Preparation of electrochemical sensor based on cobalt-covalent organic framework coupling material

Abstract

The invention belongs to the technical field of novel nano composite materials, signal amplification and biosensing, and provides a preparation method of an electrochemical sensor based on a cobalt-covalent organic framework coupling material. On the basis of an electrochemical-chemical-chemical (ECC) redox cycle amplification strategy, a large amount of Co3O4 NPs is dispersed in a unique nano structure of a covalent organic framework (COF) to form a Co3O4-COF compound, and the composite material is endowed with ultrahigh stability, good dispersity and excellent catalytic activity. At the same time, the COF provides a good sensing platform for biomolecules due to the ordered nano structure and abundant pores of the COF. The unique composition of Co3O4 provides more Co < 3 + > for HQ oxidation in ECC redox cycle, the detection signal is further amplified, and the detection sensitivity is effectively improved. The method has the advantages that high-sensitivity and high-selectivity detection on cardiac troponin I (cTnI) is realized, the immunosensor constructed by the aid of the method is excellent in analysis capability and excellent in actual detection, and a universal method is provided for manufacturing ultra-sensitive sensing systems for monitoring low-abundance targets.

Description

Technical Field

[0001] The invention belongs to the technical fields of novel nanocomposite materials, signal amplification and biosensor, and provides a preparation method of an electrochemical sensor based on a cobalt-covalent organic framework coupling material, which is applied to the detection of cardiac troponin I (cTnI). Background Art

[0002] Acute myocardial infarction (AMI) is a rare and serious cardiovascular disease that poses a serious threat to human life and health. Its incidence is still rising in recent years. As acute myocardial infarction has a high mortality rate, early, rapid and reliable diagnosis plays a vital role in the timely treatment of AMI. The identification and diagnosis of AMI mainly rely on cardiac biomarkers. Among many biomarkers, cardiac troponin I (cTnI) is considered to be a high-standard detection marker for AMI due to its ultra-specificity and high sensitivity. When AMI occurs, myocardial cells are damaged and secrete cTnI. Therefore, the detection of cTnI concentration can well reflect the degree of myocardial damage. Therefore, it is very important to develop efficient and sensitive cTnI detection methods. In recent decades, there are many cTnI detection methods, mainly including fluorescence immunoassay (FIA), radioimmunoassay (RIA), and electrochemiluminescence immunoassay (ECL). Electrochemical immunosensors have the advantages of high sensitivity, simple operation, relatively low cost, and suitability for on-site detection, and are becoming a promising method for biomarker detection. At the same time, in order to fully utilize the amplified electrochemical signals and catalytic effects and improve the sensitivity of sensors, people continue to establish various signal amplification technologies to enhance the detectable signals. The electrochemical-chemical-chemical (ECC) redox cycle amplification strategy (RCA) is an advanced cascade signal enhancement technology. Its core lies in the regeneration of electroactive substances on the electrode surface through enzyme-catalyzed reactions or specific chemical mediators and repeated coupled reduction technology, thereby enhancing the electrochemical signal and significantly improving the intensity and sensitivity of the detection signal. It is worth noting that Co3O4 is rich in Co 2+ and Co 3+ ions, and at a certain applied voltage, Co 2+ Will be oxidized to Co 3+ , can directly drive redox reactions without adding additional oxidase mimics or co-catalysts. Co3O4 nanoparticles (Co3O4NPs) have excellent physical and chemical properties, including chemical stability and conductivity, as well as good biocompatibility. The valence state changes of Co3O4 are highly dynamic and flexible, among which Co 2+ and Co 3+The reversible conversion of forms a bridge for electron transfer to the electrode, triggering the redox cycle. Meanwhile, the unique composition of Co3O4 provides more Co for HQ oxidation in the ECC redox cycle. 3+ , further amplifying the detection signal. 2+ / Co 3+ The presence of increases the number of adsorbed oxygen species and provides additional active sites on the surface of the material. However, Co3O4NPs themselves have the characteristics of high surface energy and there is a potential risk of agglomeration during the catalytic process. Therefore, encapsulating Co3O4NPs into porous materials with nanopores or nanoshells has become a promising method to overcome the instability problem. Covalent organic framework (COF) is a new type of non-metallic material. It is a porous material with a two-dimensional or three-dimensional ordered network structure formed by covalently connecting organic molecular units. It not only has a complex atomic arrangement, but also has low-density ordered channels. These channels provide efficient paths for molecular transport. At the same time, the large specific surface area and highly adjustable porosity of COF improve its application efficiency in adsorption, catalysis and sensing. Using a covalent organic framework (COF) with a large specific surface area, porosity and good stability as a sensing platform, more active sites are provided for sensor construction to connect antibodies. At the same time, the thermally stable COF was used as a carrier to encapsulate Co3O4NPs, and Co3O4NPs were anchored by Co-N bonds to form Co3O4-COF nanospheres. This method not only solved the inevitable agglomeration phenomenon of pure Co3O4NPs, but also gave Co3O4-COF excellent catalytic properties, thereby realizing sensitive detection of cTnI.

