Preparation method and application of co-reactant embedded electrochemical luminescence immunosensor

Abstract

The invention belongs to the technical field of immunoassay and electrochemical luminescence, and particularly discloses a preparation method and application of a co-reactant embedded type electrochemical luminescence immunosensor, and the method comprises the following steps: polishing a glassy carbon electrode into a mirror surface by using alumina polishing powder; dropwise adding the hollow palladium-copper nanocube dispersion liquid to the surface of the electrode A; dropwise adding a human epidermal growth factor receptor 2 captured antibody to the surface of the electrode B; dropwise adding a bovine serum albumin solution to the surface of the electrode C; dropwise adding a human epidermal growth factor receptor 2 antigen solution on the surface of the electrode D; the co-reactant embedded type electrochemical luminescence immunosensor is obtained. According to the preparation method and the application of the co-reactant embedded type electrochemical luminescence immunosensor, the co-reactant embedded type electrochemical luminescence immunosensor obtained by utilizing electrochemical luminescence and immunoassay technologies realizes rapid and sensitive detection of a low-abundance human epidermal growth factor receptor 2; the method has a good linear range and a low detection line.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay and electrochemiluminescence technology, specifically relating to a method for preparing and applying a co-reactant-embedded electrochemiluminescence immunosensor. Background Technology

[0002] Breast cancer is one of the leading malignant tumors threatening women's health, often referred to as the "pink killer" of women. Early detection and diagnosis are crucial for improving the survival rate of breast cancer patients. However, currently used clinical diagnostic methods (such as imaging examinations, tumor biopsies, and genetic testing) rely on invasive biopsies and suffer from drawbacks such as complex procedures, long processing times, patient discomfort, and poor compliance. Human epidermal growth factor receptor 2 (HGF-2) is an important molecular marker in breast cancer: approximately 20% of breast cancer patients have HGF-2 overexpression in their tumor cells, which typically indicates greater invasiveness and a poorer prognosis. Therefore, accurate detection of HGF-2 levels is essential for early screening, subtyping and treatment selection, and monitoring of breast cancer treatment efficacy.

[0003] Electrochemiluminescence sensors, which integrate electrochemistry and chemiluminescence technologies, induce redox reactions through electrochemical regulation to generate excited-state substances that emit light. They offer advantages such as high sensitivity, low background interference, and a wide dynamic range, making them an important tool in biomedicine, environmental analysis, and other fields. Therefore, developing a rapid, sensitive, and non-invasive new technology for detecting human epidermal growth factor receptor 2 (HGF2) has become a pressing challenge in the early diagnosis of breast cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a co-reactant-embedded electrochemiluminescence immunosensor and its application. This method utilizes electrochemiluminescence and immunoassay techniques to prepare a co-reactant-embedded electrochemiluminescence immunosensor. This sensor achieves rapid and sensitive detection of low-abundance human epidermal growth factor receptor 2, and has a good linear range and a low detection limit.

[0005] To achieve the above objectives, the present invention provides a method for preparing a co-reactant-intercalated electrochemiluminescence immunosensor, comprising the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion. Take 6.0 µL of 1~2 mg / mL hollow palladium-copper nanocube dispersion and drop it onto the surface of electrode A to obtain electrode B. Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at a concentration of 4.0~8.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of bovine serum albumin solution with a mass fraction of 1~2% and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution with pH=7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0006] Preferably, in step S2, the preparation of the hollow palladium-copper nanocube dispersion specifically involves: (1) Preparation of cuprous oxide nanocube dispersion; (2) Preparation of hollow palladium-copper nanocube powder; (3) Preparation of hollow palladium-copper nanocube dispersion; Take 0.35~5mg of hollow palladium copper nanocube powder and add it to 1mL of ultrapure water. Use an ultrasonic cleaner with a power of 200-300W and a frequency of 40kHz, and continue to sonicate for 30min at room temperature to obtain a hollow palladium copper nanocube dispersion.

[0007] Preferably, the preparation of the cuprous oxide nanocube dispersion specifically involves: A. Dissolve 0.05~0.1 mmol of copper chloride in 36.0 mL of ultrapure water. Add 0.03~0.12 g of sodium polyacrylate while stirring with a magnetic stirrer at a speed of 1000±100 rpm. Maintain this stirring speed for 10 min to form a uniform light blue solution. B. Add 0.2-0.8 mmol of sodium hydroxide and 0.2-0.7 mmol of ascorbic acid dropwise to the light blue solution. Hold the container and slowly tilt and rotate it at a speed of about 15 times per minute along the central axis of the container at an angle of about 30 degrees for 1-2 minutes until the solution is mixed evenly. Stir continuously at 40℃ for 30 minutes and centrifuge to obtain a cuprous oxide nanocube dispersion.

