prostate specific antigen PSA, and carbohydrate antigen .
  • CTCs markers for circulating tumor cells
  • CTCs markers for circulating tumor cells
  • CK18, CK19, CK20 markers for circulating tumor cells
  • E-cadherin an antibody that specifically recognizes an antibody that specifically recognizes tumor tissue markers such as carcino
  • a silicon dioxide nanowire array chip according to the present invention wherein a wire diameter of the silicon dioxide nanowire array is 70-300 nm, length 0.3-20 ⁇ m.
  • silica nanowire array chip wherein the polydimethylsiloxane substrate is subjected to a hydrophilization treatment.
  • the silica nanowire array chip according to the present invention wherein the silica nanowire array is formed by a microemulsion droplet under the action of ammonia water and tetraethyl orthosilicate; the microemulsion droplet is: In a n-pentanol system, polyvinylpyrrolidone (PVP), water and ethanol are blended to form a water-in-oil microemulsion.
  • PVP polyvinylpyrrolidone
  • the method for preparing the above-mentioned silicon dioxide nanowire array chip of the present invention comprises the following steps:
  • polyvinylpyrrolidone 20-300 g / L
  • water and ethanol are blended and stirred to form a water-in-oil type microemulsion
  • step (3) using the polydimethylsiloxane treated in the step (2) as a substrate, adsorbing the microemulsion prepared in the step (1), and placing it in aqueous ammonia at room temperature (mass concentration 25%, 20-400) ⁇ L) and tetraethyl orthosilicate (30-600 ⁇ L) catalyzed preparation of a silica nanowire array substrate;
  • the preparation method according to the present invention wherein the molar ratio of the n-pentanol, water and ethanol in the step (1) is 1:0.01-3:0.01-3.
  • the preparation method according to the present invention wherein the hydrophilic treatment in the step (2) is a surface plasma treatment .
  • the preparation method according to the present invention wherein the method of immobilizing an antibody that specifically recognizes a tumor cell on a silica nanowire as described in the step (4) comprises the steps of:
  • N-(4-maleimidobutyryl)succinimide is formulated to a concentration of 5-30 with dimethyl sulfoxide mM solution, the substrate obtained after drying step (a) is immersed in the above concentration of 5-30 a solution of mM N-(4-maleimidobutyryl)succinimide, placed at room temperature; the substrate was removed, rinsed with dimethyl sulfoxide and phosphate buffer, respectively, and dried;
  • step (c) Dilute streptavidin with phosphate buffer to a concentration of 10-50 ⁇ g/mL of streptavidin phosphate solution, and then the substrate obtained by drying step (b) is immersed in the above-mentioned phosphate solution of streptavidin at a concentration of 10-50 ⁇ g/mL, Placed at room temperature; the substrate was removed and washed with phosphate buffer;
  • the present invention also provides the use of the above-described silica nanowire chip, especially in circulating tumor cell capture in whole blood.
  • the present invention utilizes a polydimethylsiloxane (PDMS) interfacially grown silicon dioxide (SiO 2 ) nanowire array chip for circulating tumor cell enrichment and detection.
  • PDMS polydimethylsiloxane
  • SiO 2 silicon dioxide
  • the water-in-oil microemulsion was prepared by using the difference in solubility, and assembled on the interface of polydimethylsiloxane, which then catalyzed the in-situ growth of the silica nanowires to obtain two a silica nanowire array chip; then immobilizing a specific antibody on the surface of the circulating tumor cell on the silica nanowire to prepare a silica nanowire array chip having a specific antibody immobilized on the surface of the circulating tumor cell; A sample to be tested containing circulating tumor cells is dropped onto the surface of the chip, and a specific antibody in the surface of the circulating tumor cell cooperates with the nanowire structure to specifically capture a circulating tumor in the sample to be tested. cell
  • the invention utilizes the surface of the silica nanowire modified by the specific recognition molecule to specifically enrich and separate the circulating tumor cells (CTCs) existing in the peripheral blood.
  • the silica nanowire array chip has good biocompatibility and can be used for further non-invasive culture and clinical detection of captured circulating tumor cells (CTCs).
  • the present invention is achieved by utilizing the specificity of antibodies on the surface of circulating tumor cells (CTCs) while utilizing the properties of the nanostructures of the silica nanowire arrays to match the surface structures of circulating tumor cells (CTCs) to enhance targeting of circulating tumors. Efficient and specific capture of cells (CTCs).
