Preparation of a sandwich electrochemical immunosensor for cardiac troponin I

Abstract

The present invention belongs to the fields of novel nanocomposites, immunoassays, and biosensor technologies, and provides a method for preparing a sandwich-type electrochemical immunosensor for cardiac troponin I. The present invention uses highly conductive, gold-particle-loaded, hierarchically porous nitrogen-doped carbon nanotubes (Au NPs / N-CNTs) as a substrate and mesoporous palladium nitrogen nanocubes (meso-PdN NCs) as an electrochemical signal amplification platform to achieve quantitative detection of cardiac troponin I (CTnI) antigen. This method has the advantages of strong specificity, high sensitivity, and a low detection limit, and has important scientific significance and application value for the detection of acute myocardial infarction (AMI).

Description

Technical Field

[0001] The invention belongs to the technical fields of novel nanocomposites, immunoassays and biosensors, and provides a preparation method of a sandwich electrochemical immunosensor for cardiac troponin I, which is applied to the detection of cardiac troponin I (CTnI) antigen. Background Art

[0002] Acute myocardial infarction (AMI) is a critical illness with an incidence of 0.2% to 0.6% in China. Currently, the biomarker used in medicine to detect AMI is cardiac troponin I (CTnI). CTnI is produced in the myocardium, making it more specific for AMI. While the CTnI concentration in healthy individuals is approximately 1 pg / mL, it can rise to 100 ng / mL in the blood of AMI patients. After myocardial injury, CTnI concentrations in the patient's blood rise rapidly and persist for a long time, providing a longer window for detection. Therefore, CTnI is used as a key biomarker for diagnosing myocardial infarction.

[0003] One-dimensional carbon nanotubes (CNTs) exhibit excellent performance due to their unique physical, electronic, and chemical properties. A novel one-dimensional nitrogen-doped carbon nanotube (N-CNT) is derived from halloysite coated with nitrogen-containing polydopamine through an innovative carbonization process. During the carbonization process, micropores and mesopores are generated, forming CNTs with hierarchical pores. The presence of hierarchical pores gives N-CNTs a large surface area and excellent conductivity, enabling excellent electron transfer and rapid mass transport during reactions. Gold nanoparticles (AuNPs) are grown on the surface of N-CNTs using in situ growth. The introduction of AuNPs accelerates electron transfer and provides an optimal microenvironment for the capture and immobilization of the primary antibody (Ab1), thereby improving the stability of the sensor interface structure. The resulting AuNPs / N-CNTs, with their strong conductivity and biocompatibility, are ideal substrate materials for electrochemical immunosensors. Mesoporous palladium nitrogen nanocubes (meso-PdNNCs) exhibit excellent stability, rapid response, and high catalytic efficiency, enabling signal amplification. Meso-PdNNCs, as metal-nonmetal alloys, optimize the electronic environment to enhance reactive sites and reduce the use of precious metals during the preparation process, effectively reducing costs. The mesoporous structure offers a large surface area and abundant active sites, enhancing electrocatalytic activity and allowing for the immobilization of more antibodies via Pd-N bonds. The meso-PdNNCs, combined with a secondary antibody (Ab2), act as a signal amplifier, further enhancing the sensitivity of the immunosensor.

[0004] The present invention uses AuNPs / N-CNTs as the base material and meso-PdNNCs as the electrochemical signal amplification platform. It has the advantages of simple operation, strong specificity, high sensitivity, low detection limit, etc., and has good reproducibility, stability and selectivity, realizing ultrasensitive detection of cardiac troponin I (CTnI) antigen. Summary of the Invention

[0005] The present invention provides a preparation method for a sandwich-type electrochemical immunosensor for cardiac troponin I, which realizes ultrasensitive detection of cardiac troponin I (CTnI) antigen.

[0006] One of the purposes of the present invention is to provide a method for preparing a sandwich-type electrochemical immunosensor for cardiac troponin I.

[0007] The second purpose of the present invention is to use the prepared cardiac troponin I sandwich electrochemical immunosensor for the detection of cardiac troponin I (CTnI) antigen.