[0008] Preferably, the hollow palladium-copper nanocube powder is prepared by: A. Add 0.02~0.08 mmol potassium tetrachloropalladate and 0.003 g sodium polyacrylate sequentially to 10 mL cuprous oxide nanocube dispersion to obtain a mixed solution; B. Using a micropipette, add 0.1 mol / L sodium hydroxide solution dropwise to the mixture, gently shaking after each addition, until the pH of the mixture is 7-9; then transfer the mixture to a stainless steel container lined with polytetrafluoroethylene and heat at 100-160℃ for 6 hours to carry out the reaction. C. After the reaction is complete, cool to 25°C, adjust the pH of the mixed solution to 1-3 with nitric acid, and stir for 40 minutes to remove unreacted cuprous oxide nanocubes to obtain hollow palladium copper nanocubes. D. The hollow palladium-copper nanocubes were centrifuged at 10,000 rpm for 3 min, washed with ethanol several times, and dried in a vacuum oven at 50 ℃ for 12 h to obtain hollow palladium-copper nanocube powder.

[0009] Multifunctional hollow palladium-copper nanocubes were used as the substrate material. On the one hand, the palladium-copper alloy can act as a co-reaction promoter, significantly catalyzing the reaction between carbazide and dissolved oxygen. By lowering the activation energy, it promotes the accelerated decomposition of carbazide into more reactive intermediates, highly oxidizing free radicals, thus accelerating the generation of excited states in the covalent organic framework and amplifying the electrochemiluminescence signal. On the other hand, the palladium-nitrogen bond structure allows for the immobilization of a large number of human epidermal growth factor receptor 2 antibodies, providing sufficient recognition sites for subsequent detection and further enhancing the sensor's sensitivity.

[0010] Preferably, step S6 specifically includes: Step S61: Prepare a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody; Step S62: Drop 6 µL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 30-40 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor.

[0011] Preferably, step S61 specifically includes: (1) Preparation of co-reactant-intercalated covalent organic framework powder; (2) Preparation of a dispersion of human epidermal growth factor receptor 2 detection antibody with co-reactant intercalation type covalent organic framework labeling.

[0012] Preferably, the preparation of co-reactant-intercalated covalent organic framework powder specifically involves: A. In a 10 mL heat-resistant glass test tube, add 0.02–0.35 mmol of 1,3,6,8-tetra(4-carboxymethylphenyl)perylene, 0.05 mmol of carbazide, 2 mL of a 19:1 mixture of o-dichlorobenzene and n-butanol, and 0.2 mL of acetic acid solution to obtain a mixture. B. Place the mixture in an ultrasonic cleaner with a power of 200W and a frequency of 40kHz and sonicate for 10 minutes to ensure thorough mixing and dispersion; then, place the mixture in a test tube for degassing and freeze-vacuum-thaw. First, immerse the test tube in liquid nitrogen at -196°C to completely freeze the mixture; then connect it to a high vacuum system to evacuate and maintain the vacuum for 5 minutes; the vacuum level of the high vacuum system should be ≤10Pa; finally, remove the cold source and allow the test tube to thaw naturally at room temperature or in warm water; repeat the freezing-evacuation-thawing process 3 times. After degassing, the test tube mouths were sealed using a vacuum sealing machine, and then placed in an oven and heated continuously at 100~180℃ for 7 days to obtain the reaction mixture. C. Cool the reaction mixture to room temperature, collect the pale yellow precipitate by centrifugation, and wash it four times with water and tetrahydrofuran / chloroform; finally, dry it under vacuum at 80°C for 12 hours to obtain a pale yellow powder, which is the co-reactant intercalated covalent organic framework powder.

[0013] Preferably, the preparation of the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion specifically comprises: A. Take 0.005g of co-reactant intercalated covalent organic framework powder and disperse it in 2.0mL of phosphate buffer solution with pH=6.98. Add 100~500μL of human epidermal growth factor receptor 2 detection antibody solution and place it in a constant temperature shaking incubator at 4℃ for 8~12h. B. Centrifuge at 1500 rpm for 0.5~2.0 min to obtain the lower precipitate; C. Add 1.0 mL of phosphate buffer solution (pH=6.98) to the lower precipitate and centrifuge and wash once. Redisperse the lower precipitate in 1.0 mL of phosphate buffer solution (pH=6.98) to obtain a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody. Store at 4°C.

[0014] Using 1,3,6,8-tetra(4-carboxyphenyl)pyrene as the luminescent unit and introducing carbazide as a co-reactant via an aldehyde-amine condensation reaction, a co-reactant-intercalated covalent organic framework electrochemiluminescent device was designed. The covalent integration between the co-reactant and the luminescent unit significantly shortens the charge transfer path, avoiding the efficiency losses caused by long-distance mass transfer and short radical lifetimes in traditional co-reactant systems, thus achieving a high-intensity electrochemiluminescence signal.

