Preparation and application of a dual-mode immunosensor based on PdSn bifunctional nanozyme

Abstract

This invention belongs to the fields of novel functional nanomaterials, immunoassays, and biosensors. It provides a dual-mode immunosensor based on a PdSn bifunctional nanozyme for the sensitive detection of cardiac troponin I (cTnI). A palladium-tin alloy / molybdenum carbide composite (PdSn / Mo2C-C) as a secondary antibody beacon and a gold nanoparticle / hydrogen-bonded organic framework composite (Au NPs / HOFs) as a substrate were prepared. Based on this sandwich sensor, the sensor quantitatively measures cTnI antigen concentration in real serum samples using dual-mode (electrochemical and colorimetric) methods. This achieves visually interpretable, ultrasensitive dual-modal analysis, exhibiting high sensitivity and a low limit of detection, providing a novel approach 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 using a palladium-tin alloy / molybdenum carbide composite material (PdSn / Mo2C-C) as a marker and a gold nanoparticle / hydrogen-bonded organic framework composite material (Au NPs / HOF) as a substrate. The present invention belongs to the fields of novel functional nanomaterials, immunoassays, and biosensor technologies. Background Art

[0002] Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality worldwide, with myocardial infarction (MI) being one of the most critical and prevalent conditions. Timely and accurate diagnosis of MI is crucial for effective intervention and improved patient outcomes. Cardiac troponin I (cTnI) is a highly specific biomarker of myocardial injury. Following myocardial injury, cTnI concentrations in the blood increase rapidly and persist for a long time, providing a long window for detection. Therefore, as a key biomarker for diagnosing MI, highly sensitive detection of cTnI is essential. Recent developments in immunosensor technology have provided a promising alternative to traditional methods. Immunosensors, which combine immunoassay and sensor technologies, have the potential to provide faster, more sensitive, and lower-cost diagnostics. Dual-channel signal detection strategies not only provide stable and diverse readouts to enhance sensitivity and accuracy but also offer diverse quantification methods, broadening their applicability. Colorimetric methods are favored for their simplicity and suitability for on-site testing due to their ease of processing and visually interpretable signal readout, and are expected to replace traditional detection methods in cTnI analysis. Integrating colorimetric signals with other sensing modalities into a multi-signal platform can significantly improve the overall performance of the detection system. Electrochemical immunosensors exhibit low background interference and high sensitivity because they primarily convert the analyte content into an electrochemical signal, a feature that can help overcome the color resolution limitations of colorimetric analysis. Therefore, creating a dual-channel sensing platform combining colorimetry and electrochemistry enables visually interpretable, ultrasensitive dual-modal analysis.

[0003] Natural enzymes are quintessential biomacromolecules that accelerate various chemical reactions under extremely mild conditions in biochemical systems. To address the instability and high cost of natural enzymes, nanozymes (nanomaterials with enzyme-like properties) have emerged as potential alternatives due to their high stability, durability, and low cost. Nanozymes are generally classified into three categories: peroxidase-like (POD), oxidoreductase-like (OXD), and esterase-like. The p-d orbital hybridization strategy is considered an effective approach 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 alter the band structure, thereby adjusting the position of the d-band center. This modification has the potential to alter the interactions between the nanozyme and substrates or reaction intermediates, thereby improving catalytic activity and selectivity. Here, a bifunctional nanozyme with catalase (POD)- and oxidoreductase (OXD)-like properties was prepared using p-block metal Sn-doped Pd (PdSn) characterized by p-d orbital hybridization. First, the hybridization of the Pd orbitals causes the center of the d band to shift upward relative to the Fermi level, thereby promoting the adsorption of H2O2 and generating a significant current signal through electrochemical testing (chronoamperometry). Furthermore, the hybridization of the Pd orbitals provides high-energy electrons, which endow the PdSn nanozyme with strong reducing ability. When TMB is used as a catalytic substrate, it catalyzes the reduction of O2 to H2O via a four-electron pathway. Simultaneously, TMB is oxidized to oxTMB, resulting in a corresponding color change, which is applied to the colorimetric immunoassay of cTnI.