[0003] The present invention uses COF as the base material, Co3O4-COF as the electrochemical signal amplification platform, and utilizes the electrochemical-chemical-chemical (ECC) redox cycle amplification strategy (RCA). It fully utilizes the materials, simplifies the experimental steps, realizes the multifunctionality of a single material, and realizes the continuous amplification of the electrical signal. A sandwich-type electrochemical immunosensor with good specificity, high sensitivity and low detection limit is prepared, which realizes the ultra-sensitive detection of cTnI. Summary of the invention

[0004] The present invention provides a preparation method of an electrochemical sensor based on a cobalt-covalent organic framework coupling material, which realizes ultrasensitive detection of cTnI.

[0005] One of the purposes of the present invention is to provide a method for preparing an electrochemical sensor based on a cobalt-covalent organic framework coupling material.

[0006] The second purpose of the present invention is to use the prepared electrochemical sensor based on cobalt-covalent organic framework coupling material for the detection of cTnI.

[0007] The technical solution of the present invention comprises the following steps: 1. Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the steps are as follows: (1) Polish a glassy carbon electrode with a diameter of 4.0 mm into a mirror surface using Al2O3 polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) Add 6.0 µL of 0.5 ~ 1.5 mg / mL COF dispersion onto the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry at room temperature; (3) Add 6.0 µL of 5-15 µg / mL cardiac troponin I (cTnI) antibody to the electrode surface, rinse the electrode surface with pH 7.17 phosphate buffer, and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 1 wt% bovine serum albumin solution to the electrode surface to block the nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry it in a refrigerator at 4 ℃; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 1 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4 °C; (6) A 6.0 µL, 2.0 ~ 4.0 mg / mL Co3O4-COF-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4 ℃ refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to prepare a sandwich electrochemical immunosensor based on cobalt-covalent organic framework coupling material.

[0008] 2. Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of COF 3 ~ 5 mg 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 3 ~ 5 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in 6 ~ 10 mL acetonitrile (CH3CN) solution, respectively, and ultrasonically treated for 3 min, then vigorously stirred at room temperature, 0.1 ~ 0.3 mL glacial acetic acid solution was added while vigorously stirring, and stirring was continued at room temperature for 24 h to obtain a yellow precipitate, which was collected and washed three times by centrifugation with ethanol, and dried in a vacuum oven at 50 °C overnight to obtain a light yellow powder product; (2) Preparation of Co3O4-COF 12 ~ 20 mL of the prepared COF was dispersed in 20 ~ 28 mL of anhydrous ethanol, and ultrasonicated for 3 min to dissolve the COF completely. Then 0.8 ~ 1.6 mL of 0.2 M Co(Ac)2•4H2O aqueous solution was added to the above solution, and then 0.8 ~ 1.6 mL of water was added at room temperature, and stirred at 80 ° C for 6 h. Then the above solution was transferred to a reactor with a capacity of 50 mL, and the reactor was heated to 150 ° C for 3 h, and then cooled to room temperature. The obtained product was collected by centrifugation, washed with ethanol and water for multiple times, and the product was collected by freeze-drying; (3) Preparation of Co3O4-COF-Ab2 Add 0.8 ~ 1.2 mL Co3O4-COF (3.0 mg mL -1 ) and 0.8 ~ 1.2 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4°C by a shaker. The resulting product was centrifuged several times using phosphate buffer solution (PBS) (pH=7.17) as a dispersion solution to wash away unbound Ab2, and then re-dispersed in PBS and stored in a refrigerator at 4°C.

[0009] 3. Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material for the detection of cardiac troponin I (cTnI), the steps are as follows: (1) Using an electrochemical workstation, the test was carried out under a three-electrode system, with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared electrode as the working electrode, in 10 mL of a phosphate buffer solution containing a 2 mol / L HQ solution and a 10 mol / L TCEP solution at pH 5.0 to 8.5; (2) Analytes were detected by square wave voltammetry with a pulse amplitude of 25 mV; (3) Changing the concentration of cTnI, when the cTnI concentration was 1 fg mL -1 ~ 100 ng mL -1 The analyte is tested by a square wave voltammetry test; (4) Record the peak current of square wave voltammetry corresponding to different concentrations of cTnI; (5) Using the working curve method, the concentration of cTnI in the sample to be tested is obtained.