[0015] The present invention also provides a method for preparing a co-reactant-embedded electrochemiluminescence immunosensor and the application of the prepared co-reactant-embedded electrochemiluminescence immunosensor in the detection of human epidermal growth factor receptor 2.

[0016] Preferably, the detection of human epidermal growth factor receptor 2 specifically involves: Step S1: Use an electrochemical workstation with a three-electrode system for testing. The silver / silver chloride electrode is the reference electrode, the platinum wire electrode is the auxiliary electrode, and the prepared co-reactant embedded electrochemiluminescence immunosensor is the working electrode. The high voltage of the photomultiplier tube is set to 780V, the scanning potential is 0~1.8V, and the scanning rate is 0.1V / s. Step S2: In 10 mL of phosphate buffer solution at pH 7.38, the intensity of the electrochemiluminescence signal generated by different concentrations of analyte antigen was detected by an electrochemiluminescence system, and a working curve was plotted. Step S3: Replace the human epidermal growth factor receptor 2 antigen solution with the test sample solution for detection; Step S4: Using the working curve method, determine the human epidermal growth factor receptor 2 in the sample to be tested.

[0017] Using multifunctional hollow palladium-copper nanocubes as the substrate material, co-reactant-embedded covalent organic frameworks as the luminescent material, and human epidermal growth factor receptor 2 as the target analyte, a sensitive and rapid self-enhanced electrochemiluminescence immunosensor was constructed using layer-by-layer self-assembly technology. This sensor exhibits excellent analytical performance.

[0018] The present invention employs the above-mentioned method for preparing and applying a co-reactant-embedded electrochemiluminescence immunosensor, with the following beneficial effects: (1) This invention successfully synthesizes a co-reactant-embedded covalent organic framework luminescent material, which covalently integrates the luminescent unit and the co-reactant into the same structure, significantly improving the intermolecular charge transfer efficiency. Compared with the traditional ternary system, no other exogenous co-reactant needs to be added during the testing process, and a short-range activation reaction can be directly carried out to promote the generation of excited states, thus solving the problem of low electrochemiluminescence efficiency caused by slow intermolecular charge transfer between the co-reactant and the luminescent body.

[0019] (2) In this invention, multifunctional hollow palladium-copper nanocubes are synthesized and used as substrate materials. Their large specific surface area and abundant palladium sites not only help stabilize the binding of antibodies and enhance immune recognition capabilities, but also make full use of the catalytic active centers of the palladium-copper bimetallic compound to promote the co-reactant carbonyl hydrazine to generate more highly active free radicals, accelerate the generation of excited states of pyrene-based luminescent units, thereby achieving efficient signal amplification and improving the sensitivity of biosensors.

[0020] (3) The co-reactant-embedded covalent organic frame electrochemiluminescence sensor prepared by the present invention has a linear range of 0.005~500 ng / mL for human epidermal growth factor receptor 2 and a detection limit of 5.98 pg / mL. It also performs well in terms of stability, reproducibility and selectivity, and can realize sensitive and rapid detection of the target human epidermal growth factor receptor 2.

[0021] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0022] The technical solution of the present invention will be further described below through embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Example 1 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; (1) Preparation of cuprous oxide nanocube dispersion; A. Dissolve 0.05 mmol of copper chloride in 36.0 mL of ultrapure water. Add 0.03 g of sodium polyacrylate while stirring with a magnetic stirrer at a speed of 1000 ± 100 rpm. Maintain this stirring speed for 10 min to form a uniform light blue solution. B. Add 0.2 mmol of sodium hydroxide and 0.2 mmol of ascorbic acid dropwise to the light blue solution. Hold the container and slowly tilt and rotate it at a speed of about 15 times per minute along the central axis of the container at an angle of about 30 degrees for 1-2 minutes until the solution is mixed evenly. Stir continuously at 40°C for 30 minutes and centrifuge to obtain a cuprous oxide nanocube dispersion.

[0025] (2) Preparation of hollow palladium-copper nanocube powder; A. Add 0.02 mmol potassium tetrachloropalladate and 0.003 g sodium polyacrylate sequentially to 10 mL cuprous oxide nanocube dispersion to obtain a mixed solution; B. Using a micropipette, add 0.1 mol / L sodium hydroxide solution dropwise to the mixture, gently shaking after each addition, until the pH of the mixture is adjusted to 7; then transfer the mixture to a stainless steel container lined with polytetrafluoroethylene and heat at 100°C for 6 hours to carry out the reaction. C. After the reaction is complete, cool to 25°C, adjust the pH of the mixed solution to 1 with nitric acid, and stir for 40 min to remove unreacted cuprous oxide nanocubes to obtain hollow palladium copper nanocubes. D. The hollow palladium-copper nanocubes were centrifuged at 10,000 rpm for 3 min, washed 4 times with ethanol, and stored in a vacuum oven at 50 °C for 12 h to obtain hollow palladium-copper nanocube powder.