[0004] This study constructed a sandwich electrochemical immunosensor using a palladium-tin bifunctional nanozyme (PdSn)-loaded molybdenum carbide (Mo2C-C) composite (PdSn / Mo2C-C) as the beacon material and gold nanoparticles / hydrogen-bonding organic frameworks (Au NPs / HOFs) as the substrate for the sensing system. The unimmobilized PdSn / Mo2C-C material on the electrode surface was used for a colorimetric immunoassay. Thanks to the excellent catalase- and oxidoreductase-like dual activities of the PdSn nanozyme, the designed dual-channel signal detection sensing platform enables ultrasensitive 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:

[0009] 1. Preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme, the steps are as follows:

[0010] (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;

[0011] (2) Add 6.0 μL of 1.5-2.5 mg / mL Au NPs / HOFs dispersion onto the electrode surface and allow to dry at room temperature.

[0012] (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;

[0013] (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;

[0014] (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;

[0015] (6) 6.0 μL of 1.5 ~ 2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface, and the electrode was placed in a constant temperature drying oven at 37 °C for 25 min. The electrode was rinsed with 1 mL of phosphate buffer at pH = 7.38, and 5.0 ~ 15.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer. The electrode was shaken for 20 min, and the absorbance of the supernatant was measured. The washed electrode was dried at a constant temperature of 37 °C to prepare a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0016] 2. Preparation of a dual-mode immunosensor based on PdSn bifunctional nanozyme. The preparation of the relevant materials is as follows:

[0017] (1) Preparation of PdSn bifunctional nanozymes

[0018] 8.0-12.0 mg of palladium acetylacetonate, 70.0-90.0 mg of polyvinylpyrrolidone (MW 24000), and 1.81-2.01 mg of tin tetrachloride were dissolved in 4.0-6.0 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. The mixture was then cooled to room temperature and washed several times with ethanol and ultrapure water to obtain the PdSn bifunctional nanozyme.

[0019] (2) Preparation of Mo2C-C

[0020] At room temperature, 60.0-80.0 mg of dopamine hydrochloride and 45.0-55.0 mg of polyether F127 were added to a mixed solvent containing 7.0-9.0 mL of water and 8.0-12.0 mL of ethanol, and stirred until the solution was clear. Subsequently, 1.5-2.5 mL of 5 mg / mL ammonium molybdate was added to the solution, followed by the slow dropwise addition of 0.3-0.5 mL of 1,3,5-trimethylbenzene, and stirring was continued for 30 min. 0.4-0.6 mL of concentrated ammonia was added, and the mixture was reacted for 2 hours, centrifuged, washed three times with ethanol, and dried at 60 °C overnight to obtain Mo2C. The product was heated in a tube furnace under a nitrogen atmosphere, raised to 800 °C at 5 °C / min, and carbonized at this temperature for 3 hours to obtain the product Mo2C-C.

[0021] (3) Preparation of PdSn / Mo2C-C

[0022] 3.0-5.0 mg of PdSn / Mo2C-C and 4.0-6.0 mL of anhydrous ethanol were added to a round-bottom flask and sonicated for 15 min. Then, 40.0-60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added with stirring, and the mixture was heated under reflux at 100 °C for 2 h. After the solution was cooled naturally, it was washed with deionized water and lyophilized to obtain amino-modified Mo2C-C (NH2-Mo2C-C). Subsequently, 3.0-5.0 mg of PdSn and 3.0-5.0 mg of NH2-Mo2C-C were dispersed in 1.5-2.5 mL of deionized water and sonicated for 30 min. The mixed solution was then shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL).

[0023] (4) Preparation of Ab2-PdSn / Mo2C-C

[0024] Shake 1.5–2.5 mL of cTnI-Ab2 solution (20 μg / mL) and 1.5–2.5 mL of PdSn / Mo2C-C (2.0 mg / mL) at 4°C overnight. Centrifuge several times using phosphate buffered saline (PBS) (pH = 7.38) as the dispersion solution. Redisperse in PBS to obtain Ab2-PdSn / Mo2C-C, which is then stored in a refrigerator at 4°C.

[0025] (5) Preparation of HOFs

[0026] 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 the container and sonicate for approximately 2 hours to form a uniform milky white suspension. Transfer the resulting milky white suspension to a 50 mL autoclave and heat to 150°C for 12 hours.

[0027] (6) Preparation of Au NPs / HOFs

[0028] Au NPs were immobilized on HOFs using an in situ reduction method. Specifically, 13.0–17.0 mg of HOFs were dispersed in 16.4–20.4 mL of deionized water. 0.4–0.6 mL of 10 mmol / L sodium citrate and 0.4–0.6 mL of 10 mmol / L chloroauric acid were added with stirring. Freshly prepared 0.5–0.7 mL of 0.1 mol / L cold sodium borohydride was then quickly added. The mixture was stirred at room temperature for 20 hours and then centrifuged and freeze-dried.

