Preparation of electrochemical sensor based on nitrogen-rich interpenetrating bicrystal porous carbon material

Abstract

The invention belongs to the technical field of novel functional carbon materials, immunoassay and biosensing, and provides a preparation method of an electrochemical sensor based on a nitrogen-rich interpenetrating bicrystal porous carbon material. The sandwich type electrochemical immunosensor constructed by taking Au NPs / ITPC as a substrate material and PtCuRu NF as an electrochemical signal amplification platform shows the properties of rapidness, sensitivity and durability, has a wide dynamic linear range and an ultra-low detection limit, and can efficiently detect cTnI. Besides, the sensor also shows excellent reproducibility, high selectivity and long-term stability, is successfully applied to real serum sample detection, and shows a potential application value in early detection and prognosis of acute myocardial infarction (AMI).

Description

Technical Field

[0001] The present invention belongs to the technical fields of novel functional carbon materials, immunoassay and biosensor, and provides a preparation method of an electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material, which is applied to the detection of cardiac troponin I (cTnI). Background Art

[0002] Acute myocardial infarction (AMI) is a sudden cardiovascular disease with a high mortality rate, posing a serious threat to human life and health. Early diagnosis of AMI plays a vital role in improving the quality of life of patients, improving disease prognosis and reducing mortality. As the "golden marker" for the diagnosis of AMI, cardiac troponin I (cTnI) plays an important role in the assessment and diagnosis of myocardial necrosis and AMI due to its high specificity for myocardial tissue. Its sensitivity and specificity are higher than those of traditional markers. At present, the detection methods of cTnI mainly include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescence method (FIA) and electrochemiluminescence (ECL) colloidal gold immunochromatography. However, these methods generally have problems such as long detection cycle, complex operation steps and high equipment requirements, which limit their application in point-of-care testing or rapid diagnosis in the emergency room. Compared with traditional detection methods, electrochemical immunosensors show significant advantages in AMI detection, including excellent sensitivity, accuracy and user-friendliness. By immobilizing antibodies on a conductive substrate, electrochemical immunosensors can achieve specific binding with target analytes and convert this binding process into a quantifiable current signal, thereby achieving qualitative or quantitative evaluation of the target molecule. Electrochemical immunosensors usually have a low detection limit and a wide detection range, which are very suitable for the early diagnosis of AMI. Among them, sandwich-type electrochemical immunosensors have shown great potential in biomedical testing and clinical diagnosis due to their high sensitivity, high specificity, strong signal amplification ability and quantitative detection. Here, we carefully designed a sandwich-type electrochemical immunosensor for ultrasensitive detection of cTnI. We synthesized nitrogen-rich interpenetrating twin-crystal porous carbon materials (ITPCs) with carefully controlled morphology from ZIF-8 (ZIF-8-IT) by a simple pyrolysis method. The high nitrogen content of ITPC promoted the in situ growth of Au NPs and provided abundant binding sites for the primary antibody (Ab1), thus avoiding the use of cross-linkers to fix proteins. In addition, the large specific surface area and excellent conductivity of the material enhanced the mass and electron transport in the electrochemical reaction and improved the overall sensor performance. In addition, the synthesized PtCuRu nanoflowers (NFs) have an open flower-like structure and optimized d-orbital coupling properties, showing efficient mass and electron transfer capabilities, which endow them with excellent peroxidase (POD)-like catalytic activity.

[0003] The present invention uses Au NPs / ITPC as the substrate material and PtCuRu NF as the electrochemical signal amplification platform. The resulting sandwich electrochemical immunosensor exhibits rapid, sensitive and durable performance, has a wide dynamic linear range and ultra-low detection limit, and can efficiently detect cTnI. In addition, the sensor also demonstrated excellent reproducibility, high selectivity and long-term stability, and was successfully applied to the detection of real serum samples, showing its potential application value in the early detection and prognosis of acute myocardial infarction (AMI). Summary of the invention

[0004] The present invention provides a preparation method of an electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon 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 nitrogen-rich interpenetrating twin-crystal porous carbon material.