[0026] (3) Preparation of hollow palladium-copper nanocube dispersion; 0.35 mg of hollow palladium-copper nanocube powder was added to 1 mL of ultrapure water and ultrasonically cleaned for 30 min at room temperature using an ultrasonic cleaner with a power of 250 W and a frequency of 40 kHz to obtain a hollow palladium-copper nanocube dispersion.

[0027] 6.0 µL of a 1 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 4.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 1% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0028] Step S61: Prepare a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody; (1) Preparation of co-reactant-intercalated covalent organic framework powder; A. In a 10 mL heat-resistant glass test tube, add 0.02 mmol of 1,3,6,8-tetra(4-carboxymethylphenyl)perylene, 0.05 mmol of carbazide, 2 mL of o-dichlorobenzene / n-butanol mixture (volume ratio 19:1), and 0.2 mL of acetic acid solution to obtain a mixture. B. Place the mixture in an ultrasonic cleaner with a power of 200W and a frequency of 40kHz and sonicate for 10 minutes to ensure thorough mixing and dispersion; then, place the mixture in a test tube for degassing and freeze-vacuum-thaw. First, immerse the test tube in liquid nitrogen at -196°C to completely freeze the mixture; then connect it to a high vacuum system to evacuate and maintain the vacuum for 5 minutes; the vacuum level of the high vacuum system should be ≤10Pa; finally, remove the cold source and allow the test tube to thaw naturally at room temperature or in warm water; repeat the freezing-evacuation-thawing process 3 times. After degassing, the test tube mouths were sealed using a vacuum sealing machine, and then placed in an oven and heated at 100°C for 7 days to obtain the reaction mixture. C. Cool the reaction mixture to room temperature, collect the pale yellow precipitate by centrifugation, and wash it four times with water and tetrahydrofuran / chloroform; finally, dry it under vacuum at 80°C for 12 hours to obtain a pale yellow powder, which is the co-reactant intercalated covalent organic framework powder.

[0029] (2) Preparation of a dispersion of human epidermal growth factor receptor 2 detection antibody with co-reactant intercalation type covalent organic framework labeling.

[0030] A. Take 0.005g of co-reactant intercalated covalent organic framework powder and disperse it in 2.0mL of phosphate buffer solution with pH=6.98. Add 100μL of human epidermal growth factor receptor 2 detection antibody solution and place it in a constant temperature shaking incubator at 4℃ for 8h of shaking incubation. B. Centrifuge at 1500 rpm for 0.5 min to obtain the lower precipitate; C. Add 1.0 mL of phosphate buffer solution (pH=6.98) to the lower precipitate and centrifuge and wash once. Redisperse the lower precipitate in 1.0 mL of phosphate buffer solution (pH=6.98) to obtain a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody. Store at 4°C.

[0031] Step S62: Drop 6µL of 0.3mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 30 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0032] Example 2 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; (1) Preparation of cuprous oxide nanocube dispersion; A. Dissolve 0.07 mmol of copper chloride in 36.0 mL of ultrapure water. Add 0.08 g of sodium polyacrylate while stirring with a magnetic stirrer at a speed of 1000 ± 100 rpm. Maintain this stirring speed for 10 min to form a uniform light blue solution. B. Add 0.55 mmol of sodium hydroxide and 0.45 mmol of ascorbic acid dropwise to the light blue solution. Hold the container and slowly tilt and rotate it at a speed of about 15 times per minute along the central axis of the container at an angle of about 30 degrees for 1-2 minutes until the solution is mixed evenly. Stir continuously at 40°C for 30 minutes and centrifuge to obtain a cuprous oxide nanocube dispersion.

[0033] (2) Preparation of hollow palladium-copper nanocube powder; A. Add 0.045 mmol potassium tetrachloropalladium and 0.003 g sodium polyacrylate sequentially to 10 mL cuprous oxide nanocube dispersion to obtain a mixed solution; B. Using a micropipette, add 0.1 mol / L sodium hydroxide solution dropwise to the mixture, gently shaking after each addition, until the pH of the mixture is adjusted to 8; then transfer the mixture to a stainless steel container lined with polytetrafluoroethylene and heat at 130°C for 6 hours to carry out the reaction. C. After the reaction is complete, cool to 25°C, adjust the pH of the mixed solution to 2 with nitric acid, and stir for 40 min to remove unreacted cuprous oxide nanocubes to obtain hollow palladium copper nanocubes. D. The hollow palladium-copper nanocubes were centrifuged at 10,000 rpm for 3 min, washed 4 times with ethanol, and stored in a vacuum oven at 50 °C for 12 h to obtain hollow palladium-copper nanocube powder.

