Preparation and application of electrochemiluminescence sensor based on self-luminous europium-based metal organic framework

Abstract

The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method and application of an electrochemiluminescence sensor based on a self-luminous europium-based metal organic framework. A europium-based metal organic framework is adopted as an electrogenerated chemiluminescence material, and an organic ligand 2, 6-dipicolinic acid utilizes an antenna effect to improve an intramolecular energy transfer path, so that europium ions are efficiently sensitized, and a high-strength electrogenerated chemiluminescence signal is obtained. Nitrogen-doped carbon-coated copper-cobalt alloy balls are adopted as a co-reaction accelerant, on one hand, a high-conductivity nitrogen-doped carbon layer can improve the migration rate of charges, on the other hand, a copper-cobalt bimetallic catalyst active center can promote generation of more strong-oxidizing-property triethylamine free radicals, and the luminous efficiency of the europium-based covalent organic framework is further improved. A layer-by-layer self-assembly technology is utilized to construct a signal enhanced electrochemiluminescence sensor, and sensitive and rapid detection of carbohydrate antigen 724 is realized.

Description

Technical Field

[0001] The present invention relates to a method for preparing an electrochemiluminescent sensor based on a self-luminescent europium-based metal-organic framework. Specifically, a signal-enhanced electrochemiluminescent sensor for detecting carbohydrate antigen 724 is prepared using nitrogen-doped carbon-coated copper-cobalt alloy spheres as a coreaction promoter, a self-luminescent europium-based metal-organic framework as a luminescent material, and triethylamine as a coreactant. This method belongs to the field of immunoassay and biosensor detection technology. Background Art

[0002] Gastric cancer is a highly prevalent digestive tract malignancy worldwide. Its early symptoms are insidious and lack specificity, and most patients are already in the advanced stage at the time of diagnosis, making treatment difficult and the prognosis poor. Carbohydrate antigen 724 is a high-molecular-weight mucin-like glycoprotein secreted by tumor cells and primarily present in gastric cancer tumor tissue. In healthy individuals, carbohydrate antigen 724 levels are extremely low, with a normal reference value typically <6.9 U / mL. However, when gastric cancer develops, its serum concentration increases significantly, and the degree of increase is closely correlated with tumor progression and metastasis. Currently, carbohydrate antigen 724 has been widely used in the early screening, efficacy evaluation, and recurrence monitoring of gastric cancer, providing an important basis for the clinical development of personalized treatment plans and prognosis. Therefore, the rapid and accurate detection of carbohydrate antigen 724 is of great significance for the clinical diagnosis and treatment of related cancers and diseases.

[0003] Currently, the detection technology for carbohydrate antigen 724 mainly relies on methods such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). However, these methods generally have problems such as high risk of radioactive contamination, insufficient sensitivity, complex operating procedures, and long detection cycles. Electrochemiluminescence (ECL) sensors are a highly sensitive detection platform that combines electrochemical and chemiluminescence technologies. They have shown significant advantages in biomedical testing, environmental monitoring, and food safety. Their core principle is to trigger the excited state transition of luminescent substances through electrochemical reactions, generate light signals, and quantify and analyze them through photodetectors. ECL sensors have become a research hotspot in the field of biomedical analysis due to their advantages such as low background signal interference, excellent detection sensitivity, convenient and efficient operation, and a wide linear response range. Based on this, this study innovatively constructed a self-luminescent europium-based metal-organic framework (EUMO) ECL sensing platform, successfully achieving efficient and specific detection of carbohydrate antigen 724.