[0008] The technical solution of the present invention comprises the following steps:

[0009] 1. Preparation of a sandwich electrochemical immunosensor for cardiac troponin I, comprising the following steps:

[0010] (1) Polish a glassy carbon electrode with a diameter of 3.0 to 5.0 mm with Al2O3 polishing powder to a mirror surface and clean it with ultrasonic cleaning in anhydrous ethanol;

[0011] (2) 6.0 μL of 2.0-3.0 mg / mL Au NPs / N-CNTs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0012] (3) 6.0 μL of 5-15 μg / mL 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 4°C refrigerator.

[0013] (4) Continue to add 3.0 μL of 1-2 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH 7.17 phosphate buffer and dry in a 4°C refrigerator.

[0014] (5) Continue to add 6.0 μL of a series of cardiac troponin I (CTnI) antigen solutions with different concentrations of 10 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4°C;

[0015] (6) 6.0 μL of 1.0-3.0 mg / mL meso-PdN NCs-Ab2 dispersion was droplet-coated on the electrode surface, placed in a 4°C refrigerator for 40 min, rinsed with pH 7.17 phosphate buffer, and dried at 4°C to prepare a sandwich-type electrochemical immunosensor for cardiac troponin I.

[0016] 2. Preparation of a sandwich electrochemical immunosensor for cardiac troponin I. The preparation of the related materials is as follows: (1) Preparation of N-CNTs

[0017] 340.0-360.0 mg of halloysite was dispersed in 240-260 mL of 10 mM Tris-HCl buffer (pH = 8.5) and sonicated for 30 minutes. After that, 220.0-240.0 mg of dopamine hydrochloride was added to the above dispersion and sonicated for 5 minutes, and then stirred at room temperature for 22 hours. After centrifugation and repeated washing with water and methanol, the polydopamine-coated halloysite was obtained in an oven at 70°C overnight. The product was placed in an argon atmosphere and heated in a tube furnace at 5°C / min to 800°C for constant temperature pyrolysis for 4 hours, followed by continuous desalting with 14-16 mL of 6M HCl and 14-16 mL of 12M HF for 12 hours, and thoroughly washed with water-ethanol until neutral, and freeze-dried. The final product N-CNTs was obtained;

[0018] (2) Preparation of AuNPs

[0019] 0.5-1.5 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask containing 98.5-99.5 mL of ultrapure water. An electromagnetic rotor was placed in the flask, stirred at a constant speed, and heated to boiling. Then, 2.0-3.0 mL of sodium citrate (1.0 wt%) was added to the flask, and the flask was kept boiling for 15 minutes to obtain uniform gold nanoparticles.

[0020] (3) Preparation of Au NPs / N-CNTs

[0021] Au NPs were loaded onto N-CNTs using an in situ reduction method. 4–6 mg of N-CNTs were dispersed in 2.68–4.68 mL of deionized water. 0.05–0.15 mL of 10 mM sodium citrate and 0.05–0.15 mL of 10 mM chloroauric acid were added while stirring. This was quickly followed by the addition of 0.06–0.18 mL of freshly prepared 0.1 M cold sodium borohydride. The mixture was stirred at room temperature for 20 h and then centrifuged and freeze-dried.

[0022] (4) Preparation of PdNNCs

[0023] Mesoporous Pd nanocubes were synthesized by a one-pot solution phase method at 50°C using hexadecyltrimethylammonium chloride (CTAC) as a structure-directing surfactant, Na2PdCl4 as a metal precursor, L-ascorbic acid (AA) as a reducing agent, and H2O as a solvent. 0.30-0.35 mg of CTAC was dissolved in 4.0-6.0 mL of secondary deionized water, and 0.075-0.125 mL of 200 mM HCl was added to adjust the pH of the reaction solution. 0.20-0.28 mL of 80 mM KCl and 0.20-0.30 mL of 10 mM Na2PdCl4 solution were added to the above solution in sequence and kept at 50°C for 30 minutes. Under gentle shaking, 0.40-0.60 mL of 0.30 M freshly prepared AA was quickly injected into the above solution. After reacting for 1.0 hour, the product was collected by washing with water three times by centrifugation and then freeze-dried.

