Preparation and application of dual-mode immunosensor based on PdSn bifunctional nano-enzyme

Abstract

The invention belongs to the technical field of novel functional nano materials, immunoassay and biosensing, and provides preparation of a dual-mode immunosensor based on PdSn dual-function nano enzyme, which is used for sensitively detecting cardiac troponin I (cTnI). A palladium-tin alloy / molybdenum carbide composite material (PdSn / Mo2C-C) serving as a second antibody beacon and a gold nanoparticle / hydrogen bond organic framework composite material (Au NPs / HOFs) serving as a substrate material are prepared respectively, a sandwich type sensor is constructed based on the materials, and the cTnI antigen concentration in an actual serum sample is quantitatively detected by using dual modes (an electrochemical method and a colorimetric method). Visually explainable and ultra-sensitive bimodal analysis is realized, the advantages of high sensitivity, low detection limit and the like are shown, and a new thought is provided for cTnI detection.

Description

Technical Field

[0001] The present invention relates to the preparation and application of a dual-mode immunosensor, specifically a sandwich-type immunosensor with a palladium-tin alloy / molybdenum carbide composite material (PdSn / Mo2C-C) as a marker and a gold nanoparticle / hydrogen bond organic framework composite material (Au NPs / HOF) as a base material. The present invention belongs to the field of novel functional nanomaterials, immunoassay and biosensor technology. Background Art

[0002] Cardiovascular diseases (CVDs) remain the diseases with the highest morbidity and mortality worldwide, and myocardial infarction (MI) is one of the most critical and common diseases. Timely and accurate diagnosis of MI is crucial for effective intervention and improved patient prognosis. Cardiac troponin I (cTnI) is a highly specific biomarker of myocardial injury. After myocardial injury, the concentration of cTnI in the blood of patients increases rapidly and lasts for a long time, providing a long window period for detection. Therefore, as an important biomarker for the diagnosis of MI, it is necessary to achieve high sensitivity detection of cTnI. In recent years, the development of immunosensor technology has provided a promising alternative to traditional methods. Immunosensors combine immunoassay technology and sensor technology and have the potential to provide faster, more sensitive and lower-cost diagnosis. The dual-channel signal detection strategy not only provides stable and diverse readout signals to improve sensitivity and accuracy, but also provides different quantification methods, thereby expanding the scope of its sensing applicability. Colorimetry is favored for its simplicity and suitability for on-site detection due to its easy handling and visually interpretable signal readout, and is expected to replace traditional detection methods in cTnI analysis. Integrating colorimetric signals with other sensing modes into a multi-signal platform can significantly improve the overall performance of the detection system. Electrochemical immunosensors show low background interference and high sensitivity because electrochemical immunosensors mainly convert the content of the analyte into electrochemical signals, a feature that can help overcome the problem of color resolution limitation in colorimetric analysis. Therefore, creating a dual-channel sensing platform that combines colorimetry and electrochemistry makes visually interpretable and ultrasensitive dual-modal analysis possible.

[0003] Natural enzymes are typical biomacromolecules that can accelerate various chemical reactions under extremely mild conditions in biochemical systems. To solve the problems of instability and high cost of natural enzymes, nanozymes (denoted as nanomaterials with enzyme-like properties) have become potential candidates to replace natural enzymes due to their high stability, durability and low cost. Nanozymes are generally divided into three categories: peroxidase-like (POD), oxidoreductase-like (OXD) and esterase-like. The pd orbital hybridization strategy is considered to be an effective method to optimize the electronic structure of catalysts. The introduction of d-block transition metals doped with p-block metal atoms can introduce additional electronic states or change the band structure, thereby adjusting the position of the d-band center. This adjustment has the potential to change the interaction between the nanozyme and the substrate or reaction intermediates, thereby improving the catalytic activity and selectivity. Herein, a bifunctional nanozyme with catalase-like (POD) and oxidoreductase-like (OXD) properties of p-block metal Sn doped Pd (PdSn) characterized by pd orbital hybridization was prepared. First, the pd orbital hybridization causes the d-band center to move upward relative to the Fermi level, thereby promoting H2O2 adsorption and generating a significant current signal through electrochemical testing (chronoamperometry). In addition, the pd orbital hybridization provides high-energy electrons, which makes the PdSn nanozyme have strong reducing ability. When TMB is used as a catalytic substrate, it catalyzes the reduction of O2 to generate H2O through a four-electron pathway. At the same time, TMB is oxidized to oxTMB, resulting in a corresponding color change, which is applied to the colorimetric immunoassay of cTnI.