[0029] 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:

[0030] (1) Using an electrochemical workstation, the test was performed 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. The test was performed in 10 mL of a phosphate buffer solution containing 5 mol / L hydrogen peroxide solution at pH 5.0 to 8.5.

[0031] (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.

[0032] (3) When the background current stabilizes, 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.

[0033] (4) Record the current peak corresponding to different concentrations of cardiac troponin I (cTnI) antigen;

[0034] (5) Transfer the Ab2-PdSn / Mo2C-C not attached 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;

[0035] (6) Using the working curve method, the concentration of cardiac troponin I (cTnI) antigen in the sample to be tested is obtained.

[0036] Beneficial results of the present invention

[0037] (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 the adsorption of H2O2 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 blue oxTMB, which is used in the colorimetric immunoassay of cTnI.

[0038] (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

[0039] The present invention will now be further described through specific embodiments, but is not limited thereto.

[0040] Example 1 Preparation method of a dual-mode immunosensor based on PdSn bifunctional nanozyme

[0041] (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;

[0042] (2) 6.0 μL of 1.5 mg / mL Au NPs / HOFs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0043] (3) Add 6.0 μL of 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;

[0044] (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;

[0045] (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;

[0046] (6) 6.0 μL of 1.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface, and the mixture was placed in a constant temperature drying oven at 37 °C for 25 min. The mixture was rinsed with 1 mL of phosphate buffer at pH 7.38, and 5 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer. The mixture was shaken for 20 min, and the absorbance of the supernatant was measured. The washed electrode was dried at a constant temperature of 37 °C to prepare a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0047] Example 2 Preparation method of a dual-mode immunosensor based on PdSn bifunctional nanozyme

[0048] (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;

[0049] (2) 6.0 μL of 2.0 mg / mL Au NPs / HOFs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (6) 6.0 μL of 2.0 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface, and the mixture was placed in a constant temperature drying oven at 37 °C for 25 min. The mixture was rinsed with 1 mL of phosphate buffer at pH = 7.38, and 10.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer. The mixture was shaken for 20 min, and the absorbance of the supernatant was measured. The washed electrode was dried at a constant temperature of 37 °C to prepare a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0054] Example 3 Preparation method of a dual-mode immunosensor based on PdSn bifunctional nanozyme

[0055] (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;

[0056] (2) 6.0 μL of 2.5 mg / mL Au NPs / HOFs dispersion was added to the electrode surface, rinsed with ultrapure water, and dried at room temperature;

[0057] (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;

[0058] (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;

[0059] (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;

[0060] (6) 6.0 μL of 2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface and allowed to stand in a constant temperature drying oven at 37 °C for 25 min. The electrode was then rinsed with 1 mL of phosphate buffer at pH 7.38. 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 a constant temperature of 37 °C to prepare a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme.

[0061] Preparation of Ab2-PdSn / Mo2C-C as described in Example 4

[0062] (1) Preparation of PdSn bifunctional nanozymes

[0063] 8.0 mg of palladium acetylacetonate, 70.0 mg of polyvinylpyrrolidone (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. The mixture was then cooled to room temperature and washed several times with ethanol and ultrapure water to obtain the PdSn bifunctional nanozyme.

[0064] (2) Preparation of Mo2C-C

[0065] At room temperature, 60 mg of dopamine hydrochloride and 45 mg of polyether F127 were added to a mixed solvent containing 7 mL of water and 8 mL of ethanol, and stirred until the solution was clear. Subsequently, 1.5 mL of 5 mg / mL ammonium molybdate was added to the solution, and then 0.3 mL of 1,3,5-trimethylbenzene was slowly added dropwise, and stirring was continued for 30 min. 0.4 mL of concentrated ammonia water was added, and after reacting for 2 hours, the mixture was centrifuged, washed with ethanol three times, and dried at 60 ° C overnight to obtain Mo2C. The product was placed in a tube furnace and heated in a nitrogen atmosphere, and the temperature was increased to 800 ° C at 5 ° C / min, and carbonized at a constant temperature for 3 hours to obtain the product Mo2C-C;

[0066] (3) Preparation of PdSn / Mo2C-C

[0067] 3.0 mg of PdSn / Mo2C-C and 4.0 mL of anhydrous ethanol were added to a round-bottom flask and sonicated for 15 min. Then, 40 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated under 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 of PdSn and 3.0 mg of NH2-Mo2C-C were dispersed in 1.5 mL of deionized water and sonicated for 30 min. The mixed solution was then shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL).