[0024] (5) Preparation of meso-PdN NCs

[0025] 0.8-1.2 mL of synthesized PdNCs (1.80 mg mL -1 ), 45–55 mg of urea and 45–55 mg of polyvinylpyrrolidone (PVP) were mixed with 3.5–4.5 mL of deionized water. After sonication, the resulting solution was transferred to a 20 mL autoclave. The autoclave was heated to 180°C for 1.5 hours and then cooled to room temperature. To remove the surfactant from the mesoscopic PdN NCs, the NCs were washed three times with acetic acid and three times with ethanol / water before being freeze-dried.

[0026] (6) Preparation of meso-PdNNCs-Ab2

[0027] 0.8~1.2mL PdN NCs (3.0mg mL -1 ) and 0.8-1.2 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4° C. The resulting product was centrifuged several times using phosphate buffered saline (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.

[0028] 3. Preparation of a sandwich electrochemical immunosensor for cardiac troponin I for the detection of cardiac troponin I (CTnI) antigen, the steps are as follows:

[0029] (1) Using an electrochemical workstation, the test was performed in a 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 in 10 mL of a phosphate buffer solution containing 5 mol / L hydrogen peroxide solution at pH 5.0-8.5;

[0030] (2) Analytes were detected using chronoamperometry with an input voltage of −0.4 V, a sampling interval of 0.1 s, and a run time of 400 s;

[0031] (3) When the background current stabilized, 10 μL of 5 mol / L hydrogen peroxide solution was injected into 10 mL of pH 7.17 phosphate buffer solution every 50 s, and the current change was recorded.

[0032] (4) Record the current peak corresponding to different concentrations of cardiac troponin I (CTnI) antigen;

[0033] (5) Using the working curve method, the concentration of cardiac troponin I (CTnI) antigen in the sample to be tested is obtained.

[0034] Beneficial results of the present invention

[0035] (1) The N-CNTs prepared by the present invention have a unique multi-level porous structure and a large surface area, showing better conductivity and catalytic performance. At the same time, their high nitrogen content also helps to form anchoring sites that effectively bind to Au NPs through Au-N bonding. Au-NPs can accelerate the electron transfer rate and provide a good microenvironment for capturing and immobilizing the primary antibody (Ab1), thereby improving the stability of the sensor interface structure. Au NPs / N-CNTs as the substrate material and meso-PdN NCs as the active probe labeled with Ab2 can further achieve signal amplification. Through this synergistic effect and multiple amplification, the detection limit of the sensor is reduced, and highly sensitive and rapid electrochemical detection is achieved;

[0036] (2) A sandwich-type electrochemical immunosensor for cardiac troponin I was used to detect cardiac troponin I (CTnI) antigen. The linear detection range of the prepared sandwich-type electrochemical immunosensor for cardiac troponin I (CTnI) antigen was 10 fg / mL to 100 ng / mL, and the lowest detection limit was 13.02 fg / mL. This indicated that the prepared sandwich-type electrochemical immunosensor for cardiac troponin I can accurately and quantitatively detect cardiac troponin I (CTnI) antigen. DETAILED DESCRIPTION

[0037] The present invention will now be further described through specific embodiments, but is not limited thereto.

[0038] Example 1 Preparation Method of a Sandwich Electrochemical Immunosensor for Cardiac Troponin I

[0039] (1) A glassy carbon electrode with a diameter of 3.0 mm was polished to a mirror surface using Al2O3 polishing powder and then ultrasonically cleaned in anhydrous ethanol;

[0040] (2) 6.0 μL of 2.0 mg / mL Au NPs / N-CNTs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0041] (3) 6.0 μL of 5 μg / mL 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 4°C refrigerator;

[0042] (4) 3.0 μL of 1 mg / mL bovine serum albumin solution was added dropwise to the electrode surface to block nonspecific active sites on the electrode surface. The electrode surface was rinsed with pH 7.17 phosphate buffer and dried in a refrigerator at 4°C.