[0004] The present invention uses a molybdenum carbide (Mo2C-C) composite material (PdSn / Mo2C-C) loaded with a palladium-tin bifunctional nanozyme (PdSn) as a beacon material and gold nanoparticles / hydrogen-bonded organic frameworks (Au NPs / HOFs) as the substrate material of the sensing system to construct a sandwich electrochemical immunosensor, and the PdSn / Mo2C-C material not fixed on the electrode surface is used for colorimetric immunoassay. Thanks to the excellent catalase-like and oxidoreductase-like dual activities of PdSn nanozymes, the designed dual-channel signal detection sensing platform can achieve ultra-sensitive detection of cTnI, expanding the application of nanozymes in biosensing. Summary of the invention

[0005] The present invention provides a preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme, 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 dual-mode immunosensor of a PdSn bifunctional nanozyme.

[0007] The second purpose of the present invention is to use the prepared PdSn bifunctional nanozyme dual-mode immunosensor for the detection of cardiac troponin I (cTnI) antigen.

[0008] The technical solution of the present invention comprises the following steps: 1. Preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme, 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 1.5 ~ 2.5 mg / mL Au NPs / HOFs dispersion onto the electrode surface 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 7.38 phosphate buffer, and dry it in a 37 °C constant temperature drying oven; (4) Continue to add 6.0 μL of 0.5 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 = 7.38 phosphate buffer, and dry it in a constant temperature drying oven at 37 °C; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a constant temperature drying oven at 37 °C; (6) 6.0 μL of 1.5 ~ 2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of pH = 7.38 phosphate buffer. 5.0 ~ 15.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer and shaken for 20 min. The absorbance of the supernatant was measured and the washed electrode was dried at 37 °C to obtain a sandwich electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0009] 2. Preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme, the preparation of the relevant materials, the steps are as follows: (1) Preparation of PdSn bifunctional nanozymes 8.0 ~ 12.0 mg of palladium acetylacetonate, 70.0 ~ 90.0 mg of polyvinyl pyrrolidone (MW 24000), and 1.81 ~ 2.01 mg of tin tetrachloride were dissolved in 4.0 ~ 6.0 mL of N,N-dimethylformamide, stirred for 15 minutes, and the mixed solution was transferred to a 20 mL reactor, heated to 180 °C for 12 hours, and then cooled to room temperature. The product was washed several times with ethanol and ultrapure water, respectively, to obtain PdSn bifunctional nanozyme; (2) Preparation of Mo2C-C At room temperature, add 60.0 ~ 80.0 mg dopamine hydrochloride and 45.0 ~ 55.0 mg polyether F127 to a mixed solvent containing 7.0 ~ 9.0 mL water and 8.0 ~ 12.0 mL ethanol, and stir until the solution is clear. Subsequently, add 1.5 ~ 2.5 mL 5 mg / mL ammonium molybdate to the solution, and then slowly add 0.3 ~ 0.5 mL of 1,3,5-trimethylbenzene, and continue stirring for 30 min. Add 0.4 ~ 0.6 mL of concentrated ammonia water, react for 2 hours, centrifuge, wash with ethanol 3 times, and dry at 60 ° C overnight to obtain Mo2C. Heat the product in a tubular furnace in a nitrogen atmosphere, raise the temperature to 800 ° C at 5 ° C / min, and carbonize at a constant temperature for 3 hours to obtain the product Mo2C-C; (3) Preparation of PdSn / Mo2C-C 3.0 ~ 5.0 mg PdSn / Mo2C-C and 4.0 ~ 6.0 mL anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 min. Then, 40.0 ~ 60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated to reflux at 100 °C for 2 h. After the solution was cooled naturally, it was washed with deionized water and freeze-dried to obtain amino Mo2C-C (NH2-Mo2C-C). Subsequently, 3.0 ~ 5.0 mg PdSn and 3.0 ~ 5.0 mg NH2-Mo2C-C were dispersed in 1.5 ~ 2.5 mL deionized water and ultrasonicated for 30 min. Then, the mixed solution was shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL); (4) Preparation of Ab2-PdSn / Mo2C-C 1.5 ~ 2.5 mL cTnI-Ab2 solution (20 μg / mL) and 1.5 ~ 2.5 mL PdSn / Mo2C-C (2.0 mg / mL) were shaken overnight at 4 °C. Phosphate buffer (PBS) (pH = 7.38) was used as the dispersion solution, centrifuged several times, and redispersed in PBS to obtain Ab2-PdSn / Mo2C-C, which was stored in a refrigerator at 4 °C; (5) Preparation of HOFs Add 0.2 ~ 0.3 g of trimesic acid (BTC) and 0.1 ~ 0.2 g of melamine (MA) to 30.0 ~ 40.0 mL of methanol, seal and ultrasonicate for about 2 h to form a uniform milky white suspension. Transfer the prepared milky white suspension to a 50 mL autoclave, heat to 150 °C, and keep for 12 h; (6) Preparation of Au NPs / HOFs Au NPs were immobilized on HOFs by in situ reduction method. Specifically, 13.0-17.0 mg HOFs were dispersed in 16.4-20.4 mL deionized water, and 0.4-0.6 mL 10 mmol / L sodium citrate and 0.4-0.6 mL 10 mmol / L chloroauric acid were added while stirring, and then 0.5-0.7 mL 0.1 mol / L cold sodium borohydride was quickly added, stirred at room temperature for 20 h, and centrifuged and freeze-dried.