[0006] The second purpose of the present invention is to use the prepared electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon 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 nitrogen-rich interpenetrating twin-crystal porous carbon 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, 0.5 ~ 1.5 mg mL -1 The Au NPs / ITPC dispersion was dropped onto the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried at room temperature; (3) 6.0 µL, 5 ~ 15 µg mL -1 The cardiac troponin I (cTnI) antibody was added dropwise to the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (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 °C; (5) Continue to add 6.0 µL and 10 fg mL -1 ~ 100 ng mL -1 A series of different concentrations of cardiac troponin I (cTnI) antigen solutions were washed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (6) 6.0 µL, 2.0 ~ 4.0 mg mL -1 The PtCuRu NF-Ab2 dispersed droplets were applied to the electrode surface, placed in a 4°C refrigerator for 20 min, rinsed with phosphate buffer with pH = 7.17, and dried at 4°C to obtain a sandwich electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material.

[0008] 2. Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material, the preparation steps of the relevant materials are as follows: (1) Preparation of ITPC 41 ~ 44 mg of CTAB and 105 ~ 111 mg of Zn(CH3COO)2·2H2O were dissolved in 18 ~ 24 mL of deionized water, and 1.1 ~ 1.3 g of 2-methylimidazole was dissolved in 4 ~ 6 mL of deionized water and quickly poured into the former solution. The mixture was stirred at room temperature for 1 hour, then transferred to a stainless steel reactor and heated at 120 °C for 10 hours. After cooling, the precipitate was obtained by centrifugation and washed with methanol. Subsequently, the sample was dried in an oven at 60 °C overnight to obtain ZIF-8-IT. Subsequently, ITPC was obtained by calcination at 900 °C for 3 hours in a nitrogen atmosphere; (2) Preparation of Au NPs Add 0.5 ~ 1.5 mL HAuCl4·4H2O (1.0 wt%) to a round-bottom flask filled with 98.5 ~ 99.5 mL ultrapure water, place a magnetic rotor in the flask, stir at a certain speed, heat to boiling, then add 2.0 ~ 3.0 mL sodium citrate (1.0 wt%) to the flask and keep boiling for 15 minutes to obtain uniform gold nanoparticles; (3) Preparation of Au NPs / ITPC 13 ~ 17 mg of ITPC was dispersed in 17 ~ 19.8 mL of deionized water, and then 0.25 ~ 0.75 mL of Na3C6H5O7·2H2O solution (10 mM) and 0.25 ~ 0.75 mL of HAuCl4 solution (10 mM) were added and mixed under stirring. While stirring vigorously, 0.4 ~ 0.8 mL of freshly prepared cooled NaBH4 solution (0.1 M) was quickly added. After stirring at room temperature for 20 hours, the solution was washed with deionized water and freeze-dried to obtain Au NPs / ITPC; (4) Preparation of PtCuRu NF 70.0 ~ 74.0 mg of DDAC, 8.0 ~ 12.0 mg of Pt(acac)2, 4.0 ~ 6.36 mg of RuCl3·6H2O, 5.0 ~ 8.0 mg of Cu(acac)2, and 33.0 ~ 39.0 mg of C6H4(NH2)2 were dissolved in 8.0 ~ 12.0 mL of oleylamine and dispersed uniformly by ultrasonic treatment. Subsequently, the suspension was transferred to a stainless steel reactor and heated at 220 °C for 10 h. After cooling, the precipitate was obtained by centrifugation and washed with n-hexane, ethanol, and water in sequence. Finally, the sample was dried in a freeze dryer overnight to obtain PtCuRu NF; (5) Preparation of PtCuRu NF-Ab2 0.5 ~ 1.5 mL of PtCuRu NF dispersion (3 mg mL -1 ) and 0.5 to 1.5 mL of Ab2 solution (10 μg mL -1 ) and incubated with shaking at 4 °C overnight. The resulting product was centrifuged several times, PBS (pH = 7.17) was used as the dispersion medium, and then washed thoroughly to remove unbound Ab2. After washing, it was dispersed in PBS and stored at 4 °C until use.

