WO2018171318A1

WO2018171318A1 - Silicon dioxide nanowire array chip for gathering and detecting circulating tumor cells in whole blood and preparation method therefor

Info

Publication number: WO2018171318A1
Application number: PCT/CN2018/072966
Authority: WO
Inventors: 孟靖昕; 李冠男; 王树涛; 江雷
Original assignee: 北京赛特超润界面科技有限公司
Priority date: 2017-03-24
Filing date: 2018-01-17
Publication date: 2018-09-27
Also published as: CN108624472A

Abstract

The present invention prepares a water-in-oil type microemulsion in an organic phase system by using solubility difference, assembles same on a polydimethylsiloxane interface, then catalytically promotes in-situ growth of silicon dioxide nanowires to obtain a silicon dioxide nanowire array chip, and then fixes specific antibodies for identifying circulating tumor cells on the silicon dioxide nanowires. A circulating tumor cell-containing whole blood sample to be tested is added dropwise on the surface of the chip, because of the synergistic effect of the specific antibodie and the nanowire structure, the chip can specifically detect the circulating tumor cells in the whole blood to be tested.

Description

Silica nanowire array chip for enrichment and detection of circulating tumor cells in whole blood and preparation method thereof

Technical field

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.

Background technique

Recurrence and metastasis of the tumor is the leading cause of death. Detection of circulating tumor cells (CTCs) in peripheral blood has important application value in solving clinical problems such as recurrence and metastasis monitoring and tumor molecular characteristics. Circulating tumor cells (CTCs) 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.

With the deep development of bio-nanotechnology, 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. However, these methods still have the disadvantages of low efficiency, low purity and long capture time. Although the combination of two or three methods may become an ideal method, the research in the past decade has not made much progress.

At present, Wang Shutao, a researcher at the Institute of Physics and Chemistry of the Chinese Academy of Sciences, has developed a three-dimensional micro-nano interface material that specifically recognizes and adheres to tumor cells, and uses micro-nanoscale effects to regulate the adhesion characteristics at the solid-liquid interface, combining specific antibodies and interface nanostructures. It achieves highly sensitive and specific capture of tumor cells, and can separate living circulating tumor cells from whole blood samples, and has improved the capture efficiency of circulating tumor cells, and has received extensive attention.

The research on the separation and detection technology of circulating tumor cells (CTCs) in China is still in its infancy. 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.

technical problem

The circulating tumor cells (CTCs) in blood tests are very low in concentration and are not easy to detect. At the same time, the existing specific methods for whole blood testing, high production cost, inconvenient operation, and unsuitable for industrial mass production.

Technical solution

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 μg/cm ² .

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 .

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 μm.

The silica nanowire array chip according to the present invention, 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.

The method for preparing the above-mentioned silicon dioxide nanowire array chip of the present invention comprises the following steps:

(1) in a n-pentanol system, polyvinylpyrrolidone (PVP, 20-300 g / L), water and ethanol are blended and stirred to form a water-in-oil type microemulsion;

(2) hydrophilizing the polydimethylsiloxane;

(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) μL) and tetraethyl orthosilicate (30-600 二氧化硅L) catalyzed preparation of a silica nanowire array substrate;

(4) An antibody that specifically recognizes a tumor cell is immobilized on the silica nanowire prepared in the step (3).

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:

(a) mixing (3-mercaptopropyl)trimethoxysilane and ethanol in a volume ratio to prepare an ethanol solution of (3-mercaptopropyl)trimethoxysilane at a concentration of 5-30%; at room temperature, two The silicon oxide nanowire array substrate is immersed in an ethanol solution of (3-mercaptopropyl)trimethoxysilane having a volume concentration of 5-30%, and is allowed to stand at room temperature; the substrate is taken out, and ethanol and dimethyl groups are respectively used. Sulfone rinse, blow dry;

(b) 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;

(c) Dilute streptavidin with phosphate buffer to a concentration of 10-50 Μg/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 μg/mL, Placed at room temperature; the substrate was removed and washed with phosphate buffer;

(d) Diluting antibodies (specific antibodies on the surface of circulating tumor cells) that specifically recognize tumor cells with phosphate buffer to a concentration of 10-50 Gg/mL, which is then added dropwise to the surface of the substrate obtained by washing the phosphate buffer with the step (c), and left at room temperature to obtain a surface of the vascular endothelial cell to which the surface of the silica nanowire array substrate is fixed. Specific antibodies.