[0010] A dual-mode immunosensor based on PdSn bifunctional nanozyme was prepared for the detection of cardiac troponin I (cTnI) antigen, 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 immunosensor as the working electrode, in 10 mL of a phosphate buffer solution containing 5 mol / L hydrogen peroxide solution at pH 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, inject 10 µL of 5 mol / L hydrogen peroxide solution into 10 mL of pH = 7.38 phosphate buffer solution every 50 s and record the current change; (4) Recording the current peaks corresponding to cardiac troponin I (cTnI) antigen at different concentrations; (5) Transfer the Ab2-PdSn / Mo2C-C not connected to the electrode surface to 1 mL of pH = 7.38 phosphate buffer solution, add 3,3',5,5'-tetramethylbenzidine (TMB) to the solution, shake for 20 min, and measure the absorbance at 652 nm using a UV-visible spectrophotometer; (6) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the sample to be tested is obtained.

[0011] Beneficial results of the present invention (1) The PdSn bifunctional nanozyme prepared by the present invention has excellent catalase-like and oxidoreductase-like dual activities. The pd orbital hybridization causes the d band center to move upward relative to the Fermi level, thereby promoting H2O2 adsorption and generating obvious current signals through electrochemical testing (chronoamperometry). In addition, the pd orbital hybridization provides high-energy electrons, which makes the PdSn nanozyme have strong reducing ability. When TMB is used as a catalytic substrate, it catalyzes the reduction of O2 to generate H2O through a four-electron pathway. At the same time, TMB is oxidized to blue oxTMB, which is used in the colorimetric immunoassay of cTnI; (2) A dual-mode immunosensor based on PdSn bifunctional nanozyme was used to detect cardiac troponin I (cTnI) antigen. The linear detection range of cardiac troponin I (cTnI) antigen was 10 fg / mL ~ 100 ng / mL, the electrochemical minimum detection limit was 0.857 fg / mL and the colorimetric minimum detection limit was 0.864 ng / mL, indicating that the prepared PdSn bifunctional nanozyme dual-mode immunosensor can accurately and quantitatively detect cardiac troponin I (cTnI) antigen. DETAILED DESCRIPTION

[0012] The present invention will now be further described by way of specific embodiments, but is not limited thereto.