[0010] Beneficial results of the present invention (1) The COF prepared by the present invention has a large specific surface area, a porous structure and good stability. It is used as a sensing platform to provide more active sites for sensor construction to connect the primary antigen (Ab1), anchor Co3O4NPs through Co-N bonds to form Co3O4-COF nanospheres, and use them as signal amplifiers. This method not only realizes the multifunctionality of COF single material, but also solves the inevitable agglomeration phenomenon of Co3O4NPs and gives Co3O4-COF excellent catalytic performance. At the same time, the electrochemical-chemical-chemical (ECC) redox cycle amplification strategy (RCA) is used. In the RCA strategy, the valence state change of Co3O4 shows a high degree of dynamics and flexibility, among which Co 2+ With Co 3+ The reversible transformation between Co and N not only constitutes a bridge for electron transfer, but also promotes the effective interaction between reactants and catalysts. 2+ / Co 3+ The presence of is more likely to induce redox reactions, ensuring the continuous amplification of electrical signals; (2) An electrochemical sensor based on cobalt-covalent organic framework coupling material was used to detect cardiac troponin I (cTnI) antigen. The linear detection range of cardiac troponin I (cTnI) antigen was 1 fg / mL ~ 100 ng / mL, and the lowest detection limit was 0.88 fg / mL. This showed that the prepared covalent organic framework signal amplified electrochemical sensor can accurately and quantitatively detect cardiac troponin I (cTnI) antigen. DETAILED DESCRIPTION

[0011] The present invention will now be further described by way of specific embodiments, but is not limited thereto.

[0012] Example 1 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the steps are as follows: (1) Polish a glassy carbon electrode with a diameter of 4.0 mm into a mirror surface using Al2O3 polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) 6.0 μL of 0.5 mg / mL COF dispersion was added to the electrode surface, the electrode surface was rinsed with pH = 7.17 phosphate buffer, and dried at room temperature; (3) Add 6.0 µL, 5 µg / mL cardiac troponin I (cTnI) antibody to the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry in a 4 ℃ refrigerator; (4) Continue to add 3.0 μL of 1 wt% bovine serum albumin solution to the electrode surface to block the nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry it in a refrigerator at 4 ℃; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 1 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4 °C; (6) A 6.0 μL, 2.0 mg / mL Co3O4-COF-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4 ℃ refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to prepare a sandwich electrochemical immunosensor based on cobalt-covalent organic framework coupling material.

[0013] Example 2 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the steps are as follows: (1) Polish a glassy carbon electrode with a diameter of 4.0 mm into a mirror surface using Al2O3 polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) 6.0 μL of 1.0 mg / mL COF dispersion was added to the electrode surface, the electrode surface was rinsed with pH 7.17 phosphate buffer, and dried at room temperature; (3) Add 6.0 µL of 10 µg / mL cardiac troponin I (cTnI) antibody to the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry in a 4 ℃ refrigerator; (4) Continue to add 3.0 μL of 1 wt% bovine serum albumin solution to the electrode surface to block the nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry it in a refrigerator at 4 ℃; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 1 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4 °C; (6) A 6.0 µL, 3.0 mg / mL Co3O4-COF-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4 ℃ refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to prepare a sandwich electrochemical immunosensor based on cobalt-covalent organic framework coupling material.

[0014] Example 3 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the steps are as follows: (1) Polish a glassy carbon electrode with a diameter of 4.0 mm into a mirror surface using Al2O3 polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) 6.0 μL of 1.5 mg / mL COF dispersion was added to the electrode surface, the electrode surface was rinsed with pH 7.17 phosphate buffer, and dried at room temperature; (3) Add 6.0 µL of 15 µg / mL cardiac troponin I (cTnI) antibody to the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry in a 4 ℃ refrigerator; (4) Continue to add 3.0 μL of 1 wt% bovine serum albumin solution to the electrode surface to block the nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry it in a refrigerator at 4 ℃; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 1 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4 °C; (6) A 6.0 µL, 4.0 mg / mL Co3O4-COF-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4 ℃ refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to prepare a sandwich electrochemical immunosensor based on cobalt-covalent organic framework coupling material.