The present invention also provides the use of the above-described silica nanowire chip, especially in circulating tumor cell capture in whole blood.

Beneficial effect

The present invention utilizes a polydimethylsiloxane (PDMS) interfacially grown silicon dioxide (SiO ₂ ) nanowire array chip for circulating tumor cell enrichment and detection. In the organic phase system, 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. It achieves high capture efficiency in the patient's blood, providing a simple and quick method for clinical application.

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.

DRAWINGS

Figure 1 is a schematic illustration of capture of circulating tumor cells in Example 1 of the present invention.

2 is a modification process of the biotinylated anti-EpCAM antibody of Example 1 of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The nanowire length of the silica nanowire array chip prepared in this embodiment is 0.3 μm; 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:

(1) In a n-pentanol system, 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;

(2) subjecting the PDMS substrate to hydrophilic treatment;

(3) The 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 μm);

(4) Mixing (3-mercaptopropyl)trimethoxysilane with ethanol in a volume ratio to prepare a 5% solution of (3-mercaptopropyl)trimethoxysilane in ethanol; at room temperature, silica The nanowire array substrate was immersed in a solution of 5% (3-mercaptopropyl)trimethoxysilane in ethanol at room temperature, and the substrate was taken out, and the substrate was washed with ethanol and dimethyl sulfoxide, respectively. dry;

(5) 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

(6) The streptavidin is diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 μg/mL, and then the substrate obtained by drying the step (5) is immersed in the above concentration. Placed in a phosphate solution of 10 μg/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;

(7) The specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 μg/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;

(8) The chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(9) As a control group 1, the chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(10) As a control group 2, the chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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. The Triton-X100 aqueous solution with a mass concentration of 0.4% was immersed for 10 minutes, and the 2 μg/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

(11) As a control group 3, the chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(12) As a control group 4, the chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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%. The Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 μg/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

(13) The experimental results show that the capture efficiency of the silica nanowire array chip of the present invention for MCF7 cells is 85.6%, the capture efficiency of prostate cancer cell PC3 cells is 82%, and the capture efficiency of human lymphoid B cell cancer Daudi cells. At 0.02%, the capture efficiency of human lymphatic cancer cell Jurkat cells was 0.02%, and the capture efficiency of uterine cancer cell Hela cells was 0.02%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells and achieve very low non-specific adsorption.

Embodiments of the invention

(2) subjecting the PDMS substrate to hydrophilic treatment;

实施例Example 22

The nanowires of the silica nanowire array chip prepared in this embodiment have a length of 1.5 μm; 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:

(1) In a n-pentanol system, 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;

(2) subjecting the PDMS substrate to hydrophilic treatment;

(3) The 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 μm);

(7) The specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 μg/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.

(8) The chip obtained in the step (7) (preferred nanowire length is 1.5 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(9) As a control group 1, the chip obtained in the step (7) (preferred nanowire length of 1.5 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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.

(10) As a control group 2, the chip obtained in the step (7) (preferred nanowire length of 1.5 μm) 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. The Triton-X100 aqueous solution with a mass concentration of 0.4% was immersed for 10 minutes, and the 2 μg/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

(11) As a control group 3, the chip obtained in the step (7) (preferred nanowire length of 1.5 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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.

(12) As a control group 4, the chip obtained in the step (7) (preferred nanowire length of 1.5 μm) 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%. The Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 μg/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

(13) The experimental results show that the capture efficiency of the silica nanowire array chip of the present invention for MCF7 cells is 87.6%, the capture efficiency of prostate cancer cell PC3 cells is 80.5%, and the capture efficiency of human lymphoid B cell cancer Daudi cells. At 0.02%, the capture efficiency of human lymphoma Jurkat cells was 0.02%, and the capture efficiency of uterine cancer Hela cells was 0.01%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells and achieve very low non-specific adsorption.

实施例Example 33

The nanowire length of the silica nanowire array chip prepared in this embodiment is 10 μm; 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:

(1) In a n-pentanol system, 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: 3:3;

(2) subjecting the PDMS substrate to hydrophilic treatment;

(3) The 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 μm);

(8) Place the chip obtained in step (7) (preferred nanowire length is 10 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(9) As a control group 1, the chip obtained in the step (7) (preferred nanowire length of 10 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(10) As a control group 2, the chip obtained in the step (7) (preferred nanowire length of 10 μm) 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. The Triton-X100 aqueous solution with a mass concentration of 0.4% was immersed for 10 minutes, and the 2 μg/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

(11) As a control group 3, the chip obtained in the step (7) (preferred nanowire length of 10 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(12) As a control group 4, the chip obtained in the step (7) (preferred nanowire length of 10 μm) 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%. The Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 μg/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.