[0013] Example 1 A method for preparing a dual-mode immunosensor based on PdSn bifunctional nanozyme (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 1.5 mg / mL Au NPs / HOFs dispersion onto the electrode surface, rinse with ultrapure water, and dry 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 = 7.38 phosphate buffer, and dry it in a 37 °C constant temperature drying oven; (4) Continue to add 3.0 μL of 0.5 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 = 7.38 phosphate buffer, and dry it in a constant temperature drying oven at 37 °C; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a constant temperature drying oven at 37 °C; (6) 6.0 μL, 1.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of pH 7.38 phosphate buffer. 5 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer and shaken for 20 min. The absorbance of the supernatant was measured and the washed electrode was dried at 37 °C to obtain a sandwich electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0014] Example 2 A method for preparing a dual-mode immunosensor based on PdSn bifunctional nanozyme (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 2.0 mg / mL Au NPs / HOFs dispersion onto the electrode surface, rinse with ultrapure water, and dry 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 = 7.38 phosphate buffer, and dry it in a 37 °C constant temperature drying oven; (4) Continue to add 3.0 μL of 0.5 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 = 7.38 phosphate buffer, and dry it in a constant temperature drying oven at 37 °C; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a constant temperature drying oven at 37 °C; (6) 6.0 μL of 2.0 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of pH 7.38 phosphate buffer and 10.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer and shaken for 20 min. The absorbance of the supernatant was measured and the washed electrode was dried at 37 °C to obtain a sandwich electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0015] Example 3 A method for preparing a dual-mode immunosensor based on PdSn bifunctional nanozyme (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 2.5 mg / mL Au NPs / HOFs dispersion onto the electrode surface, rinse with ultrapure water, and dry 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 = 7.38 phosphate buffer, and dry it in a 37 °C constant temperature drying oven; (4) Continue to add 3.0 μL of 0.5 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 = 7.38 phosphate buffer, and dry it in a constant temperature drying oven at 37 °C; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a constant temperature drying oven at 37 °C; (6) 6.0 μL of 2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of pH 7.38 phosphate buffer. 15.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer and shaken for 20 min. The absorbance of the supernatant was measured and the washed electrode was dried at 37 °C to obtain a sandwich electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0016] Preparation of Ab2-PdSn / Mo2C-C as described in Example 4 (1) Preparation of PdSn bifunctional nanozymes 8.0 mg of palladium acetylacetonate, 70.0 mg of polyvinyl pyrrolidone (MW 24000), and 1.81 mg of tin tetrachloride were dissolved in 4.0 mL of N,N-dimethylformamide and stirred for 15 minutes. The mixed solution was transferred to a 20 mL reactor and heated to 180 °C for 12 hours, then cooled to room temperature. The product was washed several times with ethanol and ultrapure water, respectively, to obtain PdSn bifunctional nanozyme. (2) Preparation of Mo2C-C At room temperature, add 60 mg of dopamine hydrochloride and 45 mg of polyether F127 to a mixed solvent containing 7 mL of water and 8 mL of ethanol, and stir until the solution is clear. Subsequently, add 1.5 mL of 5 mg / mL ammonium molybdate to the solution, and then slowly add 0.3 mL of 1,3,5-trimethylbenzene and continue stirring for 30 min. Add 0.4 mL of concentrated ammonia water, react for 2 hours, centrifuge, wash with ethanol 3 times, and dry at 60 ° C overnight to obtain Mo2C. Heat the product in a tubular furnace in a nitrogen atmosphere, raise the temperature to 800 ° C at 5 ° C / min, and carbonize at a constant temperature for 3 hours to obtain the product Mo2C-C; (3) Preparation of PdSn / Mo2C-C 3.0 mg PdSn / Mo2C-C and 4.0 mL anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 min. Then, 40 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated to reflux at 100 °C for 2 h. After the solution was cooled naturally, it was washed with deionized water and freeze-dried to obtain amino Mo2C-C (NH2-Mo2C-C). Subsequently, 3.0 mg PdSn and 3.0 mg NH2-Mo2C-C were dispersed in 1.5 mL deionized water and ultrasonicated for 30 min. Then, the mixed solution was shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL); (4) Preparation of Ab2-PdSn / Mo2C-C 1.5 mL of cTnI-Ab2 solution (20 μg / mL) and 1.5 mL of PdSn / Mo2C-C (2.0 mg / mL) were shaken overnight at 4 °C. Phosphate buffered saline (PBS) (pH = 7.38) was used as the dispersion solution, centrifuged several times, and redispersed in PBS to obtain Ab2-PdSn / Mo2C-C, which was stored in a refrigerator at 4 °C.