[0004] In the present invention, europium ions are selected as metal nodes and 2,6-pyridinedicarboxylic acid is selected as an organic ligand to design and synthesize a self-luminescent europium-based metal-organic framework. 2,6-pyridinedicarboxylic acid utilizes the terminal carboxyl group to coordinate with europium ions to form a stable metal-organic framework, and utilizes the "antenna effect" to improve the intramolecular energy transfer path, thereby achieving efficient sensitization of europium ions and displaying a high-intensity electrochemiluminescence signal. Nitrogen-doped carbon-coated copper-cobalt alloy balls are used as co-reaction promoters. The highly conductive nitrogen-doped carbon layer can increase the charge migration rate, and the copper-cobalt bimetallic catalyst active center can promote the generation of more strongly oxidizing triethylamine radicals, accelerating the generation of excited states of the europium-based metal-organic framework. The present invention utilizes layer-by-layer self-assembly technology to prepare an electrochemiluminescence sensor based on a europium-based metal-organic framework to achieve sensitive and rapid detection of carbohydrate antigen 724. The sensor exhibits a wide detection range, a low detection line, and good analytical performance. Summary of the Invention

[0005] One of the purposes of the present invention is to synthesize a europium-based metal organic framework as a luminophore, and the material has excellent electrochemiluminescence performance.

[0006] The second purpose of the present invention is to synthesize nitrogen-doped carbon-coated copper-cobalt alloy balls as a co-reaction promoter. This material exhibits good conductivity and can accelerate the decomposition of triethylamine into more active intermediates, thereby accelerating the generation of excited states of europium-based metal-organic frameworks and achieving electrochemiluminescence signal amplification.

[0007] The third purpose of the present invention is to use nitrogen-doped carbon-coated copper-cobalt alloy balls as the base material to fix the antibody, use europium-based metal organic frameworks as the luminophore to combine with the detection antibody, use carbohydrate antigen 724 as the target analyte, and utilize the specific immune reaction between antigen and antibody to construct a sensitive and rapid enhanced electrochemiluminescence biosensor to achieve quantitative analysis of the concentration of carbohydrate antigen 724.

[0008] The technical solutions of the present invention are as follows: 1. A method for preparing an electrochemiluminescence sensor based on a self-luminescent europium-based metal-organic framework, comprising the following steps: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) 3.0-5.0 μL of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion was added dropwise to the electrode surface and stored at room temperature to dry; (3) Continue to add 6.0 μL, 5.0-10.0 μg / mL carbohydrate antigen 724 capture antibody dropwise to the electrode surface, store in a 4°C refrigerator to dry, and rinse with ultrapure water; (4) 3.0 μL of 1% 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 7.38 phosphate buffer and stored in a refrigerator at 4°C to dry. (5) Add 6.0 μL of a series of carbohydrate antigen 724 antigen solutions with different concentrations, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a refrigerator at 4°C; (6) Finally, 6 μL of 0.3-3.0 mg / mL europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30-40 min, and rinsed with pH = 7.38 phosphate buffer to prepare an europium-based metal organic framework-based electrochemiluminescence sensor, which was then stored in a 4°C refrigerator for subsequent use.

[0009] 2. The nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion is prepared in the following steps: (1) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy balls Dissolve 4.5-7.0 mmol of cobalt nitrate hexahydrate and 0.54 mmol of copper nitrate hexahydrate in 60 mL of N,N-dimethylformamide and stir continuously for 15 minutes until a uniformly dispersed mixed solution 1 is formed. Subsequently, dissolve 0.6 g of trimesic acid and 4.0 g of polyvinylpyrrolidone in 60 mL of N,N-dimethylformamide and stir continuously for 15 minutes until a uniformly dispersed mixed solution 2 is formed. Finally, solution 1 is slowly added dropwise to solution 2 using a peristaltic pump (4 mL / min). After stirring continuously for 30 minutes, the mixed solution is transferred to a stainless steel container lined with polytetrafluoroethylene and then heated at 60-150°C for 10 hours. The solid material after the reaction was washed three times with ethanol and N,N-dimethylformamide, respectively, and then dried in a vacuum freeze dryer for 48 hours. After the reaction was completed, a solid powder was obtained. 0.1g of the solid powder was thoroughly ground and mixed with 0.1-0.25g of melamine. The mixture was placed in a tube furnace and, under a flowing nitrogen atmosphere, heated to 550°C (2°C / min) and kept warm for 3 hours. Then, the temperature was raised to 650°C (3°C / min) and kept warm for 3 hours. The mixture was then cooled naturally to room temperature to obtain nitrogen-doped carbon-coated copper-cobalt alloy balls. (2) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion 0.35-5 mg of the above materials were added to 1 mL of ultrapure water and ultrasonicated for 30 min to obtain a nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion.