  • the method for specifically capturing circulating tumor cells (CTCs) using a silica nanowire array chip for capturing blood cancer cells by whole blood according to the present invention, the method (1) demonstrating a silica nanowire array modified with specific recognition molecules
  • the surface can achieve efficient capture of circulating tumor cells (CTCs); (2) high specificity, high sensitivity, can capture specific tumor cells, and reduce the adhesion of non-specific tumor cells.
  • the method for specifically capturing the circulating tumor cells (CTCs) on the surface of the silica nanowire array modified by the specific recognition molecule of the present invention achieves the capture of targeted cancer cells in whole blood.
  • the method can significantly improve the capture efficiency of circulating tumor cells (CTCs), has low cost, is simple to operate, and can be used for clinical early diagnosis and postoperative monitoring of cancer.
  • Figure 1 is a schematic illustration of capture of circulating tumor cells in Example 1 of the present invention.
  • Example 2 is a modification process of the biotinylated anti-EpCAM antibody of Example 1 of the present invention.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 0.3 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by utilizing the synergistic action of the surface micro-nano structure of the silica nanowire array and the specific recognition molecule comprises the following steps:
  • n-pentanol polyvinylpyrrolidone (PVP, 20 g/L), water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein n-pentanol, water and ethanol are used in a ratio of 1: 0.01:0.01;
  • PVP polyvinylpyrrolidone
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 0.3 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) is placed face up in a sterile six-well plate, and 3 mL of breast cancer at a concentration of 1 ⁇ 10 5 cells/mL is added.
  • the cell MCF7 suspension was placed in a cell culture incubator and the reaction was preferentially carried out for 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL prostate cancer cell PC3 suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added. /mL of human lymphatic B cell carcinoma Daudi suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes. Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL human lymphoma Jurkat suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL uterine cancer cell Hela suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 0.3 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by utilizing the synergistic action of the surface micro-nano structure of the silica nanowire array and the specific recognition molecule comprises the following steps:
  • n-pentanol polyvinylpyrrolidone (PVP, 20 g/L), water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein n-pentanol, water and ethanol are used in a ratio of 1: 0.01:0.01;
  • PVP polyvinylpyrrolidone
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 0.3 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) is placed face up in a sterile six-well plate, and 3 mL of breast cancer at a concentration of 1 ⁇ 10 5 cells/mL is added.
  • the cell MCF7 suspension was placed in a cell culture incubator and the reaction was preferentially carried out for 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL prostate cancer cell PC3 suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added. /mL of human lymphatic B cell carcinoma Daudi suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes. Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL human lymphoma Jurkat suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL uterine cancer cell Hela suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the nanowires of the silica nanowire array chip prepared in this embodiment have a length of 1.5 ⁇ m; taking the circulating tumor cells EpCAM-specific cells and EpCAM non-specific cells as the circulating tumor cells to be captured, for example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by utilizing the synergistic action of the surface micro-nano structure of the silica nanowire array and the specific recognition molecule comprises the following steps:
  • n-pentanol polyvinylpyrrolidone (PVP, 300 g/L), water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein n-pentanol, water and ethanol are used in a ratio of 1: 0.01:0.01;
  • PVP polyvinylpyrrolidone
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 1.5 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature.
  • the lower layer was placed to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells was immobilized.
  • the chip obtained in the step (7) (preferred nanowire length is 1.5 ⁇ m) is placed face up in a sterile six-well plate, and 3 mL of breast cancer at a concentration of 1 ⁇ 10 5 cells/mL is added.
  • the cell MCF7 suspension was placed in a cell culture incubator and the reaction was preferentially carried out for 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 1.5 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL prostate cancer cell PC3 suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the chip obtained in the step (7) (preferred nanowire length of 1.5 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • /mL of human lymphatic B cell carcinoma Daudi suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 1.5 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL human lymphoma Jurkat suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the chip obtained in the step (7) (preferred nanowire length of 1.5 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL uterine cancer cell Hela suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 10 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by utilizing the synergistic action of the surface micro-nano structure of the silica nanowire array and the specific recognition molecule comprises the following steps:
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 10 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • step (7) Place the chip obtained in step (7) (preferred nanowire length is 10 ⁇ m) face up in a sterile six-well plate and add 3 mL of breast cancer at a concentration of 1 ⁇ 10 5 cells/mL.