[0009] 3. Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon 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, and cyclic voltammetry was performed in a phosphate buffer solution (PBS) with a pH of 7.17 and a concentration of 0.1 M; (2) Detect the analytes using cyclic voltammetry at a voltage of -0.4 V to 0.6 V; (3) Changing the concentration of cTnI, when the cTnI concentration was 10 fg mL -1 ~ 100 ng mL -1 The analytes are tested by cyclic voltammetry. (4) Record the oxidation peak current of the cyclic voltammetry curve corresponding to cTnI at different concentrations; (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 high nitrogen content of ITPC not only promotes the in situ growth of Au NPs, but also provides sufficient binding sites for the primary antibody (Ab1), eliminating the step of fixing the protein with a crosslinker. Its large specific surface area and excellent conductivity significantly improve the mass and electron transfer efficiency in electrochemical reactions, thereby enhancing the overall performance of the sensor. In addition, the carefully prepared PtCuRu NF has an open structure and optimized d-orbital coupling properties, which ensures efficient mass and electron transfer and endows it with excellent peroxidase-like (POD) catalytic activity; (2) An electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material for the detection of cardiac troponin I (cTnI) antigen, with a linear detection range of 10 fg mL -1 ~ 100 ng mL -1 The minimum detection limit is 0.58 fg mL -1 ; It shows that the prepared electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material 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 nitrogen-rich interpenetrating twin-crystal porous carbon 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, 0.5 mg mL -1 The Au NPs / ITPC dispersion was dropped onto the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried at room temperature; (3) 6.0 µL, 5 µg mL -1 The cardiac troponin I (cTnI) antibody was added dropwise to the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (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 °C; (5) Continue to add 6.0 µL and 10 fg mL -1 ~ 100 ng mL -1A series of different concentrations of cardiac troponin I (cTnI) antigen solutions were washed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (6) 6.0 µL, 2.0 mg mL -1 The PtCuRu NF-Ab2 dispersion droplets were applied to the electrode surface, placed in a 4 ℃ refrigerator for 20 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to obtain a sandwich electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material.

[0013] Example 2 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon 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, 1.0 mg mL -1 The Au NPs / ITPC dispersion was dropped onto the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried at room temperature; (3) 6.0 µL and 10.0 µg mL -1 The cardiac troponin I (cTnI) antibody was added dropwise to the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (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 °C; (5) Continue to add 6.0 µL and 10 fg mL -1 ~ 100 ng mL -1 A series of different concentrations of cardiac troponin I (cTnI) antigen solutions were washed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (6) 6.0 µL, 3.0 mg mL -1 The PtCuRu NF-Ab2 dispersion droplets were applied to the electrode surface, placed in a 4 ℃ refrigerator for 20 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to obtain a sandwich electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material.

[0014] Example 3 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twin-crystal porous carbon 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, 1.5 mg mL -1 The Au NPs / ITPC dispersion was dropped onto the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried at room temperature; (3) 6.0 µL, 15.0 µg mL -1 The cardiac troponin I (cTnI) antibody was added dropwise to the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (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 °C; (5) Continue to add 6.0 µL and 10 fg mL -1 ~ 100 ng mL -1 A series of different concentrations of cardiac troponin I (cTnI) antigen solutions were washed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (6) 6.0 µL, 4.0 mg mL -1 The PtCuRu NF-Ab2 dispersion droplets were applied to the electrode surface, placed in a 4 ℃ refrigerator for 20 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to obtain a sandwich electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material.

[0015] Example 4 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of ITPC 41 mg of CTAB and 105 mg of Zn(CH3COO)2·2H2O were dissolved in 18 mL of deionized water, and 1.1 g of 2-methylimidazole was dissolved in 4 mL of deionized water and quickly poured into the former solution. The mixture was stirred at room temperature for 1 hour, then transferred to a stainless steel reactor and heated at 120 °C for 10 hours. After cooling, the precipitate was obtained by centrifugation and washed with methanol. Subsequently, the sample was dried in an oven at 60 °C overnight to obtain ZIF-8-IT. Subsequently, ITPC was obtained by calcination at 900 °C for 3 hours in a nitrogen atmosphere; (2) Preparation of Au NPs 0.5 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 98.5 mL of ultrapure water, a magnetic rotor was placed in the flask, stirred at a certain speed, and heated to boiling. Then, 2.0 mL of sodium citrate (1.0 wt%) was added to the flask and kept boiling for 15 minutes to obtain uniform gold nanoparticles. (3) Preparation of Au NPs / ITPC 13 mg of ITPC was dispersed in 17 mL of deionized water, and then 0.25 mL of Na3C6H5O7·2H2O solution (10 mM) and 0.25 mL of HAuCl4 solution (10 mM) were added and mixed under stirring. While stirring vigorously, 0.4 mL of freshly prepared cooled NaBH4 solution (0.1 M) was quickly added. After stirring at room temperature for 20 h, the solution was washed with deionized water and freeze-dried to obtain Au NPs / ITPC; (4) Preparation of PtCuRu NF 70.0 mg of DDAC, 8.0 mg of Pt(acac)2, 4.0 mg of RuCl3·6H2O, 5.0 mg of Cu(acac)2, and 33.0 mg of C6H4(NH2)2 were dissolved in 8.0 mL of oleylamine and uniformly dispersed by ultrasonic treatment. Subsequently, the suspension was transferred to a stainless steel reactor and heated at 220 °C for 10 h. After cooling, the precipitate was obtained by centrifugation and washed with n-hexane, ethanol, and water in sequence. Finally, the sample was dried in a freeze dryer overnight to obtain PtCuRu NF; (5) Preparation of PtCuRu NF-Ab2 0.5 mL of PtCuRu NF dispersion (3 mg mL -1 ) and 0.5 mL of Ab2 solution (10 μg mL -1 ) and incubated with shaking at 4 °C overnight. The resulting product was centrifuged several times, PBS (pH = 7.17) was used as the dispersion medium, and then washed thoroughly to remove unbound Ab2. After washing, it was dispersed in PBS and stored at 4 °C until use.