[0015] Example 4 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of the COF 3 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 3 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in 6 mL of acetonitrile (CH3CN) solution, respectively, and ultrasonically treated for 3 min, then vigorously stirred at room temperature, 0.1 mL of glacial acetic acid solution was added while vigorously stirring, and stirring was continued at room temperature for 24 h to obtain a yellow precipitate, which was collected and washed three times by centrifugation with ethanol, and dried in a vacuum oven at 50 °C overnight to obtain a light yellow powder product; (2) Preparation of the Co3O4-COF 12 mL of the COF prepared above was dispersed in 20 mL of anhydrous ethanol, and ultrasonicated for 3 min to dissolve the COF completely. Then 0.8 mL of 0.2 M Co(Ac)2•4H2O aqueous solution was added to the above solution, and then 0.8 mL of water was added at room temperature. The mixture was stirred at 80 °C for 6 h. The above solution was transferred to a 50 mL reactor, and the reactor was heated to 150 °C for 3 h, and then cooled to room temperature. The obtained product was collected by centrifugation, washed with ethanol and water for multiple times, and the product was collected by freeze drying. (3) Preparation of Co3O4-COF-Ab2 Add 0.8 mL Co3O4-COF (3.0 mg mL -1 ) and 0.8 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4°C by a shaker. The resulting product was centrifuged several times using phosphate buffer solution (PBS) (pH=7.17) as a dispersion solution to wash away unbound Ab2, and then re-dispersed in PBS and stored in a refrigerator at 4°C.

[0016] Example 5 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of the COF 4 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 4 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in 8 mL of acetonitrile (CH3CN) solution, respectively, and ultrasonically treated for 3 min, then vigorously stirred at room temperature, 0.2 mL of glacial acetic acid solution was added while vigorously stirring, and stirring was continued at room temperature for 24 h to obtain a yellow precipitate, which was collected and washed three times by centrifugation with ethanol, and dried in a vacuum oven at 50 °C overnight to obtain a light yellow powder product; (2) Preparation of the Co3O4-COF 16 mL of the COF prepared above was dispersed in 24 mL of anhydrous ethanol, and ultrasonicated for 3 min to dissolve the COF completely. Then 1.2 mL of 0.2 M Co(Ac)2•4H2O aqueous solution was added to the above solution, and then 1.2 mL of water was added at room temperature. The mixture was stirred at 80 °C for 6 h. The above solution was transferred to a reactor with a capacity of 50 mL, and the reactor was heated to 150 °C for 3 h, and then cooled to room temperature. The obtained product was collected by centrifugation, washed with ethanol and water for multiple times, and the product was collected by freeze-drying. (3) Preparation of Co3O4-COF-Ab2 Add 1.0 mL Co3O4-COF (3.0 mg mL -1 ) and 1.0 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4°C by a shaker. The resulting product was centrifuged several times using phosphate buffer solution (PBS) (pH=7.17) as a dispersion solution to wash away unbound Ab2, and then re-dispersed in PBS and stored in a refrigerator at 4°C.

[0017] Example 6 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of the COF 5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 5 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in 10 mL of acetonitrile (CH3CN) solution, respectively, and ultrasonically treated for 3 min, then vigorously stirred at room temperature, 0.3 mL of glacial acetic acid solution was added while vigorously stirring, and stirring was continued at room temperature for 24 h to obtain a yellow precipitate, which was collected and washed three times by centrifugation with ethanol, and dried in a vacuum oven at 50 °C overnight to obtain a light yellow powder product; (2) Preparation of the Co3O4-COF 20 mL of the prepared COF was dispersed in 28 mL of anhydrous ethanol, and ultrasonicated for 3 min to dissolve the COF. Then 1.6 mL of 0.2M Co(Ac)2•4H2O aqueous solution was added to the solution, and then 1.6 mL of water was added at room temperature. The mixture was stirred at 80 °C for 6 h. The solution was transferred to a 50 mL reactor, and the reactor was heated to 150 °C for 3 h. The reaction was then cooled to room temperature. The product was collected by centrifugation, washed with ethanol and water for several times, and lyophilized. (3) Preparation of Co3O4-COF-Ab2 Add 1.2 mL Co3O4-COF (3.0 mg mL -1 ) and 1.2 mL of Ab2 solution (10 μg mL -1 ) was shaken overnight at 4°C by a shaker. The resulting product was centrifuged several times using phosphate buffer solution (PBS) (pH=7.17) as a dispersion solution to wash away unbound Ab2, and then re-dispersed in PBS and stored in a refrigerator at 4°C.