  • the cell MCF7 suspension was placed in a cell culture incubator and the reaction was preferentially carried out for 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 10 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL prostate cancer cell PC3 suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 10 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • /mL of human lymphatic B cell carcinoma Daudi suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 10 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL human lymphoma Jurkat suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 10 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL uterine cancer cell Hela suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 20 ⁇ m; taking the circulating tumor cell EpCAM-specific cells and the EpCAM non-specific cells as the circulating tumor cells to be captured as an example, the capture system of the present invention is used. Further elaboration and verification.
  • the method for performing specific capture of circulating tumor cells by utilizing the synergistic action of the surface micro-nano structure of the silica nanowire array and the specific recognition molecule comprises the following steps:
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 20 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (preferred nanowire length is 20 ⁇ m) is placed face up in a sterile six-well plate, and 3 mL of breast cancer at a concentration of 1 ⁇ 10 5 cells/mL is added.
  • the cell MCF7 suspension was placed in a cell culture incubator and the reaction was preferentially carried out for 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 20 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL prostate cancer cell PC3 suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 20 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • /mL of human lymphatic B cell carcinoma Daudi suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 20 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL human lymphoma Jurkat suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (7) (preferred nanowire length of 20 ⁇ m) was placed face up in a sterile six-well plate, and 3 mL of a concentration of 1 ⁇ 10 5 cells was added.
  • the /mL uterine cancer cell Hela suspension was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes.
  • the uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the capture system of the present invention is further illustrated and verified by taking circulating tumor cell EpCAM-specific cells and EpCAM non-specific cells as circulating tumor cells to be captured.
  • a method for circulating tumor cell capture using a flat silicon dioxide chip includes the following steps:
  • a flat silica substrate is selected
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (2) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin was diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (3) was immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (4), room temperature. Placed underneath to obtain a specific antibody on the surface of the silica substrate to which the surface of the circulating tumor cells is immobilized;
  • step (6) Place the chip obtained in step (5) face up in a sterile six-well plate, add 3 mL of MCF7 suspension of breast cancer cells at a concentration of 1 ⁇ 10 5 cells/mL, and place in a cell culture incubator. The priority of the reaction is 45 minutes.
  • the breast cancer cell MCF7 suspension was removed, and the chip capturing the breast cancer cell MCF7 was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (5) was placed face up in a sterile six-well plate, and 3 mL of a prostate cancer cell PC3 suspension having a concentration of 1 ⁇ 10 5 cells/mL was added. In a cell culture incubator, placed in a cell culture incubator, the reaction time was 45 minutes. The prostate cancer cell PC3 suspension was removed, and the chip capturing the prostate cancer cell PC3 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.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (5) was placed face up in a sterile six-well plate, and 3 mL of human lymphatic B-cell cancer cells Daudi suspended at a concentration of 1 ⁇ 10 5 cells/mL was added. The solution was placed in a cell culture incubator and placed in a cell culture incubator with a preferential reaction time of 45 minutes. Human lymphoid B-cell cancer cell Daudi was removed, and the chip capturing human lymphoid B-cell cancer cell Daudi was washed three times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde.
  • PBS phosphate buffered saline
  • the Triton-X100 aqueous solution with a mass 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 were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (5) was placed face up in a sterile six-well plate, and 3 mL of a human lymphoma Jurkat suspension having a concentration of 1 ⁇ 10 5 cells/mL was added. Place in a cell culture incubator and place in a cell culture incubator. The reaction time is preferably 45 minutes.
  • the human lymphatic cancer cell Jurkat was removed, and the human lymphatic cancer cell Jurkat chip was washed three times with phosphate buffered saline (PBS), and then immersed in a 4% mass% aqueous solution of formaldehyde for 20 minutes at a mass concentration of 0.4.
  • PBS phosphate buffered saline
  • the solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the chip obtained in the step (5) was placed face up in a sterile six-well plate, and 3 mL of a suspension of uterine cancer cells HeLa at a concentration of 1 ⁇ 10 5 cells/mL was added. In a cell culture incubator, placed in a cell culture incubator, the reaction time was 45 minutes. The uterine cancer cell line Hela was removed, and the chip capturing the uterine cancer cell line Hela 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%.