(13) The experimental results show that the capture efficiency of the silica nanowire array chip of the present invention for MCF7 cells is 83.3%, the capture efficiency of prostate cancer cell PC3 cells is 74.5%, and the capture efficiency of human lymphoid B cell cancer Daudi cells. At 0.01%, the capture efficiency of human lymphoma Jurkat cells was 0.01%, and the capture efficiency of uterine cancer Hela cells was 0.01%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells and achieve very low non-specific adsorption.

实施例Example 44

The nanowire length of the silica nanowire array chip prepared in this embodiment is 20 μm; 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:

(1) In a n-pentanol system, 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: 3:3;

(2) subjecting the PDMS substrate to hydrophilic treatment;

(3) The 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 μm);

(8) The chip obtained in the step (7) (preferred nanowire length is 20 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(9) As a control group 1, the chip obtained in the step (7) (preferred nanowire length of 20 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(10) As a control group 2, the chip obtained in the step (7) (preferred nanowire length of 20 μm) 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. The Triton-X100 aqueous solution with a mass concentration of 0.4% was immersed for 10 minutes, and the 2 μg/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

(11) As a control group 3, the chip obtained in the step (7) (preferred nanowire length of 20 μm) 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(12) As a control group 4, the chip obtained in the step (7) (preferred nanowire length of 20 μm) 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%. The Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 μg/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

(13) The experimental results show that the capture efficiency of the silica nanowire array chip of the present invention on MCF7 cells is 85%, the capture efficiency of prostate cancer cell PC3 cells is 81.5%, and the capture efficiency of human lymphoid B cell cancer Daudi cells. At 0.01%, the capture efficiency of human lymphatic cancer cell Jurkat cells was 0.02%, and the capture efficiency of uterine cancer cell Hela cells was 0.01%. These data indicate that this method can achieve efficient and specific capture of circulating tumor cells and achieve very low non-specific adsorption.

实施例Example 55

In the flat silicon dioxide chip prepared in this embodiment, 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:

(1) As a control group, a flat silica substrate is selected;

(2) Mixing (3-mercaptopropyl)trimethoxysilane with ethanol in a volume ratio to prepare a 5% solution of (3-mercaptopropyl)trimethoxysilane in ethanol; at room temperature, silica The substrate is immersed in a solution of 5% (3-mercaptopropyl)trimethoxysilane in ethanol, and placed at room temperature; the substrate is taken out, washed with ethanol and dimethyl sulfoxide, and dried;

(3) 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

(4) The streptavidin was diluted with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10 μg/mL, and then the substrate obtained by drying the step (3) was immersed in the above concentration. Placed in a phosphate solution of 10 μg/mL streptavidin at room temperature; the substrate was removed and washed with phosphate buffer;

(5) The specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 μg/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;

(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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(7) As a control group 1, 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(8) As a control group 2, 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. The Triton-X100 aqueous solution with a mass concentration of 0.4% was immersed for 10 minutes, and the 2 μg/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

(9) As a control group 3, 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. The solution was soaked for 10 minutes in an aqueous solution of Triton-X100 and soaked in a 2 μg/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

(10) As a control group 4, 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%. The Triton-X100 aqueous solution was immersed for 10 minutes, and immersed in a 2 μg/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

(11) The experimental results show that the capture efficiency of the smoothed silica chip on MCF7 cells is 3.0%, the capture efficiency of prostate cancer cell PC3 cells is 3.0%, and the capture efficiency of human lymphoid B cell cancer Daudi cells is 0.01%. The capture efficiency of lymphoma Jurkat cells was 0.01%, and the capture efficiency of uterine cancer Hela cells was 0.01%. These data indicate that this method demonstrates that a flat silica chip is unable to achieve efficient specific capture of circulating tumor cells.