[0043] (5) Continue to add 6.0 μL of a series of cardiac troponin I (CTnI) antigen solutions with different concentrations of 10 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4°C;

[0044] (6) A 6.0 μL, 1.0 mg / mL meso-PdN NCs-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4°C refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4°C to prepare a sandwich electrochemical immunosensor based on Au NPs / N-CNTs;

[0045] Example 2 Preparation Method of a Sandwich-Type Electrochemical Immunosensor for Cardiac Troponin I

[0046] (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using Al2O3 polishing powder and then ultrasonically cleaned in anhydrous ethanol;

[0047] (2) 6.0 μL of 2.5 mg / mL Au NPs / N-CNTs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0048] (3) 6.0 μL of 10 μg / mL 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 4°C refrigerator.

[0049] (4) 3.0 μL of 1.5 mg / mL bovine serum albumin solution was added dropwise to the electrode surface to block nonspecific active sites on the electrode surface. The electrode surface was rinsed with pH 7.17 phosphate buffer and dried in a refrigerator at 4°C.

[0050] (5) Continue to add 6.0 μL of a series of cardiac troponin I (CTnI) antigen solutions with different concentrations of 10 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4°C;

[0051] (6) A 6.0 μL, 2.0 mg / mL meso-PdN NCs-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4°C refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4°C to prepare a sandwich electrochemical immunosensor based on Au NPs / N-CNTs.

[0052] Example 3 Preparation Method of a Sandwich Electrochemical Immunosensor for Cardiac Troponin I

[0053] (1) A glassy carbon electrode with a diameter of 5.0 mm was polished to a mirror surface using Al2O3 polishing powder and then ultrasonically cleaned in anhydrous ethanol;

[0054] (2) 6.0 μL of 3.0 mg / mL Au NPs / N-CNTs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0055] (3) 6.0 μL of 15 μg / mL 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 4°C refrigerator.

[0056] (4) 3.0 μL of 2 mg / mL bovine serum albumin solution was added dropwise to the electrode surface to block nonspecific active sites on the electrode surface. The electrode surface was rinsed with pH 7.17 phosphate buffer and dried in a refrigerator at 4°C.

[0057] (5) Continue to add 6.0 μL of a series of cardiac troponin I (CTnI) antigen solutions with different concentrations of 10 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4°C;

[0058] (6) A 6.0 μL, 3.0 mg / mL meso-PdN NCs-Ab2 dispersion droplet was applied to the electrode surface, placed in a 4°C refrigerator for 40 min, rinsed with pH = 7.17 phosphate buffer, and dried at 4°C to prepare a sandwich electrochemical immunosensor based on Au NPs / N-CNTs.

[0059] Preparation of Au NPs / N-CNTs as described in Example 4

[0060] (1) Preparation of N-CNTs

[0061] 340.0 mg of halloysite was dispersed in 240 mL of 10 mM Tris-HCl buffer (pH = 8.5) and sonicated for 30 minutes. After that, 220.0 mg of dopamine hydrochloride was added to the above dispersion and sonicated for 5 minutes, and then stirred at room temperature for 22 hours. After centrifugation and repeated washing with water and methanol, the polydopamine-coated halloysite was obtained in an oven at 70 ° C overnight. The product was placed in an argon atmosphere and heated in a tube furnace at 5 ° C / min, heated to 800 ° C and pyrolyzed at a constant temperature for 4 hours, and then desalted with 14 mL of 6M HCl and 14 mL of 12M HF for 12 hours with continuous stirring, and thoroughly washed with water-ethanol until neutral, and freeze-dried. The final product N-CNTs was obtained;

[0062] (2) Preparation of AuNPs

[0063] 0.5 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 99.5 mL of ultrapure water. An electromagnetic rotor was placed in the flask, stirred at a constant speed, and heated to boiling. Then, 2.0 mL of sodium citrate (1.0 wt%) was added to the flask, and the flask was kept boiling for 15 minutes to obtain uniform gold nanoparticles.

[0064] (3) Preparation of Au NPs / N-CNTs

[0065] Au NPs were loaded onto N-CNTs using an in situ reduction method. 4 mg of N-CNTs were dispersed in 2.68 mL of deionized water. 0.05 mL of 10 mM sodium citrate and 0.05 mL of 10 mM chloroauric acid were added while stirring. This was quickly followed by the addition of 0.06 mL of freshly prepared 0.1 M cold sodium borohydride. The mixture was stirred at room temperature for 20 h and then centrifuged and freeze-dried.