[0034] (3) Preparation of hollow palladium-copper nanocube dispersion; 2.5 mg of hollow palladium-copper nanocube powder was added to 1 mL of ultrapure water and ultrasonically cleaned for 30 min at room temperature using an ultrasonic cleaner with a power of 250 W and a frequency of 40 kHz to obtain a hollow palladium-copper nanocube dispersion.

[0035] 6.0 µL of a 1 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 4.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 1% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0036] Step S61: Prepare a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody; (1) Preparation of co-reactant-intercalated covalent organic framework powder; A. In a 10 mL heat-resistant glass test tube, add 0.15 mmol of 1,3,6,8-tetra(4-carboxymethylphenyl)perylene, 0.05 mmol of carbazide, 2 mL of o-dichlorobenzene / n-butanol mixture (volume ratio 19:1), and 0.2 mL of acetic acid solution to obtain a mixture. B. Place the mixture in an ultrasonic cleaner with a power of 200W and a frequency of 40kHz and sonicate for 10 minutes to ensure thorough mixing and dispersion; then, place the mixture in a test tube for degassing and freeze-vacuum-thaw. First, immerse the test tube in liquid nitrogen at -196°C to completely freeze the mixture; then connect it to a high vacuum system to evacuate and maintain the vacuum for 5 minutes; the vacuum level of the high vacuum system should be ≤10Pa; finally, remove the cold source and allow the test tube to thaw naturally at room temperature or in warm water; repeat the freezing-evacuation-thawing process 3 times. After degassing, the test tube mouths were sealed using a vacuum sealing machine, and then placed in an oven and heated at 140°C for 7 days to obtain the reaction mixture. C. Cool the reaction mixture to room temperature, collect the pale yellow precipitate by centrifugation, and wash it four times with water and tetrahydrofuran / chloroform; finally, dry it under vacuum at 80°C for 12 hours to obtain a pale yellow powder, which is the co-reactant intercalated covalent organic framework powder.

[0037] (2) Preparation of a dispersion of human epidermal growth factor receptor 2 detection antibody with co-reactant intercalation type covalent organic framework labeling.

[0038] A. Take 0.005g of co-reactant intercalated covalent organic framework powder and disperse it in 2.0mL of phosphate buffer solution with pH=6.98. Add 250μL of human epidermal growth factor receptor 2 detection antibody solution and place it in a constant temperature shaking incubator at 4℃ for 10h. B. Centrifuge at 1500 rpm for 1.0 min to obtain the lower precipitate; C. Add 1.0 mL of phosphate buffer solution (pH=6.98) to the lower precipitate and centrifuge and wash once. Redisperse the lower precipitate in 1.0 mL of phosphate buffer solution (pH=6.98) to obtain a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody. Store at 4°C.

[0039] Step S62: Drop 6µL of 0.3mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 30 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0040] Example 3 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; (1) Preparation of cuprous oxide nanocube dispersion; A. Dissolve 0.1 mmol of copper chloride in 36.0 mL of ultrapure water. Add 0.12 g of sodium polyacrylate while stirring with a magnetic stirrer at a speed of 1000 ± 100 rpm. Maintain this stirring speed for 10 min to form a uniform light blue solution. B. Add 0.8 mmol of sodium hydroxide and 0.7 mmol of ascorbic acid dropwise to the light blue solution. Hold the container and slowly tilt and rotate it at a speed of about 15 times per minute along the central axis of the container at an angle of about 30 degrees for 1-2 minutes until the solution is mixed evenly. Stir continuously at 40°C for 30 minutes and centrifuge to obtain a cuprous oxide nanocube dispersion.

[0041] (2) Preparation of hollow palladium-copper nanocube powder; A. Add 0.08 mmol potassium tetrachloropalladium and 0.003 g sodium polyacrylate sequentially to 10 mL cuprous oxide nanocube dispersion to obtain a mixed solution; B. Using a micropipette, add 0.1 mol / L sodium hydroxide solution dropwise to the mixture, gently shaking after each addition, until the pH of the mixture is adjusted to 8; then transfer the mixture to a stainless steel container lined with polytetrafluoroethylene and heat at 160°C for 6 hours to carry out the reaction. C. After the reaction is complete, cool to 25°C, adjust the pH of the mixed solution to 3 with nitric acid, and stir for 40 min to remove unreacted cuprous oxide nanocubes to obtain hollow palladium copper nanocubes. D. The hollow palladium-copper nanocubes were centrifuged at 10,000 rpm for 3 min, washed 4 times with ethanol, and stored in a vacuum oven at 50 °C for 12 h to obtain hollow palladium-copper nanocube powder.

[0042] (3) Preparation of hollow palladium-copper nanocube dispersion; Take 5 mg of hollow palladium copper nanocube powder and add it to 1 mL of ultrapure water. Use an ultrasonic cleaner with a power of 250 W and a frequency of 40 kHz to continuously sonicate for 30 min at room temperature to obtain a hollow palladium copper nanocube dispersion.