[0068] (4) Preparation of Ab2-PdSn / Mo2C-C

[0069] 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 medium, and the mixture was centrifuged several times. The resulting Ab2-PdSn / Mo2C-C was redispersed in PBS and stored in a refrigerator at 4°C.

[0070] Preparation of Ab2-PdSn / Mo2C-C as described in Example 5

[0071] (1) Preparation of PdSn bifunctional nanozymes

[0072] 10.0 mg of palladium acetylacetonate, 80.0 mg of polyvinylpyrrolidone (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. The mixture was then cooled to room temperature and washed several times with ethanol and ultrapure water to obtain the PdSn bifunctional nanozyme.

[0073] (2) Preparation of Mo2C-C

[0074] At room temperature, 70.0 mg of dopamine hydrochloride and 50.0 mg of polyether F127 were added to a mixed solvent containing 8.0 mL of water and 10.0 mL of ethanol, and stirred until the solution was clear. Subsequently, 2.0 mL of 5 mg / mL ammonium molybdate was added to the solution, and then 0.4 mL of 1,3,5-trimethylbenzene was slowly added dropwise, and stirring was continued for 30 min. 0.5 mL of concentrated ammonia water was added, and the mixture was centrifuged after reacting for 2 hours, washed with ethanol three times, and dried at 60 ° C overnight to obtain Mo2C. The product was placed in a tube furnace and heated at 5 ° C / min to 800 ° C in a nitrogen atmosphere, and carbonized at constant temperature for 3 hours to obtain the product Mo2C-C;

[0075] (3) Preparation of PdSn / Mo2C-C

[0076] 4.0 mg of PdSn / Mo2C-C and 5.0 mL of anhydrous ethanol were added to a round-bottom flask and sonicated for 15 min. Then, 50.0 μL of 3-aminopropyltriethoxysilane (APTES) was added with stirring and heated under 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 of PdSn and 4.0 mg of NH2-Mo2C-C were dispersed in 2.0 mL of deionized water and sonicated for 30 min. The mixed solution was then shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL).

[0077] (4) Preparation of Ab2-PdSn / Mo2C-C

[0078] 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, and the mixture was centrifuged several times. The resulting Ab2-PdSn / Mo2C-C was redispersed in PBS and stored in a refrigerator at 4°C.

[0079] Preparation of Ab2-PdSn / Mo2C-C as described in Example 6

[0080] (1) Preparation of PdSn bifunctional nanozymes

[0081] 12.0 mg of palladium acetylacetonate, 90.0 mg of polyvinylpyrrolidone (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. The mixture was then cooled to room temperature and washed several times with ethanol and ultrapure water to obtain the PdSn bifunctional nanozyme.

[0082] (2) Preparation of Mo2C-C

[0083] At room temperature, 80.0 mg of dopamine hydrochloride and 55.0 mg of polyether F127 were added to a mixed solvent containing 9.0 mL of water and 12.0 mL of ethanol, and stirred until the solution was clear. Subsequently, 2.5 mL of 5 mg / mL ammonium molybdate was added to the solution, and then 0.5 mL of 1,3,5-trimethylbenzene was slowly added dropwise, and stirring was continued for 30 min. 0.6 mL of concentrated ammonia water was added, and after reacting for 2 hours, the mixture was centrifuged, washed with ethanol three times, and dried at 60 ° C overnight to obtain Mo2C. The product was placed in a tube furnace in a nitrogen atmosphere and heated at 5 ° C / min to 800 ° C. It was carbonized at this constant temperature for 3 hours to obtain the product Mo2C-C;

[0084] (3) Preparation of PdSn / Mo2C-C

[0085] 5.0 mg of PdSn / Mo2C-C and 6.0 mL of anhydrous ethanol were added to a round-bottom flask and sonicated for 15 min. Then, 60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added with stirring and heated under 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 of PdSn and 5.0 mg of NH2-Mo2C-C were dispersed in 2.5 mL of deionized water and sonicated for 30 min. The mixed solution was then shaken at 25 °C for 8 h to obtain PdSn / Mo2C-C (2.0 mg / mL).