[0066] Preparation of Au NPs / N-CNTs as described in Example 5

[0067] (1) Preparation of N-CNTs

[0068] 350.0 mg of halloysite was dispersed in 250 mL of 10 mM Tris-HCl buffer (pH = 8.5) and sonicated for 30 minutes. After that, 230.0 mg of dopamine hydrochloride was added to the above dispersion and sonicated for 5 minutes, and then stirred at room temperature for 22 hours. After centrifugation and repeated washing with water and methanol, the polydopamine-coated halloysite was obtained in an oven at 70 ° C overnight. The product was placed in an argon atmosphere and heated in a tube furnace at 5 ° C / min, heated to 800 ° C and pyrolyzed at a constant temperature for 4 hours, and then desalted with 15 mL of 6M HCl and 15 mL of 12M HF for 12 hours with continuous stirring, and thoroughly washed with water-ethanol until neutral, and freeze-dried. The final product N-CNTs was obtained;

[0069] (2) Preparation of AuNPs

[0070] 1.0 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask containing 99 mL of ultrapure water. An electromagnetic rotor was placed in the flask, stirred at a constant speed, and heated to boiling. Then, 2.5 mL of sodium citrate (1.0 wt%) was added to the flask, and the flask was kept boiling for 15 minutes to obtain uniform gold nanoparticles.

[0071] (3) Preparation of Au NPs / N-CNTs

[0072] Au NPs were loaded onto N-CNTs using an in situ reduction method. 5.0 mg of N-CNTs were dispersed in 3.68 mL of deionized water. 0.1 mL of 10 mM sodium citrate and 0.1 mL of 10 mM chloroauric acid were added while stirring. This was quickly followed by the addition of 0.12 mL of freshly prepared 0.1 M cold sodium borohydride. The mixture was stirred at room temperature for 20 h and then centrifuged and freeze-dried.

[0073] Preparation of Au NPs / N-CNTs as described in Example 6

[0074] (1) Preparation of N-CNTs

[0075] 360.0 mg of halloysite was dispersed in 260 mL of 10 mM Tris-HCl buffer (pH = 8.5) and sonicated for 30 minutes. After that, 240.0 mg of dopamine hydrochloride was added to the above dispersion and sonicated for 5 minutes, and then stirred at room temperature for 22 hours. After centrifugation and repeated washing with water and methanol, the polydopamine-coated halloysite was obtained in an oven at 70 ° C overnight. The product was placed in an argon atmosphere and heated in a tube furnace at 5 ° C / min, heated to 800 ° C and pyrolyzed at a constant temperature for 4 hours, and then desalted with 16 mL of 6M HCl and 16 mL of 12M HF for 12 hours, and thoroughly washed with water-ethanol until neutral, and freeze-dried. The final product N-CNTs was obtained;

[0076] (2) Preparation of AuNPs

[0077] 1.5 mL of HAuCl4·4H2O (1.0 wt%) was added to a round-bottom flask filled with 98.5 mL of ultrapure water. An electromagnetic rotor was placed in the flask, stirred at a constant speed, and heated to boiling. Then, 3.0 mL of sodium citrate (1.0 wt%) was added to the flask, and the flask was kept boiling for 15 minutes to obtain uniform gold nanoparticles.

[0078] (3) Preparation of Au NPs / N-CNTs

[0079] Au NPs were loaded onto N-CNTs using an in situ reduction method. 6.0 mg of N-CNTs were dispersed in 4.68 mL of deionized water. 0.15 mL of 10 mM sodium citrate and 0.15 mL of 10 mM chloroauric acid were added while stirring. This was quickly followed by the addition of 0.18 mL of freshly prepared 0.1 M cold sodium borohydride. The mixture was stirred at room temperature for 20 h and then centrifuged and freeze-dried.