[0017] Preparation of Ab2-PdSn / Mo2C-C as described in Example 5 (1) Preparation of PdSn bifunctional nanozymes 10.0 mg of palladium acetylacetonate, 80.0 mg of polyvinyl pyrrolidone (MW 24000), and 1.91 mg of tin tetrachloride were dissolved in 5.0 mL of N,N-dimethylformamide and stirred for 15 minutes. The mixed solution was transferred to a 20 mL reactor and heated to 180 °C for 12 hours, then cooled to room temperature. The product was washed several times with ethanol and ultrapure water, respectively, to obtain PdSn bifunctional nanozyme. (2) Preparation of Mo2C-C At room temperature, add 70.0 mg dopamine hydrochloride and 50.0 mg polyether F127 to a mixed solvent containing 8.0 mL water and 10.0 mL ethanol, and stir until the solution is clear. Subsequently, add 2.0 mL 5 mg / mL ammonium molybdate to the solution, and then slowly add 0.4 mL of 1,3,5-trimethylbenzene, and continue stirring for 30 min. Add 0.5 mL of concentrated ammonia water, react for 2 hours, centrifuge, wash with ethanol 3 times, and dry at 60 ° C overnight to obtain Mo2C. Heat the product in a tubular furnace in a nitrogen atmosphere, raise the temperature to 800 ° C at 5 ° C / min, and carbonize at a constant temperature for 3 hours to obtain the product Mo2C-C; (3) Preparation of PdSn / Mo2C-C 4.0 mg PdSn / Mo2C-C and 5.0 mL anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 min. Then, 50.0 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated to reflux at 100 °C for 2 h. After the solution was cooled naturally, it was washed with deionized water and freeze-dried to obtain amino Mo2C-C (NH2-Mo2C-C). Subsequently, 4.0 mg PdSn and 4.0 mg NH2-Mo2C-C were dispersed in 2.0 mL deionized water and ultrasonicated for 30 min. Then, the mixed solution was shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL); (4) Preparation of Ab2-PdSn / Mo2C-C 2.0 mL of cTnI-Ab2 solution (20 μg / mL) and 2.0 mL of PdSn / Mo2C-C (2.0 mg / mL) were shaken overnight at 4 °C. Phosphate buffered saline (PBS) (pH = 7.38) was used as the dispersion solution, centrifuged several times, and redispersed in PBS to obtain Ab2-PdSn / Mo2C-C, which was stored in a refrigerator at 4 °C.