[0086] (4) Preparation of Ab2-PdSn / Mo2C-C

[0087] 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, and the mixture was centrifuged several times. The Ab2-PdSn / Mo2C-C was redispersed in PBS and stored in a refrigerator at 4°C.

[0088] Example 7 Preparation of the Au NPs / HOFs

[0089] (1) Preparation of HOFs

[0090] Add 0.2 g of trimesic acid (BTC) and 0.1 g of melamine (MA) to 30.0 mL of methanol, seal the container, and sonicate for approximately 2 hours to form a uniform milky white suspension. Transfer the resulting milky white suspension to a 50 mL autoclave and heat to 150°C for 12 hours.

[0091] (2) Preparation of Au NPs / HOFs

[0092] Au NPs were immobilized on HOFs using an in situ reduction method. Specifically, 13.0 mg of HOFs were dispersed in 16.4 mL of deionized water. 0.4 mL of 10 mmol / L sodium citrate and 0.4 mL of 10 mmol / L chloroauric acid were added with stirring. Then, 0.5 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added. The mixture was stirred at room temperature for 20 hours and then centrifuged and freeze-dried.

[0093] Preparation of Au NPs / HOFs as described in Example 8

[0094] (1) Preparation of HOFs

[0095] Add 0.25 g of trimesic acid (BTC) and 0.15 g of melamine (MA) to 35.0 mL of methanol, seal the container, and sonicate for approximately 2 hours to form a uniform milky white suspension. Transfer the resulting milky white suspension to a 50 mL autoclave and heat to 150°C for 12 hours.

[0096] (2) Preparation of Au NPs / HOFs

[0097] Au NPs were immobilized on HOFs using an in situ reduction method. Specifically, 15.0 mg of HOFs were dispersed in 18.4 mL of deionized water. 0.5 mL of 10 mmol / L sodium citrate and 0.5 mL of 10 mmol / L chloroauric acid were added while stirring. Then, 0.6 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added. The mixture was stirred at room temperature for 20 hours and then centrifuged and freeze-dried.

[0098] Preparation of Au NPs / HOFs as described in Example 9

[0099] (1) Preparation of HOFs

[0100] Add 0.3 g of trimesic acid (BTC) and 0.2 g of melamine (MA) to 40.0 mL of methanol, seal the container, and sonicate for approximately 2 hours to form a uniform milky white suspension. Transfer the resulting milky white suspension to a 50 mL autoclave and heat to 150°C for 12 hours.

[0101] (2) Preparation of Au NPs / HOFs

[0102] Au NPs were immobilized on HOFs using an in situ reduction method. Specifically, 17.0 mg of HOFs were dispersed in 20.4 mL of deionized water. 0.6 mL of 10 mmol / L sodium citrate and 0.6 mL of 10 mmol / L chloroauric acid were added with stirring. Then, 0.7 mL of freshly prepared 0.1 mol / L cold sodium borohydride was quickly added. The mixture was stirred at room temperature for 20 hours and then centrifuged and freeze-dried.

[0103] Example 10 Detection of cardiac troponin I (cTnI) antigen by the dual-mode immunosensor based on PdSn dual-functional nanozyme

[0104] (1) Using an electrochemical workstation, the test was performed 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 pH 5.0-8.5 phosphate buffer solution;

[0105] (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.

[0106] (3) When the background current stabilizes, 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.

[0107] (4) Record the current peak corresponding to different concentrations of cardiac troponin I (cTnI) antigen;

[0108] (5) Transfer the Ab2-PdSn / Mo2C-C not attached 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;

[0109] (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 calculated to be 0.864 ng / mL.