  • PBS phosphate buffered saline
  • Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 ⁇ g/mL DAPI aqueous solution for 15 minutes to achieve the purpose of dyeing. Photographs were taken 10 times with a Nikon inverted fluorescence microscope (10 different positions in the middle of each chip that captured the circulating tumor cells), and the circulating tumor cells captured on the chip capturing the circulating tumor cells were counted and calculated. Capture efficiency
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 0.3 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a silica nanowire array chip includes the following steps:
  • n-pentanol polyvinylpyrrolidone (PVP, 20 g/L), water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein n-pentanol, water and ethanol are used in a ratio of 1: 0.01:0.01;
  • PVP polyvinylpyrrolidone
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 0.3 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (preferred nanowire length is 0.3 ⁇ m) is placed face up in a sterile six-well plate, placed in a microplate, and 1 mL of breast cancer patient blood is added. Place in the cell culture incubator and give priority to the reaction for 45 minutes. Excess blood was removed, and the chip capturing breast cancer cell MCF7 was washed 3 times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde in a mass concentration of 0.4% Triton.
  • PBS phosphate buffered saline
  • the -X100 aqueous solution was immersed for 10 minutes, and 200 ⁇ g of a blocking agent (5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer) was added and allowed to stand at room temperature for one hour.
  • a blocking agent 5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer
  • the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 1.5 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a silica nanowire array chip includes the following steps:
  • n-pentanol polyvinylpyrrolidone (PVP, 300 g/L), water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein n-pentanol, water and ethanol are used in a ratio of 1: 0.01:0.01;
  • PVP polyvinylpyrrolidone
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 1.5 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (preferred nanowire length is 1.5 ⁇ m) is placed face up in a sterile six-well plate, placed in a wafer culture dish, and 1 mL of breast cancer patient blood is added. Place in the cell culture incubator and give priority to the reaction for 45 minutes. Excess blood was removed, and the chip capturing breast cancer cell MCF7 was washed 3 times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde in a mass concentration of 0.4% Triton.
  • PBS phosphate buffered saline
  • the -X100 aqueous solution was immersed for 10 minutes, and 200 ⁇ g of a blocking agent (5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer) was added and allowed to stand at room temperature for one hour.
  • a blocking agent 5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer
  • the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 10 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a silica nanowire array chip includes the following steps:
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 10 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (the preferred length of the nanowire is 10 ⁇ m) is placed face up in a sterile six-well plate, placed in a microplate, and the blood of a 1 mL breast cancer patient is added. Place in the cell culture incubator and give priority to the reaction for 45 minutes. Excess blood was removed, and the chip capturing breast cancer cell MCF7 was washed 3 times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde in a mass concentration of 0.4% Triton.
  • PBS phosphate buffered saline
  • the -X100 aqueous solution was immersed for 10 minutes, and 200 ⁇ g of a blocking agent (5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer) was added and allowed to stand at room temperature for one hour.
  • a blocking agent 5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer
  • the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 20 ⁇ m; the capture system of the present invention is further illustrated and verified by taking the capture of breast cancer cells in the blood of breast cancer patients as an example.
  • a method for performing specific capture of circulating tumor cells using a silica nanowire array chip includes the following steps:
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 20 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (the preferred nanowire length is 20 ⁇ m) is placed face up in a sterile six-well plate, placed in a microplate, and the blood of a 1 mL breast cancer patient is added. Place in the cell culture incubator and give priority to the reaction for 45 minutes. Excess blood was removed, and the chip capturing breast cancer cell MCF7 was washed 3 times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde in a mass concentration of 0.4% Triton.
  • PBS phosphate buffered saline
  • the -X100 aqueous solution was immersed for 10 minutes, and 200 ⁇ g of a blocking agent (5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer) was added and allowed to stand at room temperature for one hour.
  • a blocking agent 5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer
  • the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times.
  • the flat silicon dioxide chip prepared in this embodiment is taken as an example to capture the breast cancer cells in the blood of breast cancer patients, and the capture system of the present invention is further elaborated and verified.
  • a method for circulating tumor cell capture using a flat silicon dioxide chip includes the following steps:
  • a flat silica substrate is selected
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (2) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin was diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (3) was immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (4), room temperature. Placed underneath to obtain a specific antibody on the surface of the flat silica substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (5) is placed face up in a sterile six-well plate, placed in a microplate, and the blood of a 1 mL breast cancer patient is added and placed in a cell culture incubator to preferentially react. The time is 45 minutes. Excess blood was removed, and the chip capturing breast cancer cell MCF7 was washed 3 times with phosphate buffered saline (PBS), and then soaked for 20 minutes with a 4% aqueous solution of paraformaldehyde in a mass concentration of 0.4% Triton.