[0080] Preparation of meso-PdN NCs-Ab2 dispersion described in Example 7

[0081] (1) Preparation of PdNNCs

[0082] Mesoporous Pd nanocubes were synthesized by a one-pot solution phase method at 50°C using hexadecyltrimethylammonium chloride (CTAC) as a structure-directing surfactant, Na2PdCl4 as a metal precursor, L-ascorbic acid (AA) as a reducing agent, and H2O as a solvent. 0.30 mg of CTAC was dissolved in 4.0 mL of secondary deionized water, and 0.075 mL of 200 mM HCl was added to adjust the pH of the reaction solution. 0.20 mL of 80 mM KCl and 0.20 mL of 10 mM Na2PdCl4 solution were added to the above solution in sequence and kept at 50°C for 30 minutes. Under gentle shaking, 0.40 mL of 0.30 M freshly prepared AA was quickly injected into the above solution. After reacting for 1.0 hour, the product was collected by centrifugation, washing with water 3 times, and then freeze-drying.

[0083] (2) Preparation of meso-PdN NCs

[0084] 0.8 mL of synthesized mesoscopic PdNCs (1.80 mg mL -1), 45 mg of urea, and 45 mg of polyvinylpyrrolidone (PVP) were mixed with 3.5 mL of deionized water. After sonication, the resulting solution was transferred to a 20 mL autoclave. The autoclave was heated to 180°C for 1.5 hours and then cooled to room temperature. To remove the surfactant from the mesoscopic PdN NCs, the NCs were washed three times with acetic acid and three times with ethanol / water before being lyophilized.

[0085] (3) Preparation of meso-PdNNCs-Ab2

[0086] 0.8 mL PdN NCs (3.0 mg mL -1 ) and 0.8 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4° C. The resulting product was centrifuged several times using phosphate buffered saline (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.

[0087] Preparation of meso-PdN NCs-Ab2 dispersion described in Example 8

[0088] (1) Preparation of PdNNCs

[0089] Mesoporous Pd nanocubes were synthesized by a one-pot solution phase method at 50°C using hexadecyltrimethylammonium chloride (CTAC) as a structure-directing surfactant, Na2PdCl4 as a metal precursor, L-ascorbic acid (AA) as a reducing agent, and H2O as a solvent. 0.325 mg of CTAC was dissolved in 5.0 mL of secondary deionized water, and 0.1 mL of 200 mM HCl was added to adjust the pH of the reaction solution. 0.24 mL of 80 mM KCl and 0.25 mL of 10 mM Na2PdCl4 solution were added to the above solution in sequence and kept at 50°C for 30 minutes. Under gentle shaking, 0.50 mL of 0.30 M freshly prepared AA was quickly injected into the above solution. After reacting for 1.0 hour, the product was collected by centrifugation, washing with water 3 times, and then freeze-drying.

[0090] (2) Preparation of meso-PdN NCs

[0091] 1.0 mL of synthesized mesoscopic PdNCs (1.80 mg mL -1 ), 50 mg of urea, and 50 mg of polyvinylpyrrolidone (PVP) were mixed with 4.0 mL of deionized water. After sonication, the resulting solution was transferred to a 20 mL autoclave. The autoclave was heated to 180°C for 1.5 hours and then cooled to room temperature. To remove the surfactant from the mesoscopic PdN NCs, the NCs were washed three times with acetic acid and three times with ethanol / water before being freeze-dried.

[0092] (3) Preparation of meso-PdNNCs-Ab2

[0093] 1.0 mL PdN NCs (3.0 mg mL -1 ) and 1.0 mL Ab2 solution (10 μg mL -1 ) was shaken overnight at 4° C. The resulting product was centrifuged several times using phosphate buffered saline (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.

[0094] Preparation of meso-PdN NCs-Ab2 dispersion described in Example 9

[0095] (1) Preparation of PdNNCs

[0096] Mesoporous Pd nanocubes were synthesized by a one-pot solution phase method at 50°C using hexadecyltrimethylammonium chloride (CTAC) as a structure-directing surfactant, Na2PdCl4 as a metal precursor, L-ascorbic acid (AA) as a reducing agent, and H2O as a solvent. 0.35 mg of CTAC was dissolved in 6.0 mL of secondary deionized water, and 0.125 mL of 200 mM HCl was added to adjust the pH of the reaction solution. 0.28 mL of 80 mM KCl and 0.30 mL of 10 mM Na2PdCl4 solution were added to the above solution in sequence and kept at 50°C for 30 minutes. Under gentle shaking, 0.60 mL of 0.30 M freshly prepared AA was quickly injected into the above solution. After reacting for 1.0 hour, the product was collected by centrifugation, washing with water 3 times, and then freeze-drying.