[0010] 3. The preparation steps of the Europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution are as follows: (1) Preparation of Europium-based Metal-Organic Frameworks Dissolve 0.40-0.60 mmol of dipyridinecarboxylic acid and 1 mmol of triethylamine in 15 mL of ethanol, and dissolve 0.25 mmol of europium chloride hexahydrate in 5 mL of water. The solutions were then mixed and sealed in a 50 mL round-bottom flask and stirred continuously in a 40°C oil bath for 2 hours. The resulting white precipitate was collected and purified three times with water and ethanol, respectively, and then dried in a vacuum oven at 40°C overnight to obtain a white europium-based metal-organic framework. (2) Preparation of Europium-based Metal-Organic Framework-labeled Carbohydrate Antigen 724 Detection Antibody Solution 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1-1 mol / L N-hydroxysuccinimide and 1 mL of europium metal organic framework were evenly mixed and incubated at 4°C for 5-10 hours. The solution was centrifuged at 12000 rpm, 100-500 μL of carbohydrate antigen 724 detection antibody solution was added, and the mixture was shaken at room temperature for 10-40 minutes to obtain a europium metal organic framework-labeled carbohydrate antigen 724 detection antibody solution.

[0011] 4. For the detection of carbohydrate antigen 724, the steps are as follows: (1) The test was performed using an electrochemical workstation with a three-electrode system. A silver / silver chloride electrode was used as the reference electrode, a platinum wire electrode was used as the auxiliary electrode, and the prepared biosensor based on the self-luminescent europium-based metal-organic framework was used as the working electrode. The high voltage of the photomultiplier tube was set to 600 V, the scanning potential was 0-1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 150 mmol / L triethylamine at pH 7.38, the electrochemiluminescence signal intensity generated by the electrochemiluminescence system for different concentrations of the analyte antigen was detected, and a working curve was drawn; (3) replacing the carbohydrate antigen 724 antigen solution with the sample solution to be tested; (4) Using the working curve method, the concentration of carbohydrate antigen 724 in the sample to be tested is calculated.

[0012] Beneficial results of the present invention (1) The present invention successfully synthesized a europium-based metal-organic framework with high luminescence efficiency. The organic ligand 2,6-pyridinedicarboxylic acid uses the antenna effect to transfer energy to europium ions in a non-radiative form to achieve self-luminescence. At the same time, the highly ordered framework structure avoids the stacking of luminophores, further improving the luminescence efficiency and solving the problem of low electrochemiluminescence signal of lanthanide metal ions.

[0013] (2) In the present invention, nitrogen-doped carbon-coated copper-cobalt alloy spheres are used as co-reactant promoters. The large area of nitrogen-doped carbon layer greatly improves the electron transfer efficiency of the sensing interface. The uniformly dispersed copper-cobalt catalytic active centers can promote the co-reactant triethylamine to generate more strongly oxidizing triethylamine free radicals, accelerate the generation of excited states of the europium-based metal-organic framework, further amplify the electrochemiluminescence signal, and enhance the sensitivity of the biosensor.

[0014] (3) The electrochemiluminescence sensor based on the self-luminescent europium-based metal-organic framework prepared by the present invention has a linear range of 0.005 to 100 U / mL for carbohydrate antigen 724, a minimum detection limit of 0.0039 U / mL, good specificity, stability and reproducibility, simple operation, and can achieve sensitive and rapid detection of the target carbohydrate antigen 724. DETAILED DESCRIPTION (The present invention will now be further described by way of specific embodiments, but is not limited thereto) Example 1. A method for preparing an electrochemiluminescence sensor based on a self-luminescent europium-based metal-organic framework, comprising the following steps: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) 3.0 μL of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion was added dropwise to the electrode surface and stored at room temperature to dry; (3) Continue to add 6.0 μL, 5.0 μg / mL carbohydrate antigen 724 capture antibody dropwise to the electrode surface, store in a 4°C refrigerator to dry, and rinse with ultrapure water; (4) 3.0 μL of 1% 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 7.38 phosphate buffer and stored in a refrigerator at 4°C to dry. (5) Add 6.0 μL of a series of carbohydrate antigen 724 antigen solutions with different concentrations, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a refrigerator at 4°C; (6) Finally, 6 μL of 0.3 mg / mL europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30 min, and rinsed with pH = 7.38 phosphate buffer to prepare an europium-based metal organic framework-based electrochemiluminescence sensor, which was then stored in a 4°C refrigerator for subsequent use.