[0018] Example 7 Preparation of an electrochemical sensor based on cobalt-covalent organic framework coupling material for the detection of cardiac troponin I (cTnI), the steps are as follows: (1) Using an electrochemical workstation, the test was carried out under a three-electrode system, with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared electrode as the working electrode, in 10 mL of a phosphate buffer solution containing a 2 mol / L HQ solution and a 10 mol / L TCEP solution at pH 5.0 to 8.5; (2) Analytes were detected by square wave voltammetry with a pulse amplitude of 25 mV; (3) Changing the concentration of cTnI, and performing square wave voltammetry testing on the analytes at cTnI concentrations ranging from 1 fg / mL to 100 ng / mL; (4) Record the peak current of square wave voltammetry corresponding to different concentrations of cTnI; (5) Using the working curve method, the linear detection range of cardiac troponin I (cTnI) antigen in the test samples was 1 fg / mL ~ 100 ng / mL, and the lowest detection limit was 0.88 fg / mL.

Claims

1. Preparation of an electrochemical immunosensor based on cobalt-covalent organic framework coupling material, characterized in that: The following steps are involved: (1) Preparation of Co3O4-COF-Ab2: Co3O4 NPs and COF were synthesized into Co3O4-COF nanospheres through Co-N bonds, and then the separately prepared Co3O4-COF and Ab2 were combined; (2) Construct a COF-based sandwich electrochemical immunosensor to measure the cardiac troponin I (cTnI) antigen content and draw a working curve.

2. The electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen and the preparation method thereof according to claim 1, characterized in that: The preparation of Co3O4-COF and Ab2 described in step (1) is as follows: (1) Preparation of COF 3 ~ 5 mg 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 3 ~ 5 mg 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP) were dissolved in 6 ~ 10 mL acetonitrile (CH3CN) solution, respectively, and ultrasonically treated for 3 min, then vigorously stirred at room temperature, 0.1 ~ 0.3 mL glacial acetic acid solution was added while vigorously stirring, and stirring was continued at room temperature for 24 h to obtain a yellow precipitate, which was collected and washed three times by centrifugation with ethanol, and dried in a vacuum oven at 50 °C overnight to obtain a light yellow powder product; (2) Preparation of Co3O4-COF 12 ~ 20 mL of the prepared COF was dispersed in 20 ~ 28 mL of anhydrous ethanol, and ultrasonicated for 3 min to dissolve the COF completely. Then 0.8 ~ 1.6 mL of 0.2 M Co(Ac)2•4H2O aqueous solution was added to the above solution, and then 0.8 ~ 1.6 mL of water was added at room temperature, and stirred at 80 ° C for 6 h. Then the above solution was transferred to a reactor with a capacity of 50 mL, and the reactor was heated to 150 ° C for 3 h, and then cooled to room temperature. The obtained product was collected by centrifugation, washed with ethanol and water for multiple times, and the product was collected by freeze-drying; (3) Preparation of Co3O4-COF-Ab2 0.8 ~ 1.2 mL Co3O4-COF (3.0 mg / mL) and 0.8 ~ 1.2 mL Ab2 solution (10 μg / mL) were shaken overnight at 4 °C by a shaker. The resulting product was centrifuged several times using phosphate buffer solution (PBS) (pH = 7.17) as a dispersion solution to wash away unbound Ab2, then redispersed in PBS and stored in a refrigerator at 4 °C.

3. The electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen and the preparation method thereof according to claim 1, characterized in that: The construction of the electrochemical immunosensor in step (2) is as follows: (1) Polish a glassy carbon electrode with a diameter of 4.0 mm into a mirror surface using Al2O3 polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) Add 6.0 µL of 0.5 ~ 1.5 mg / mL COF dispersion onto the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry at room temperature; (3) Add 6.0 µL of 5-15 µg / mL cardiac troponin I (cTnI) antibody to the electrode surface, rinse the electrode surface with pH 7.17 phosphate buffer, and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 1 wt% bovine serum albumin solution to the electrode surface to block the nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.17 phosphate buffer, and dry it in a refrigerator at 4 ℃; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 1 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4 °C; (6) A 6.0 µL, 2.0 ~ 4.0 mg / mL Co3O4-COF-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4 ℃ refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to prepare a sandwich electrochemical immunosensor based on cobalt-covalent organic framework coupling material.

4. The electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen and the preparation method thereof according to claim 1, characterized in that: The step (2) of measuring cardiac troponin I (cTnI) antigen and drawing a working curve is as follows: (1) 6 µL of cardiac troponin I (cTnI) antigen solution of different concentrations was dripped onto the electrode surface and reacted for 0.5 to 2 h. After drying, the electrode was connected to the electrochemical workstation and immersed in 10 mL of phosphate buffer solution containing 2 mol / L HQ solution and 10 mol / L TCEP solution at pH 5.0 to 8.5 for testing. The analyte was detected by square wave voltammetry with a pulse amplitude of 25 mV. (2) Drawing a working curve based on the linear relationship between the obtained current difference and the cardiac troponin I (cTnI) antigen concentration; (3) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the sample to be tested is obtained.