[0018] Preparation of Ab2-PdSn / Mo2C-C as described in Example 6 (1) Preparation of PdSn bifunctional nanozymes 12.0 mg of palladium acetylacetonate, 90.0 mg of polyvinyl pyrrolidone (MW 24000), and 2.01 mg of tin tetrachloride were dissolved in 6.0 mL of N,N-dimethylformamide and stirred for 15 minutes. The mixed solution was transferred to a 20 mL reactor and heated to 180 °C for 12 hours, then cooled to room temperature. The product was washed several times with ethanol and ultrapure water, respectively, to obtain PdSn bifunctional nanozyme. (2) Preparation of Mo2C-C At room temperature, add 80.0 mg dopamine hydrochloride and 55.0 mg polyether F127 to a mixed solvent containing 9.0 mL water and 12.0 mL ethanol, and stir until the solution is clear. Subsequently, add 2.5 mL 5 mg / mL ammonium molybdate to the solution, and then slowly add 0.5 mL of 1,3,5-trimethylbenzene and continue stirring for 30 min. Add 0.6 mL of concentrated ammonia water, react for 2 hours, centrifuge, wash with ethanol 3 times, and dry at 60 ° C overnight to obtain Mo2C. Heat the product in a tubular furnace in a nitrogen atmosphere, raise the temperature to 800 ° C at 5 ° C / min, and carbonize at a constant temperature for 3 hours to obtain the product Mo2C-C; (3) Preparation of PdSn / Mo2C-C 5.0 mg PdSn / Mo2C-C and 6.0 mL anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 min. Then, 60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated to reflux at 100 °C for 2 h. After the solution was cooled naturally, it was washed with deionized water and freeze-dried to obtain amino Mo2C-C (NH2-Mo2C-C). Subsequently, 5.0 mg PdSn and 5.0 mg NH2-Mo2C-C were dispersed in 2.5 mL deionized water and ultrasonicated for 30 min. Then, the mixed solution was shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL); (4) Preparation of Ab2-PdSn / Mo2C-C 2.5 mL of cTnI-Ab2 solution (20 μg / mL) and 2.5 mL of PdSn / Mo2C-C (2.0 mg / mL) were shaken overnight at 4 °C. Phosphate buffered saline (PBS) (pH = 7.38) was used as the dispersion solution, centrifuged several times, and redispersed in PBS to obtain Ab2-PdSn / Mo2C-C, which was stored in a refrigerator at 4 °C.

[0019] Example 7 Preparation of the Au NPs / HOFs (1) Preparation of HOFs 0.2 g of trimesic acid (BTC) and 0.1 g of melamine (MA) were added to 30.0 mL of methanol, sealed and ultrasonicated for about 2 h to form a uniform milky white suspension. The prepared milky white suspension was transferred to a 50 mL autoclave, heated to 150 °C, and maintained for 12 h; (2) Preparation of Au NPs / HOFs Au NPs were immobilized on HOFs by in situ reduction method. Specifically, 13.0 mg HOFs were dispersed in 16.4 mL deionized water, 0.4 mL 10 mmol / L sodium citrate and 0.4 mL 10 mmol / L chloroauric acid were added while stirring, and then 0.5 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added, stirred at room temperature for 20 h, and centrifuged and freeze-dried.

[0020] Preparation of Au NPs / HOFs as described in Example 8 (1) Preparation of HOFs 0.25 g of trimesic acid (BTC) and 0.15 g of melamine (MA) were added to 35.0 mL of methanol, sealed and ultrasonicated for about 2 h to form a uniform milky white suspension. The prepared milky white suspension was transferred to a 50 mL autoclave, heated to 150 °C, and maintained for 12 h; (2) Preparation of Au NPs / HOFs Au NPs were immobilized on HOFs by in situ reduction method. Specifically, 15.0 mg HOFs were dispersed in 18.4 mL deionized water, 0.5 mL 10 mmol / L sodium citrate and 0.5 mL 10 mmol / L chloroauric acid were added while stirring, and then 0.6 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added, stirred at room temperature for 20 h, and centrifuged and freeze-dried.