[0043] 6.0 µL of a 1 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 4.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 1% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0044] Step S61: Prepare a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody; (1) Preparation of co-reactant-intercalated covalent organic framework powder; A. In a 10 mL heat-resistant glass test tube, add 0.35 mmol of 1,3,6,8-tetra(4-carboxymethylphenyl)perylene, 0.05 mmol of carbazide, 2 mL of o-dichlorobenzene / n-butanol mixture (volume ratio 19:1), and 0.2 mL of acetic acid solution to obtain a mixture. B. Place the mixture in an ultrasonic cleaner with a power of 200W and a frequency of 40kHz and sonicate for 10 minutes to ensure thorough mixing and dispersion; then, place the mixture in a test tube for degassing and freeze-vacuum-thaw. First, immerse the test tube in liquid nitrogen at -196°C to completely freeze the mixture; then connect it to a high vacuum system to evacuate and maintain the vacuum for 5 minutes; the vacuum level of the high vacuum system should be ≤10Pa; finally, remove the cold source and allow the test tube to thaw naturally at room temperature or in warm water; repeat the freezing-evacuation-thawing process 3 times. After degassing, the test tube mouths were sealed using a vacuum sealing machine, and then placed in an oven and heated at 180°C for 7 days to obtain the reaction mixture. C. Cool the reaction mixture to room temperature, collect the pale yellow precipitate by centrifugation, and wash it four times with water and tetrahydrofuran / chloroform; finally, dry it under vacuum at 80°C for 12 hours to obtain a pale yellow powder, which is the co-reactant intercalated covalent organic framework powder.

[0045] (2) Preparation of a dispersion of human epidermal growth factor receptor 2 detection antibody with co-reactant intercalation type covalent organic framework labeling.

[0046] A. Take 0.005g of co-reactant intercalated covalent organic framework powder and disperse it in 2.0mL of phosphate buffer solution with pH=6.98. Add 500μL of human epidermal growth factor receptor 2 detection antibody solution and place it in a constant temperature shaking incubator at 4℃ for 12h. B. Centrifuge at 1500 rpm for 2.0 min to obtain the lower precipitate; C. Add 1.0 mL of phosphate buffer solution (pH=6.98) to the lower precipitate and centrifuge and wash once. Redisperse the lower precipitate in 1.0 mL of phosphate buffer solution (pH=6.98) to obtain a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody. Store at 4°C.

[0047] Step S62: Drop 6µL of 0.3mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 30 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0048] Example 4 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as those in Example 1. 6.0 µL of a 1.5 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 6.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of bovine serum albumin solution with a mass fraction of 1.5% and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution with pH=7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0049] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 1; Step S62: Drop 6µL of 1.5mg / mL co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 35 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0050] Example 5 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as in Example 2. 6.0 µL of a 1.5 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 6.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of bovine serum albumin solution with a mass fraction of 1.5% and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution with pH=7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0051] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 2; Step S62: Drop 6µL of 1.5mg / mL co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 35 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0052] Example 6 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as those in Example 3. 6.0 µL of a 1.5 mg / mL hollow palladium copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 6.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of bovine serum albumin solution with a mass fraction of 1.5% and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution with pH=7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0053] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 3; Step S62: Drop 6µL of 1.5mg / mL co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 35 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0054] Example 7 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as those in Example 1. 6.0 µL of a 2 mg / mL hollow palladium-copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 8.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 2% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0055] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 1; Step S62: Drop 6µL of 3.0mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4℃ refrigerator for 40min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0056] Example 8 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as in Example 2. 6.0 µL of a 2 mg / mL hollow palladium-copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 8.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 2% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0057] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 2; Step S62: Drop 6µL of 3.0mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4℃ refrigerator for 40min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0058] Example 9 A method for preparing a co-reactant-embedded electrochemiluminescence immunosensor includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion; The preparation processes and parameters for (1) preparing cuprous oxide nanocube dispersion, (2) preparing hollow palladium copper nanocube powder, and (3) preparing hollow palladium copper nanocube dispersion are the same as those in Example 3. 6.0 µL of a 2 mg / mL hollow palladium-copper nanocube dispersion was dropped onto the surface of electrode A to obtain electrode B; Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at 8.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of 2% bovine serum albumin solution and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution at pH 7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

[0059] Step S61: The preparation process and parameters for the co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion are the same as in Example 3; Step S62: Drop 6µL of 3.0mg / mL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4℃ refrigerator for 40min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor, and store it in a 4°C refrigerator for subsequent use.