[0016] Example 5 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of ITPC 42.5 mg of CTAB and 108 mg of Zn(CH3COO)2·2H2O were dissolved in 21 mL of deionized water, and 1.2 g of 2-methylimidazole was dissolved in 5 mL of deionized water and quickly poured into the former solution. The mixture was stirred at room temperature for 1 hour, then transferred to a stainless steel reactor and heated at 120 °C for 10 hours. After cooling, the precipitate was obtained by centrifugation and washed with methanol. Subsequently, the sample was dried in an oven at 60 °C overnight to obtain ZIF-8-IT. Subsequently, ITPC was obtained by calcination at 900 °C for 3 hours in a nitrogen atmosphere; (2) Preparation of Au NPs 1.0 mL HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 99.0 mL ultrapure water, a magnetic rotor was placed in the flask, stirred at a certain speed, heated to boiling, then, 2.5 mL sodium citrate (1.0 wt%) was added to the flask, and kept boiling for 15 min to obtain uniform gold nanoparticles; (3) Preparation of Au NPs / ITPC 15 mg of ITPC was dispersed in 18.4 mL of deionized water, and then 0.5 mL of Na3C6H5O7·2H2O solution (10 mM) and 0.5 mL of HAuCl4 solution (10 mM) were added and mixed under stirring. While stirring vigorously, 0.6 mL of freshly prepared cooled NaBH4 solution (0.1 M) was quickly added. After stirring at room temperature for 20 h, the solution was washed with deionized water and freeze-dried to obtain Au NPs / ITPC; (4) Preparation of PtCuRu NF 72.0 mg of DDAC, 10.0 mg of Pt(acac)2, 5.18 mg of RuCl3·6H2O, 6.5 mg of Cu(acac)2, and 36.0 mg of C6H4(NH2)2 were dissolved in 10.0 mL of oleylamine and dispersed uniformly by ultrasonic treatment. Subsequently, the suspension was transferred to a stainless steel reactor and heated at 220 °C for 10 h. After cooling, the precipitate was obtained by centrifugation and washed with n-hexane, ethanol, and water in sequence. Finally, the sample was dried in a freeze dryer overnight to obtain PtCuRu NF; (5) Preparation of PtCuRu NF-Ab2 1.0 mL of PtCuRu NF dispersion (3 mg mL -1 ) and 1.0 mL of Ab2 solution (10 μg mL -1) and incubated with shaking at 4 °C overnight. The resulting product was centrifuged several times, PBS (pH = 7.17) was used as the dispersion medium, and then washed thoroughly to remove unbound Ab2. After washing, it was dispersed in PBS and stored at 4 °C until use.