实施例Example 66

The nanowire length of the silica nanowire array chip prepared in this embodiment is 0.3 μm; 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:

(2) subjecting the PDMS substrate to hydrophilic treatment;

(8) The chip obtained in the step (7) (preferred nanowire length is 0.3 μm) 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. The -X100 aqueous solution was immersed for 10 minutes, and 200 μg 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC anti-human CD45), then the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(9) Experimental results show that the number of captures of the silica nanowire array chip of the present invention applied to whole blood-captured cancer cells is 5. These results indicate that the silica nanowire array chip of the present invention has highly efficient and sensitive capture performance and extremely low non-specific adsorption for circulating tumor cells in whole blood. Clinical trial results are significant.

实施例Example 77

The nanowire length of the silica nanowire array chip prepared in this embodiment is 1.5 μm; 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:

(2) subjecting the PDMS substrate to hydrophilic treatment;

(8) The chip obtained in the step (7) (preferred nanowire length is 1.5 μm) 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. The -X100 aqueous solution was immersed for 10 minutes, and 200 μg 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC anti-human CD45), then the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(9) The experimental results show that the number of captures of the silica nanowire array chip of the present invention applied to whole blood-captured cancer cells is 6. These results indicate that the silica nanowire array chip of the present invention has highly efficient and sensitive capture performance and extremely low non-specific adsorption for circulating tumor cells in whole blood. Clinical trial results are significant.

实施例Example 88

The nanowire length of the silica nanowire array chip prepared in this embodiment is 10 μm; 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:

(2) subjecting the PDMS substrate to hydrophilic treatment;

(8) The chip obtained in the step (7) (the preferred length of the nanowire is 10 μm) 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. The -X100 aqueous solution was immersed for 10 minutes, and 200 μg 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC anti-human CD45), then the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(9) Experimental results show that the number of captures of the silica nanowire array chip of the present invention applied to whole blood-captured cancer cells is 6. These results indicate that the silica nanowire array chip of the present invention has highly efficient and sensitive capture performance and extremely low non-specific adsorption for circulating tumor cells in whole blood. Clinical trial results are significant.

实施例Example 99

The nanowire length of the silica nanowire array chip prepared in this embodiment is 20 μm; 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:

(2) subjecting the PDMS substrate to hydrophilic treatment;

(8) The chip obtained in the step (7) (the preferred nanowire length is 20 μm) 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. The -X100 aqueous solution was immersed for 10 minutes, and 200 μg 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC anti-human CD45), then the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(9) Experimental results show that the number of captures of the silica nanowire array chip of the present invention applied to whole blood-captured cancer cells is 8. These results indicate that the silica nanowire array chip of the present invention has highly efficient and sensitive capture performance and extremely low non-specific adsorption for circulating tumor cells in whole blood. Clinical trial results are significant.

实施例Example 1010

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:

(1) As a control group, a flat silica substrate is selected;

(2) Mixing (3-mercaptopropyl)trimethoxysilane and ethanol in a volume ratio to prepare a 5% solution of (3-mercaptopropyl)trimethoxysilane in ethanol; at room temperature, the flattened two The silicon oxide substrate is immersed in the above-mentioned 5% (3-mercaptopropyl)trimethoxysilane in ethanol solution, and placed at room temperature; the substrate is taken out, washed with ethanol and dimethyl sulfoxide, and dried;

(5) The specific antibody on the surface of the circulating tumor cells was diluted with phosphate buffer to a concentration of 10 μg/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;

(6) 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. The -X100 aqueous solution was immersed for 10 minutes, and 200 μg 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC anti-human CD45), then the substrate was stored at 4 degrees C for 12 hours in the dark, and washed with phosphate buffer for 3 times. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(7) Experimental results show that the number of captures of the flat silica chip of the present invention applied to whole blood-captured cancer cells is 1. These results indicate that the flat silica nanochip of the present invention fails to achieve efficient and specific capture of circulating tumor cells in whole blood.

实施例Example 1111

The nanowire length of the silica nanowire array chip prepared in this embodiment is 20 μm; 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:

(2) subjecting the PDMS substrate to hydrophilic treatment;

(4) Mixing (3-mercaptopropyl)trimethoxysilane with ethanol in a volume ratio to prepare a 5% solution of (3-mercaptopropyl)trimethoxysilane in ethanol; at room temperature, silica The nanowire array substrate was immersed in an ethanol solution of the above-mentioned 10% (3-mercaptopropyl)trimethoxysilane in an ethanol solution at room temperature; the substrate was taken out and rinsed with ethanol and dimethyl sulfoxide, respectively. dry;

(8) The chip obtained in the step (7) (the preferred nanowire length is 20 μm) 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. 200 μg of 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. Add 200 μg of fluorescently labeled antibody solution (20 μg/mL initial concentration, antibody anti-cytokeratin PE and FITC respectively) Anti-human CD45), the substrate was then stored at 4 degrees C for 12 hours in the dark, and phosphate buffer was added for 3 washes. Add 10 μg / mL DAPI aqueous solution for 5 minutes, and add phosphate buffer for 3 times 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

(9) Experimental results show that the number of captures of the silica nanowire array chip of the present invention applied to whole blood-captured cancer cells is zero. These results indicate that the silica nanowire array chip of the present invention has highly efficient and sensitive capture performance and extremely low non-specific adsorption for circulating tumor cells in whole blood. Clinical trial results are significant.