[0097] (2) Preparation of meso-PdN NCs

[0098] 1.2 mL of synthesized mesoscopic PdNCs (1.80 mg mL -1 ), 55 mg of urea, and 55 mg of polyvinylpyrrolidone (PVP) were mixed with 4.5 mL of deionized water. After sonication, the resulting solution was transferred to a 20 mL autoclave. The autoclave was heated to 180°C for 1.5 hours and then cooled to room temperature. To remove the surfactant from the mesoscopic PdN NCs, the NCs were washed three times with acetic acid and three times with ethanol / water before being freeze-dried.

[0099] (3) Preparation of meso-PdNNCs-Ab2

[0100] 1.2 mL of PdN NCs (3.0 mg mL -1 ) and 1.2 mL of Ab2 solution (10 μg mL-1 ) was shaken overnight at 4° C. The resulting product was centrifuged several times using phosphate buffered saline (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.

[0101] Detection of cardiac troponin I (CTnI) antigen by the sandwich electrochemical immunosensor for cardiac troponin I described in Example 10

[0102] (1) Using an electrochemical workstation, the test was performed in a 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 in 10 mL of a phosphate buffer solution containing 5 mol / L hydrogen peroxide solution at pH 5.0-8.5;

[0103] (2) Analytes were detected using chronoamperometry with an input voltage of −0.4 V, a sampling interval of 0.1 s, and a run time of 400 s;

[0104] (3) When the background current stabilized, 10 μL of 5 mol / L hydrogen peroxide solution was injected into 10 mL of pH 7.17 phosphate buffer solution every 50 s, and the current change was recorded.

[0105] (4) Record the current peak corresponding to different concentrations of cardiac troponin I (CTnI) antigen;

[0106] (5) Using the working curve method, the linear detection range of cardiac troponin I (CTnI) antigen in the test sample was 10 fg / mL to 100 ng / mL, and the lowest detection limit was 13.02 fg / mL.