  • PBS phosphate buffered saline
  • the -X100 aqueous solution was immersed for 10 minutes, and 200 ⁇ g of a blocking agent (5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer) was added and allowed to stand at room temperature for one hour.
  • a blocking agent 5% goat serum, 0.1% Tween 20, 3% bovine serum albumin phosphate buffer
  • the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times.
  • the nanowire length of the silica nanowire array chip prepared in this embodiment is 20 ⁇ m; the capture system of the present invention is further elaborated and verified by taking breast cancer cells in normal human blood as an example.
  • a method for performing specific capture of circulating tumor cells using a silica nanowire array chip includes the following steps:
  • step (2) substrate is uniformly adsorbed to the microemulsion prepared in the step (1), and is catalyzed to obtain a silica nanowire array substrate under the atmosphere of ammonia water and tetraethyl orthosilicate at room temperature (preferred choice)
  • the length of the nanowire is 20 ⁇ m
  • N-(4-maleimidobutyryl)succinimide is formulated into a solution having a concentration of 5 mM with dimethyl sulfoxide, and the substrate obtained by drying step (4) is immersed in The above solution having a concentration of 5 mM of N-(4-maleimidobutyryl)succinimide was placed at room temperature; the substrate was taken out and rinsed with dimethyl sulfoxide and phosphate buffer, respectively. Blow dry
  • the streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 ⁇ g/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 ⁇ g/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;
  • the specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 ⁇ g/mL, and then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (6), at room temperature. Placed underneath to obtain a specific antibody on the surface of the silica nanowire array substrate to which the surface of the circulating tumor cells is immobilized;
  • the chip obtained in the step (7) (the preferred nanowire length is 20 ⁇ m) is placed face up in a sterile six-well plate, placed in a microplate, and 1 mL of normal human blood is added. In the cell culture incubator, the priority of the reaction was 45 minutes. Excess blood was removed, and the chip was washed 3 times with phosphate buffered saline (PBS), then immersed in a 4% aqueous solution of paraformaldehyde for 20 minutes, and immersed in a 0.4% aqueous Triton-X100 solution for 10 minutes.
  • PBS phosphate buffered saline
  • the method for specifically capturing the circulating tumor cells (CTCs) on the surface of the silica nanowire array modified by the specific recognition molecule of the present invention achieves the capture of targeted cancer cells in whole blood.
  • the method can significantly improve the capture efficiency of circulating tumor cells (CTCs), has low cost, is simple to operate, and can be used for clinical early diagnosis and postoperative monitoring of cancer.

Abstract

La présente invention prépare une microémulsion de type eau dans huile dans un système de phase organique à l'aide d'une différence de solubilité, la rassemble sur une interface de polydiméthylsiloxane, puis favorise catalytiquement la croissance in situ de nanofils de dioxyde de silicium pour obtenir une puce à réseau de nanofils de dioxyde de silicium, puis fixe, sur les nanofils de dioxyde de silicium, des anticorps spécifiques afin d'identifier des cellules tumorales circulantes. Un échantillon de sang total contenant des cellules tumorales circulantes à tester est ajouté goutte à goutte sur la surface de la puce, en raison de l'effet synergique de l'anticorps spécifique et de la structure de nanofil, la puce permet de détecter spécifiquement les cellules tumorales circulantes dans le sang total à tester.
PCT/CN2018/072966 2017-03-24 2018-01-17 Puce à réseau de nanofils de dioxyde de silicium destinée à collecter et à détecter des cellules tumorales circulantes dans le sang total et son procédé de préparation WO2018171318A1 (fr)

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CN111137897B (zh) * 2020-01-14 2023-08-15 苏州大学 一种二氧化硅纳米棒阵列的制备方法
CN111423971A (zh) * 2020-04-17 2020-07-17 南京鼓楼医院 一种用于循环肿瘤细胞捕获的聚合物微球及其制备方法
CN112011435B (zh) * 2020-09-11 2023-03-21 徐州工程学院 一种用于精准捕获循环肿瘤细胞的微流控系统及制备方法
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