[0021] Preparation of Au NPs / HOFs as described in Example 9 (1) Preparation of HOFs 0.3 g of trimesic acid (BTC) and 0.2 g of melamine (MA) were added to 40.0 mL of methanol, sealed and ultrasonicated for about 2 h to form a uniform milky white suspension. The prepared milky white suspension was transferred to a 50 mL autoclave, heated to 150 °C, and maintained for 12 h; (2) Preparation of Au NPs / HOFs Au NPs were immobilized on HOFs by in situ reduction method. Specifically, 17.0 mg HOFs were dispersed in 20.4 mL deionized water, 0.6 mL 10 mmol / L sodium citrate and 0.6 mL 10 mmol / L chloroauric acid were added while stirring, and then 0.7 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added, stirred at room temperature for 20 h, and centrifuged and freeze-dried.

[0022] Example 10 Detection of cardiac troponin I (cTnI) antigen by the dual-mode immunosensor based on PdSn bifunctional nanozyme (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 immunosensor as the working electrode in 10 mL of a pH 5.0-8.5 phosphate buffer solution; (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, inject 10 µL of 5 mol / L hydrogen peroxide solution into 10 mL of pH = 7.38 phosphate buffer solution every 50 s and record the current change; (4) Recording the current peaks corresponding to cardiac troponin I (cTnI) antigen at different concentrations; (5) Transfer the Ab2-PdSn / Mo2C-C not connected to the electrode surface to 1 mL of pH = 7.38 phosphate buffer solution, add 3,3',5,5'-tetramethylbenzidine (TMB) to the solution, shake for 20 min, and measure the absorbance at 652 nm using a UV-visible spectrophotometer; (6) The linear detection range of cardiac troponin I (cTnI) antigen in the test sample is 10 fg / mL ~ 100 ng / mL. Using the working curve method, the electrochemical minimum detection limit is calculated to be 0.857 fg / mL, and the colorimetric minimum detection limit is 0.864 ng / mL.

Claims

1. Preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme, characterized in that: The following steps are involved: (1) Preparation of Ab2-PdSn / Mo2C-C: PdSn bifunctional nanozyme and molybdenum carbide (Mo2C-C) were prepared separately, and the two materials were combined with cTnI-Ab2; (2) Preparation of Au NPs / HOFs: In situ growth of gold nanoparticles (Au NPs) on the surface of separately prepared hydrogen-bonded organic frameworks (HOFs); (3) A sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme was constructed. The cardiac troponin I (cTnI) antigen content was determined by electrochemical and colorimetric dual modes, and a working curve was drawn.