[0017] Example 6 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material, the preparation of the relevant materials, the steps are as follows: (1) Preparation of ITPC 44 mg of CTAB and 111 mg of Zn(CH3COO)2·2H2O were dissolved in 24 mL of deionized water, and 1.2 g of 2-methylimidazole was dissolved in 5 mL of deionized water and quickly poured into the former solution. The mixture was stirred at room temperature for 1 hour, then transferred to a stainless steel reactor and heated at 120 °C for 10 hours. After cooling, the precipitate was obtained by centrifugation and washed with methanol. Subsequently, the sample was dried in an oven at 60 °C overnight to obtain ZIF-8-IT. Subsequently, ITPC was obtained by calcination at 900 °C for 3 hours in a nitrogen atmosphere; (2) Preparation of Au NPs 1.5 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 99.5 mL of ultrapure water, a magnetic rotor was placed in the flask, stirred at a certain speed, and heated to boiling. Then, 3.0 mL of sodium citrate (1.0 wt%) was added to the flask and kept boiling for 15 minutes to obtain uniform gold nanoparticles. (3) Preparation of Au NPs / ITPC 15 mg of ITPC was dispersed in 19.8 mL of deionized water, and then 0.5 mL of Na3C6H5O7·2H2O solution (10 mM) and 0.5 mL of HAuCl4 solution (10 mM) were added and mixed under stirring. While stirring vigorously, 0.6 mL of freshly prepared cooled NaBH4 solution (0.1 M) was quickly added. After stirring at room temperature for 20 h, the solution was washed with deionized water and freeze-dried to obtain Au NPs / ITPC; (4) Preparation of PtCuRu NF 74.0 mg of DDAC, 12.0 mg of Pt(acac)2, 6.36 mg of RuCl3·6H2O, 8.0 mg of Cu(acac)2, and 39.0 mg of C6H4(NH2)2 were dissolved in 12.0 mL of oleylamine and uniformly dispersed by ultrasonic treatment. Subsequently, the suspension was transferred to a stainless steel reactor and heated at 220 °C for 10 h. After cooling, the precipitate was obtained by centrifugation and washed with n-hexane, ethanol, and water in sequence. Finally, the sample was dried in a freeze dryer overnight to obtain PtCuRu NF; (5) Preparation of PtCuRu NF-Ab2 1.5 mL of PtCuRu NF dispersion (3 mg mL -1 ) and 1.5 mL of Ab2 solution (10 μg mL -1 ) and incubated with shaking at 4 °C overnight. The resulting product was centrifuged several times, PBS (pH = 7.17) was used as the dispersion medium, and then washed thoroughly to remove unbound Ab2. After washing, it was dispersed in PBS and stored at 4 °C until use.

[0018] Example 7 Preparation of an electrochemical sensor based on nitrogen-rich interpenetrating twinned porous carbon material for the detection of cardiac troponin I (cTnI), the steps are as follows: (1) An electrochemical workstation was used to test the three-electrode system, with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared immunosensor as the working electrode. -1 The pH value of hydrogen peroxide solution was tested in phosphate buffer solution with a pH of 5.0 to 8.5; (2) Analytes were detected by chronoamperometry with an input voltage of -0.4 V, a sampling interval of 0.1 s, and a running time of 400 s. (3) When the background current tends to be stable, 10 μL of 5 mol L-1 phosphate buffer solution with a pH of 7.17 was injected into the 10 mL solution every 50 s. -1 of hydrogen peroxide solution and record the current change; (4) Recording the current peaks corresponding to cardiac troponin I (cTnI) antigen at different concentrations; (5) Using the working curve method, the linear detection range of cardiac troponin I (cTnI) antigen in the sample to be tested was 10 fg mL -1 ~ 100 ng mL -1 The minimum detection limit is 0.58 fg mL -1 .