Claims

1. A method for preparing a sandwich-type electrochemical immunosensor for cardiac troponin I, characterized in that: The steps include: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using Al2O3 polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) 6.0 μL of 0.5-1.5 mg / mL AuNPs / N-CNTs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature; (3) 6.0 μL of 5-15 μg / mL 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 4°C refrigerator. (4) 3.0 μL of 1 wt% bovine serum albumin solution was added dropwise to the electrode surface to block nonspecific active sites on the electrode surface. The electrode surface was rinsed with pH 7.17 phosphate buffer and dried in a refrigerator at 4°C. (5) Continue to add 6.0 μL of a series of cardiac troponin I (cTnI) antigen solutions with different concentrations of 10 fg / mL to 100 ng / mL, rinse with pH = 7.17 phosphate buffer, and dry in a refrigerator at 4°C; (6) 6.0 μL of 2.0-4.0 mg / mL meso-PdNNCs-Ab2 dispersion was dropwise applied to the electrode surface, placed in a 4°C refrigerator for 40 min, rinsed with pH 7.17 phosphate buffer, and dried at 4°C to prepare a sandwich-type chemical immunosensor for cardiac troponin I. The preparation of AuNPs / N-CNTs in step (1) is as follows: ① Preparation of N-CNTs 340.0-360.0 mg of halloysite was dispersed in 240-260 mL of 10 mM pH = 8.5 Tris-HCl buffer and sonicated for 30 minutes; then, 220.0-240.0 mg of dopamine hydrochloride was added to the above dispersion and sonicated for 5 minutes, and then stirred at room temperature for 22 hours; after centrifugation and repeated washing with water and methanol, the mixture was dried in an oven at 70°C overnight to obtain polydopamine-coated halloysite; the product was placed in an argon atmosphere and heated in a tube furnace at 5°C / min to 800°C, and pyrolyzed at this temperature for 4 hours, and then desalted with 14-16 mL of 6M HCl and 14-16 mL of 12M HF with continuous stirring for 12 hours, and thoroughly washed with water-ethanol until neutral, and freeze-dried to obtain the final product N-CNTs; ②Preparation of AuNPs 0.5-1.5 mL of HAuCl4·4H2O was added to a round-bottom flask containing 98.5-99.5 mL of ultrapure water. An electromagnetic rotor was placed in the flask, and the mixture was stirred at a constant speed and heated to boiling. Then, 2.0-3.0 mL of sodium citrate was added to the flask, and the mixture was kept boiling for 15 minutes to obtain uniform gold nanoparticles. ③ Preparation of AuNPs / N-CNTs AuNPs were loaded on N-CNTs by in situ reduction method; 4-6 mg N-CNTs were dispersed in 2.68-4.68 mL secondary deionized water, and 0.05-0.15 mL 10 mM sodium citrate and 0.05-0.15 mL 10 mM chloroauric acid were added while stirring, followed by rapid addition of 0.06-0.18 mL 0.1 M cold sodium borohydride, stirred at room temperature for 20 h, and centrifuged and freeze-dried; the preparation of meso-PdNNCs-Ab2 described in step (6) was as follows: ① Preparation of PdNNCs PdNCs were synthesized via a one-pot solution-phase method at 50°C using hexadecyltrimethylammonium chloride (CTAC) as a structure-directing surfactant, Na2PdCl4 as a metal precursor, L-ascorbic acid (AA) as a reducing agent, and H2O as a solvent. 0.30-0.35 mg of CTAC was dissolved in 4.0-6.0 mL of deionized water, and the pH of the reaction solution was adjusted by adding 0.075-0.125 mL of 200 mM HCl. 0.20-0.28 mL of 80 mM KCl and 0.20-0.30 mL of 10 mM Na2PdCl4 were sequentially added to the above solution, and the mixture was maintained at 50°C for 30 min. With gentle shaking, 0.40-0.60 mL of 0.30 M freshly prepared AA was rapidly injected into the above solution. After a 1.0 hour reaction, the product was washed three times with water by centrifugation and then lyophilized. ② Preparation of meso-PdNNCs 0.8–1.2 mL of synthesized PdNCs, 45–55 mg of urea, and 45–55 mg of polyvinylpyrrolidone (PVP) were mixed with 3.5–4.5 mL of deionized water. After sonication, the resulting solution was transferred to a 20 mL autoclave. The autoclave was heated to 180°C for 1.5 hours and then cooled to room temperature. To remove the surfactant from the meso-PdNNCs, the meso-PdNNCs were washed three times with acetic acid and three times with ethanol / water before being freeze-dried. ③ Preparation of meso-PdNNCs-Ab2 0.8-1.2 mL of meso-PdNNCs and 0.8-1.2 mL of Ab2 solution were shaken overnight at 4°C using a shaker; the resulting product was centrifuged several times using phosphate buffered saline (PBS) as a dispersion solution to wash away unbound Ab2, and then re-dispersed in PBS and stored in a refrigerator at 4°C.

2. A sandwich electrochemical immunosensor for cardiac troponin I prepared by the preparation method according to claim 1, used in a method for detecting cardiac troponin I for non-diagnostic and / or therapeutic purposes, characterized in that: The detection method comprises the following steps: (1) 6 μL of cardiac troponin I (cTnI) antigen solution of different concentrations was drop-coated on the electrode surface, reacted for 0.5 to 2 h, and then connected to an electrochemical workstation after drying. The electrodes were immersed in 10 mL of pH = 7.17 phosphate buffer solution to measure the current changes. When the background current stabilized, 10 μL of 5 mol / L hydrogen peroxide solution was injected into the 10 mL of pH = 7.17 phosphate buffer solution every 50 s, and the current changes were recorded. (2) based on the linear relationship between the obtained current difference and the cardiac troponin I (cTnI) antigen concentration, a working curve was drawn; (3) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the sample to be tested is obtained.