[0060] Experimental Example The co-reactant-embedded electrochemiluminescence immunosensors prepared in Examples 1-9 were applied to the detection of human epidermal growth factor receptor 2, specifically as follows: Step S1: The electrochemical workstation was used to perform the test in a three-electrode system. The silver / silver chloride electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the co-reactant embedded electrochemiluminescence immunosensors prepared in Examples 1 to 9 were used as the working electrodes. The high voltage of the photomultiplier tube was set to 780V, the scanning potential was 0~1.8V, and the scanning rate was 0.1V / s. Step S2: In 10 mL of phosphate buffer solution at pH 7.38, the intensity of the electrochemiluminescence signal generated by different concentrations of analyte antigen was detected by an electrochemiluminescence system, and a working curve was plotted. Step S3: Replace the human epidermal growth factor receptor 2 antigen solution with the test sample solution for detection; Step S4: Using the working curve method, determine the human epidermal growth factor receptor 2 in the sample to be tested.

[0061] The co-reactant-embedded electrochemiluminescence immunosensor used in this experiment was applied to the detection of human epidermal growth factor receptor 2. The linear range of the working curves was 0.005~500 ng / mL, and the detection limit was 5.98 pg / mL.

[0062] Therefore, the present invention adopts the above-mentioned preparation method and application of a co-reactant-embedded electrochemiluminescence immunosensor. The preparation method utilizes electrochemiluminescence and immunoassay technology to prepare a co-reactant-embedded electrochemiluminescence immunosensor. This sensor achieves rapid and sensitive detection of low-abundance human epidermal growth factor receptor 2, and has a good linear range and a low detection limit.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a co-reactant-intercalated electrochemiluminescence immunosensor, characterized in that, Includes the following steps: Step S1: Polish the glassy carbon electrode with a diameter of 4.0 mm to a mirror surface with alumina polishing powder, and ultrasonically clean it in anhydrous ethanol for 30 seconds to remove residual polishing powder on the surface, thus obtaining electrode A; Step S2: Prepare hollow palladium-copper nanocube dispersion. Take 6.0 µL of 1~2 mg / mL hollow palladium-copper nanocube dispersion and drop it onto the surface of electrode A to obtain electrode B. Step S3: Take 6.0 µL of human epidermal growth factor receptor 2 capture antibody at a concentration of 4.0~8.0 µg / mL and drop it onto the surface of electrode B. Store it in a refrigerator at 4°C until it is dry. Then wash it with ultrapure water to obtain electrode C. Step S4: Take 3.0 µL of bovine serum albumin solution with a mass fraction of 1~2% and add it to the surface of electrode C to block non-specific active sites on the surface of electrode C. Rinse the surface of electrode C with phosphate buffer solution with pH=7.38 and store it in a refrigerator at 4°C until it is dried to obtain electrode D. Step S5: Add 6.0 µL of several different concentrations of human epidermal growth factor receptor 2 antigen solution to the surface of electrode D, rinse the surface of electrode D with phosphate buffer solution at pH 7.38, and store in a refrigerator at 4°C until dried to obtain several types of electrode E. Step S6: Prepare a co-reactant-embedded electrochemiluminescence immunosensor and store it in a 4°C refrigerator for later use.

2. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 1, characterized in that, In step S2, the preparation of the hollow palladium-copper nanocube dispersion specifically involves: (1) Preparation of cuprous oxide nanocube dispersion; (2) Preparation of hollow palladium-copper nanocube powder; (3) Preparation of hollow palladium-copper nanocube dispersion; Take 0.35~5mg of hollow palladium copper nanocube powder and add it to 1mL of ultrapure water. Use an ultrasonic cleaner with a power of 200-300W and a frequency of 40kHz, and continue to sonicate for 30min at room temperature to obtain a hollow palladium copper nanocube dispersion.

3. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 2, characterized in that, The specific steps for preparing the cuprous oxide nanocube dispersion are as follows: A. Dissolve 0.05~0.1 mmol of copper chloride in 36.0 mL of ultrapure water. Add 0.03~0.12 g of sodium polyacrylate while stirring with a magnetic stirrer at a speed of 1000±100 rpm. Maintain this stirring speed for 10 min to form a uniform light blue solution. B. Add 0.2-0.8 mmol of sodium hydroxide and 0.2-0.7 mmol of ascorbic acid dropwise to the light blue solution. Hold the container and slowly tilt and rotate it at a speed of about 15 times per minute along the central axis of the container at an angle of about 30 degrees for 1-2 minutes until the solution is mixed evenly. Stir continuously at 40℃ for 30 minutes and centrifuge to obtain a cuprous oxide nanocube dispersion.

4. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 3, characterized in that, The specific steps for preparing hollow palladium-copper nanocube powder are as follows: A. Add 0.02~0.08 mmol potassium tetrachloropalladate and 0.003 g sodium polyacrylate sequentially to 10 mL cuprous oxide nanocube dispersion to obtain a mixed solution; B. Using a micropipette, add 0.1 mol / L sodium hydroxide solution dropwise to the mixture, shaking well after each addition, until the pH of the mixture is 7-9; then transfer the mixture to a stainless steel container lined with polytetrafluoroethylene and heat at 100-160℃ for 6 hours to carry out the reaction. C. After the reaction is complete, cool to 25°C, adjust the pH of the mixed solution to 1-3 with nitric acid, and stir for 40 minutes to remove unreacted cuprous oxide nanocubes to obtain hollow palladium copper nanocubes. D. The hollow palladium-copper nanocubes were centrifuged at 10,000 rpm for 3 min, washed 4 times with ethanol, and dried in a vacuum oven at 50 °C for 12 h to obtain hollow palladium-copper nanocube powder.

5. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 1, characterized in that, Step S6 is as follows: Step S61: Prepare a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody; Step S62: Drop 6 µL of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody dispersion onto electrode E and let it stand in a 4°C refrigerator for 30-40 min. Step S63: Rinse with phosphate buffer solution at pH 7.38 to obtain the co-reactant-embedded electrochemiluminescence immunosensor.

6. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 5, characterized in that, Step S61 is as follows: (1) Preparation of co-reactant-intercalated covalent organic framework powder; (2) Preparation of a dispersion of human epidermal growth factor receptor 2 detection antibody with co-reactant intercalation type covalent organic framework labeling.

7. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 6, characterized in that, The preparation of co-reactant-intercalated covalent organic framework powders specifically involves: A. In a 10 mL heat-resistant glass test tube, add 0.02–0.35 mmol of 1,3,6,8-tetra(4-carboxymethylphenyl)perylene, 0.05 mmol of carbazide, 2 mL of a 19:1 mixture of o-dichlorobenzene and n-butanol, and 0.2 mL of acetic acid solution to obtain a mixture. B. Place the mixture in an ultrasonic cleaner with a power of 200W and a frequency of 40kHz and sonicate for 10 minutes to ensure thorough mixing and dispersion; then, place the mixture in a test tube for degassing and freeze-vacuum-thaw. First, immerse the test tube in liquid nitrogen at -196°C to completely freeze the mixture; then connect it to a high vacuum system to evacuate and maintain the vacuum for 5 minutes; the vacuum level of the high vacuum system should be ≤10Pa; finally, remove the cold source and allow the test tube to thaw naturally at room temperature or in warm water; repeat the freezing-evacuation-thawing process 3 times. After degassing, the test tube mouths were sealed using a vacuum sealing machine, and then placed in an oven and heated continuously at 100~180℃ for 7 days to obtain the reaction mixture. C. Cool the reaction mixture to room temperature, collect the pale yellow precipitate by centrifugation, and wash it four times with water and tetrahydrofuran / chloroform; finally, dry it under vacuum at 80°C for 12 hours to obtain a pale yellow powder, which is the co-reactant intercalated covalent organic framework powder.

8. The method for preparing a co-reactant-embedded electrochemiluminescence immunosensor according to claim 7, characterized in that, The preparation of a co-reactant-intercalated, covalently organic framework-labeled human epidermal growth factor receptor 2 (HGF2) antibody dispersion is as follows: A. Take 0.005g of co-reactant intercalated covalent organic framework powder and disperse it in 2.0mL of phosphate buffer solution with pH=6.

98. Add 100~500μL of human epidermal growth factor receptor 2 detection antibody solution and place it in a constant temperature shaking incubator at 4℃ for 8~12h. B. Centrifuge at 1500 rpm for 0.5~2.0 min to obtain the lower precipitate; C. Add 1.0 mL of phosphate buffer solution (pH=6.98) to the lower precipitate and centrifuge and wash once. Redisperse the lower precipitate in 1.0 mL of phosphate buffer solution (pH=6.98) to obtain a dispersion of co-reactant-intercalated covalent organic framework-labeled human epidermal growth factor receptor 2 detection antibody. Store at 4°C.

9. The application of the co-reactant-intercalated electrochemiluminescence immunosensor prepared by the method of any one of claims 1-8 in the detection of human epidermal growth factor receptor 2.

10. The application according to claim 9, characterized in that, The specific detection of human epidermal growth factor receptor 2 is as follows: Step S1: Use an electrochemical workstation with a three-electrode system for testing. The silver / silver chloride electrode is the reference electrode, the platinum wire electrode is the auxiliary electrode, and the prepared co-reactant embedded electrochemiluminescence immunosensor is the working electrode. The high voltage of the photomultiplier tube is set to 780V, the scanning potential is 0~1.8V, and the scanning rate is 0.1V / s. Step S2: In 10 mL of phosphate buffer solution at pH 7.38, the intensity of the electrochemiluminescence signal generated by different concentrations of analyte antigen was detected by an electrochemiluminescence system, and a working curve was plotted. Step S3: Replace the human epidermal growth factor receptor 2 antigen solution with the test sample solution for detection; Step S4: Using the working curve method, determine the human epidermal growth factor receptor 2 in the sample to be tested.