Claims

1. Preparation of an electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material, characterized in that: The following steps are involved: (1) Preparation of Au NPs / ITPC: In situ growth of gold nanoparticles (Au NPs) on the surface of separately prepared nitrogen-rich interpenetrating twin-crystalline porous carbon (ITPC); (2) Preparation of PtCuRu NF-Ab2: combining the separately prepared PtCuRu NF and Ab2; (3) Construct an ITPC-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 Au NPs / ITPC described in step (1) is specifically as follows: (1) Preparation of ITPC 41 ~ 44 mg of CTAB and 105 ~ 111 mg of Zn(CH3COO)2·2H2O were dissolved in 18 ~ 24 mL of deionized water, and 1.1 ~ 1.3 g of 2-methylimidazole was dissolved in 4 ~ 6 mL of deionized water and quickly poured into the former solution. The mixture was stirred at room temperature for 1 hour, then transferred to a stainless steel reactor and heated at 120 °C for 10 hours. After cooling, the precipitate was obtained by centrifugation and washed with methanol. Subsequently, the sample was dried in an oven at 60 °C overnight to obtain ZIF-8-IT. Subsequently, ITPC was successfully obtained by calcination at 900 °C for 3 hours in a nitrogen atmosphere; (2) Preparation of Au NPs 0.5 ~ 1.5 mL HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 98.5 ~ 99.5 mL ultrapure water, a magnetic rotor was placed in the flask, stirred at a certain speed, heated to boiling, then, 2.0 ~ 3.0 mL sodium citrate (1.0 wt%) was added to the flask, and kept boiling for 15 minutes to obtain uniform gold nanoparticles; (3) Preparation of Au NPs / ITPC 13 ~ 17 mg of ITPC was dispersed in 17 ~ 19.8 mL of deionized water, and then 0.25 ~ 0.75 mL of Na3C6H5O7·2H2O solution (10 mM) and 0.25 ~ 0.75 mL of HAuCl4 solution (10 mM) were added and mixed under stirring. While stirring vigorously, 0.4 ~ 0.8 mL of freshly prepared cooled NaBH4 solution (0.1 M) was quickly added. After stirring at room temperature for 20 hours, the solution was washed with deionized water and freeze-dried to obtain Au NPs / ITPC.

3. 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 PtCuRu NF-Ab2 described in step (2) is specifically as follows: (1) Preparation of PtCuRu NF 70.0 ~ 74.0 mg of DDAC, 8.0 ~ 12.0 mg of Pt(acac)2, 4.0 ~ 6.36 mg of RuCl3·6H2O, 5.0 ~ 8.0 mg of Cu(acac)2, and 33.0 ~ 39.0 mg of C6H4(NH2)2 were dissolved in 8.0 ~ 12.0 mL of oleylamine and dispersed uniformly by ultrasonic treatment. Subsequently, the suspension was transferred to a stainless steel reactor and heated at 220 °C for 10 h. After cooling, the precipitate was obtained by centrifugation and washed with n-hexane, ethanol, and water in sequence. Finally, the sample was dried in a freeze dryer overnight to obtain PtCuRu NF; (2) Preparation of PtCuRu NF-Ab2 0.5 ~ 1.5 mL of PtCuRu NF dispersion (3 mg mL -1 ) and 0.5 to 1.5 mL of Ab2 solution (10 μg mL -1 ) and incubated with shaking at 4 °C overnight. The resulting product was centrifuged several times, PBS (pH = 7.17) was used as the dispersion medium, and then washed thoroughly to remove unbound Ab2. After washing, it was dispersed in PBS and stored at 4 °C until use.

4. 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) 6.0 µL, 0.5 ~ 1.5 mg mL -1 The Au NPs / ITPC dispersion was dropped onto the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried at room temperature; (3) 6.0 µL, 5 ~ 15 µg mL -1 The cardiac troponin I (cTnI) antibody was added dropwise to the electrode surface, and the electrode surface was rinsed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (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 °C; (5) Continue to add 6.0 µL and 10 fg mL -1 ~ 100 ng mL -1 A series of different concentrations of cardiac troponin I (cTnI) antigen solutions were washed with pH = 7.17 phosphate buffer and dried in a refrigerator at 4 °C; (6) 6.0 µL, 2.0 ~ 4.0 mg mL -1 The PtCuRu NF-Ab2 dispersion droplets were applied to the electrode surface, placed in a 4 ℃ refrigerator for 20 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4 ℃ to obtain a sandwich electrochemical immunosensor based on nitrogen-rich interpenetrating twin-crystal porous carbon material.

5. The electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen and the preparation method thereof according to claim 1, characterized in that: The cardiac troponin I (cTnI) antigen is measured in step (2) and a working curve is drawn as follows: (1) 6 µL of cardiac troponin I (cTnI) antigen solution of different concentrations was dripped onto the electrode surface and allowed to react for 0.5 to 2 h. After drying, the electrode was connected to an electrochemical workstation and the current change was measured by immersing the electrode in 10 mL of pH = 7.17 phosphate buffer solution. When the background current tended to be stable, 10 µL of 5 mol L cTnI was injected into 10 mL of pH = 7.17 phosphate buffer solution every 50 s. -1 of hydrogen peroxide solution and record the current change; (2) based on the linear relationship between the obtained current difference and the concentration of cardiac troponin I (cTnI) antigen, a working curve is drawn; (3) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the sample to be tested is obtained.