Claims

1. A method for preparing 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 composited and combined with cTnI-Ab2; (2) Preparation of Au NPs / HOFs: In situ growth of gold nanoparticles (AuNPs) on the surface of separately prepared hydrogen-bonded organic frameworks (HOFs); (3) Construct a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozymes, use electrochemical and colorimetric methods to determine the content of cardiac troponin I (cTnI) antigen, and draw a working curve; The preparation of Ab2-PdSn / Mo2C-C 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 polyvinylpyrrolidone (MW24000), and 1.81-2.01 mg of tin tetrachloride were dissolved in 4.0-6.0 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. The mixture was then cooled to room temperature and the product was washed several times with ethanol and ultrapure water to obtain a PdSn bifunctional nanozyme. ②Preparation of Mo2C-C At room temperature, 60.0-80.0 mg of dopamine hydrochloride and 45.0-55.0 mg of polyether F127 were added to a mixed solvent containing 7.0-9.0 mL of water and 8.0-12.0 mL of ethanol, and stirred until the solution was clear. Subsequently, 1.5-2.5 mL of 5 mg / mL ammonium molybdate was added to the solution, and then 0.3-0.5 mL of 1,3,5-trimethylbenzene was slowly added dropwise, and stirring was continued for 30 minutes; 0.4-0.6 mL of concentrated ammonia water was added, and the mixture was reacted for 2 hours, then centrifuged, washed with ethanol three times, and dried at 60°C overnight to obtain Mo2C; the product was placed in a tube furnace and heated in a nitrogen atmosphere, and the temperature was increased to 800°C at 5°C / min, and carbonized at this temperature for 3 hours to obtain the product Mo2C-C; ③ Preparation of PdSn / Mo2C-C 3.0-5.0 mg of PdSn / Mo2C-C and 4.0-6.0 mL of anhydrous ethanol were added to a round-bottom flask and ultrasonicated for 15 minutes. Then, 40.0-60.0 μL of 3-aminopropyltriethoxysilane (APTES) was added under stirring and heated under 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 of PdSn and 3.0-5.0 mg of NH2-Mo2C-C were dispersed in 1.5-2.5 mL of deionized water and ultrasonicated for 30 minutes. The mixed solution was then shaken at 25°C for 8 hours to obtain 2.0 mg / mL of PdSn / Mo2C-C. ④ Preparation of Ab2-PdSn / Mo2C-C 1.5-2.5 mL of 20 μg / mL cTnI-Ab2 solution and 1.5-2.5 mL of 2.0 mg / mL PdSn / Mo2C-C 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 then stored in a refrigerator at 4°C. The preparation of AuNPs / 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 the container, and sonicate for approximately 2 hours to form a uniform milky white suspension. Transfer the resulting milky white suspension to a 50 mL autoclave and heat to 150°C for 12 hours. ②Preparation of AuNPs / HOFs The Au NPs were immobilized on HOFs using an in situ reduction method. Specifically, 13.0–17.0 mg of HOFs were dispersed in 16.4–20.4 mL of deionized water. 0.4–0.6 mL of 10 mmol / L sodium citrate and 0.4–0.6 mL of 10 mmol / L chloroauric acid were added while stirring. Freshly prepared 0.5–0.7 mL of 0.1 mol / L cold sodium borohydride was then quickly added. The mixture was stirred at room temperature for 20 hours and then centrifuged and freeze-dried. The electrochemical immunosensor constructed in step (3) is specifically 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 with 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 nonspecific active sites on the electrode surface, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a 37°C constant temperature drying oven; ⑤ Continue to add 6.0 μL of a series of cardiac troponin I (cTnI) antigen solutions of different concentrations ranging from 10 fg / mL to 100 ng / mL, rinse with pH = 7.38 phosphate buffer, and dry in a 37°C constant temperature drying oven; ⑥ 6.0 μL, 1.5-2.5 mg / mL Ab2-PdSn / Mo2C-C dispersion was dropwise applied to the electrode surface, allowed to stand in a constant temperature drying oven at 37°C for 25 min, rinsed with 1 mL of phosphate buffer at pH = 7.38, 5.0-15.0 μL of 20 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added to the phosphate buffer, shaken for 20 minutes, the absorbance of the supernatant was measured, and the washed electrode was dried at a constant temperature of 37°C to prepare a sandwich-type electrochemical immunosensor based on PdSn bifunctional nanozyme.

2. The method for preparing an electrochemical immunosensor for detecting cardiac troponin I (cTnI) antigen according to claim 1, characterized in that: The cardiac troponin I (cTnI) antigen is measured in step (3) and a working curve is drawn as follows: ① 6 μL of cardiac troponin I (cTnI) antigen solution of different concentrations was drop-coated onto the electrode surface, incubated for 20–40 min, and connected to an 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 stabilized, 10 μL of 5 mol / L hydrogen peroxide solution was injected into the 10 mL of pH 7.38 phosphate buffer solution every 50 s, and the current peaks corresponding to the different concentrations of cardiac troponin I (cTnI) antigen were recorded. ② Transfer the Ab2-PdSn / Mo2C-C not attached to the electrode surface to 1 mL of pH 7.38 phosphate buffer solution. Add 3,3',5,5'-tetramethylbenzidine (TMB) to the solution and shake for 20 minutes. 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.