Industrial applicability

Claims

A silica nanowire array chip for enrichment and detection of circulating tumor cells in whole blood, characterized in that the silicon dioxide nanowire array chip comprises: based on polydimethylsiloxane, A polydimethylsiloxane interface in situ grown silica nanowire array, and an antibody that specifically recognizes tumor cells is modified on the surface of the silica nanowire array.
The silica nanowire array chip according to claim 1, wherein the silica nanowire array has a line diameter of 70 to 300 nm and a length of 0.3 to 20 μm.
The silica nanowire array chip according to claim 1, wherein the antibody specifically recognizing the tumor cell comprises: a specific antibody that binds to an EpCAM-specific antigen on the surface of the tumor cell, and a common antigen on the surface of the leukocyte. A specific antibody that binds to CD45, an antibody that specifically recognizes a circulating tumor cell marker, or an antibody that specifically recognizes a tumor tissue marker .
The silica nanowire array chip according to claim 1, wherein the antibody specifically recognizing the tumor cell is immobilized in an amount of not less than 0.1 μg/cm 2 on the silica nanowire.
A method for preparing a silica nanowire array chip according to any one of claims 1 to 4, comprising the steps of:

(1) In a n-pentanol system, polyvinylpyrrolidone, water and ethanol are blended and stirred to form a water-in-oil microemulsion, wherein the molar ratio of n-pentanol, water and ethanol is 1:0.01-3:0.01- 3;

(2) hydrophilizing the polydimethylsiloxane;

(3) using the polydimethylsiloxane treated in the step (2) as a substrate, adsorbing the microemulsion prepared in the step (1), and catalyzing it under an atmosphere of ammonia water and tetraethyl orthosilicate at room temperature. a silicon dioxide nanowire array substrate;

(4) An antibody that specifically recognizes a tumor cell is immobilized on the silica nanowire prepared in the step (3).
The method according to claim 5, wherein the step of immobilizing the antibody specifically recognizing the tumor cell on the silica nanowire according to the step (4) comprises the steps of:

(a) mixing (3-mercaptopropyl)trimethoxysilane and ethanol in a volume ratio to prepare an ethanol solution of (3-mercaptopropyl)trimethoxysilane at a concentration of 5-30%; at room temperature, two The silicon oxide nanowire array substrate is immersed in an ethanol solution of (3-mercaptopropyl)trimethoxysilane having a volume concentration of 5-30%, and is allowed to stand at room temperature; the substrate is taken out, and ethanol and dimethyl groups are respectively used. Sulfone rinse, blow dry;

(b) a substrate obtained by formulating N-(4-maleimidobutyryl)succinimide with dimethyl sulfoxide to a concentration of 5 to 30 mM, and drying the step (a) Soak in a solution of N-(4-maleimidobutyryl)succinimide at a concentration of 5-30 mM, and place at room temperature; remove the substrate and buffer with dimethyl sulfoxide and phosphate, respectively. Liquid rinse, blow dry;

(c) diluting streptavidin with phosphate buffer to a phosphate solution of streptavidin at a concentration of 10-50 μg/mL, and then immersing the substrate obtained after drying step (b) The above-mentioned phosphate solution of streptavidin having a concentration of 10-50 μg/mL is placed at room temperature; the substrate is taken out and washed with a phosphate buffer;

(d) diluting the antibody specifically recognizing the tumor cells with a phosphate buffer to a concentration of 10-50 μg/mL, and then dropping it onto the surface of the substrate obtained by washing the phosphate buffer with the step (c), After standing at room temperature, a specific antibody having a surface of a circulating tumor cell immobilized on the surface of the silica nanowire array substrate was obtained.
Use of the silica nanowire chip according to any one of claims 1 to 4 for circulating tumor cell capture in whole blood.