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 Ab2-PdSn / Mo2C-C described in step (1) is as follows: (1) Preparation of PdSn bifunctional nanozymes 8.0 ~ 12.0 mg of palladium acetylacetonate, 70.0 ~ 90.0 mg of polyvinyl pyrrolidone (MW 24000), and 1.81 ~ 2.01 mg of tin tetrachloride were dissolved in 4.0 ~ 6.0 mL of N,N-dimethylformamide, stirred for 15 minutes, and the mixed solution was transferred to a 20 mL reactor, heated to 180 ° C for 12 hours, and then cooled to room temperature. The product was washed several times with ethanol and ultrapure water, respectively, to obtain PdSn bifunctional nanozyme; (2) Preparation of Mo2C-C At room temperature, add 60.0 ~ 80.0 mg dopamine hydrochloride and 45.0 ~ 55.0 mg polyether F127 to a mixed solvent containing 7.0 ~ 9.0 mL water and 8.0 ~ 12.0 mL ethanol, and stir until the solution is clear. Subsequently, add 1.5 ~ 2.5 mL 5 mg / mL ammonium molybdate to the solution, and then slowly add 0.3 ~ 0.5 mL of 1,3,5-trimethylbenzene, and continue stirring for 30 min. Add 0.4 ~ 0.6 mL of concentrated ammonia water, react for 2 hours, centrifuge, wash with ethanol 3 times, and dry at 60 ° C overnight to obtain Mo2C. Heat the product in a tubular furnace in a nitrogen atmosphere, raise the temperature to 800 ° C at 5 ° C / min, and carbonize at a constant temperature for 3 hours to obtain the product Mo2C-C; (3) Preparation of PdSn / Mo2C-C 3.0 ~ 5.0 mg PdSn / Mo2C-C and 4.0 ~ 6.0 mL anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 min. Then, 40.0 ~ 60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated to reflux at 100 °C for 2 hours. After the solution was cooled naturally, it was washed with deionized water and freeze-dried to obtain amino Mo2C-C (NH2-Mo2C-C). Subsequently, 3.0 ~ 5.0 mg PdSn and 3.0 ~ 5.0 mg NH2-Mo2C-C were dispersed in 1.5 ~ 2.5 mL deionized water and ultrasonicated for 30 min. Then, the mixed solution was shaken at 25 °C for 8 hours to obtain PdSn / Mo2C-C (2.0 mg / mL); (4) Preparation of Ab2-PdSn / Mo2C-C 1.5 ~ 2.5 mL cTnI-Ab2 solution (20 μg / mL) and 1.5 ~ 2.5 mL PdSn / Mo2C-C (2.0 mg / mL) were shaken overnight at 4 °C. Phosphate buffered saline (PBS) (pH = 7.38) was used as the dispersion solution, centrifuged several times, and redispersed in PBS to obtain Ab2-PdSn / Mo2C-C, which was 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 preparation of Au NPs / HOFs described in step (2) is specifically as follows: (1) Preparation of HOFs Add 0.2 ~ 0.3 g of trimesic acid (BTC) and 0.1 ~ 0.2 g of melamine (MA) to 30.0 ~ 40.0 mL of methanol, seal and ultrasonicate for about 2 h to form a uniform milky white suspension. Transfer the prepared milky white suspension to a 50 mL autoclave, heat to 150 °C, and keep for 12 h; (2) Preparation of Au NPs / HOFs Au NPs were immobilized on HOFs by in situ reduction method. Specifically, 13.0-17.0 mg HOFs were dispersed in 16.4-20.4 mL deionized water, and 0.4-0.6 mL 10 mmol / L sodium citrate and 0.4-0.6 mL 10 mmol / L chloroauric acid were added while stirring, and then 0.5-0.7 mL 0.1 mol / L cold sodium borohydride was quickly added, stirred at room temperature for 20 h, and centrifuged and freeze-dried.

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 (3) 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 1.5 ~ 2.5 mg / mL Au NPs / HOFs dispersion onto the electrode surface, rinse with ultrapure water, 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 7.38 phosphate buffer, and dry it in a 37 °C constant temperature drying oven; (4) Continue to add 3.0 μL of 0.5 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 = 7.38 phosphate buffer, and dry it in a constant temperature drying oven at 37 °C; (5) Continue to add 6.0 µL of a series of different concentrations of cardiac troponin I (cTnI) antigen solutions ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a constant temperature drying oven at 37 °C; (6) 6.0 μL of 1.5 ~ 2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of pH = 7.38 phosphate buffer. 5.0 ~ 15.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer and shaken for 20 min. The absorbance of the supernatant was measured and the washed electrode was dried at 37 °C to obtain a sandwich electrochemical immunosensor based on PdSn bifunctional nanozyme.

5. The electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen and the preparation method thereof according to claim 1, characterized in that: The step (3) 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 were dripped onto the electrode surface, incubated for 20-40 min, and connected to the electrochemical workstation after drying. The electrodes were immersed in 10 mL of pH=7.38 phosphate buffer solution to measure the current changes. When the background current tended to be stable, 10 µL of 5 mol / L hydrogen peroxide solution was injected into 10 mL of pH=7.38 phosphate buffer solution every 50 s, and the current peaks corresponding to cardiac troponin I (cTnI) antigen at different concentrations were recorded. (2) Transfer the Ab2-PdSn / Mo2C-C not connected to the electrode surface to 1 mL of pH = 7.38 phosphate buffer solution, add 3,3',5,5'-tetramethylbenzidine (TMB) to the solution, shake for 20 minutes, and measure the absorbance at 652 nm using a UV-visible spectrophotometer; (3) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the test samples under the two modes was obtained.