[0015] Example 2. A method for preparing an electrochemiluminescence sensor based on a self-luminescent europium-based metal-organic framework, comprising the following steps: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) 4.0 μL of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion was added dropwise to the electrode surface and stored at room temperature to dry; (3) Continue to add 6.0 μL, 7.0 μg / mL carbohydrate antigen 724 capture antibody dropwise to the electrode surface, store in a 4°C refrigerator to dry, and rinse with ultrapure water; (4) 3.0 μL of 1% 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 7.38 phosphate buffer and stored in a refrigerator at 4°C to dry. (5) Add 6.0 μL of a series of carbohydrate antigen 724 antigen solutions with different concentrations, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a refrigerator at 4°C; (6) Finally, 6 μL of 1.5 mg / mL europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 35 min, and rinsed with pH = 7.38 phosphate buffer to prepare an europium-based metal organic framework-based electrochemiluminescence sensor, which was then stored in a 4°C refrigerator for subsequent use.

[0016] Example 3. A method for preparing an electrochemiluminescence sensor based on a self-luminescent europium-based metal-organic framework, comprising the following steps: (1) Polish a 4.0 mm diameter glassy carbon electrode to a mirror finish using alumina polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) Add 5.0 μL of nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion dropwise to the electrode surface and store at room temperature to dry; (3) Continue to add 6.0 μL, 10.0 μg / mL carbohydrate antigen 724 capture antibody dropwise to the electrode surface, store in a 4°C refrigerator to dry, and rinse with ultrapure water; (4) 3.0 μL of 1% 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 7.38 phosphate buffer and stored in a refrigerator at 4°C to dry. (5) Add 6.0 μL of a series of carbohydrate antigen 724 antigen solutions with different concentrations, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a refrigerator at 4°C; (6) Finally, 6 μL of 3.0 mg / mL europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 40 min, and rinsed with pH = 7.38 phosphate buffer to prepare an europium-based metal organic framework-based electrochemiluminescence sensor, which was then stored in a 4°C refrigerator for subsequent use.

[0017] Example 4. The nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion is prepared in the following steps: (1) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy balls 4.5 mmol of cobalt nitrate hexahydrate and 0.54 mmol of copper nitrate hexahydrate were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 1 was formed. Subsequently, 0.6 g of trimesic acid and 4.0 g of polyvinylpyrrolidone were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 2 was formed. Finally, solution 1 was slowly added dropwise to solution 2 using a peristaltic pump (4 mL / min). After stirring continuously for 30 minutes, the mixed solution was transferred to a stainless steel container lined with polytetrafluoroethylene and then heated at 60°C for 10 hours. The solid material after the reaction was washed three times with ethanol and N,N-dimethylformamide, respectively, and then dried in a vacuum freeze dryer for 48 hours to obtain a solid powder after the reaction was completed. 0.1 g of solid powder was thoroughly ground and mixed with 0.1 g of melamine. The mixture was placed in a tube furnace and heated to 550°C (2°C / min) and kept for 3 h under a flowing nitrogen atmosphere. The temperature was then raised to 650°C (3°C / min) and kept for 3 h. The mixture was then naturally cooled to room temperature to obtain nitrogen-doped carbon-coated copper-cobalt alloy balls. (2) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion 0.35 mg of the above material was added to 1 mL of ultrapure water and ultrasonicated for 30 min to obtain a nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion.

[0018] Example 5. The nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion is prepared in the following steps: (1) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy balls 5.5 mmol of cobalt nitrate hexahydrate and 0.54 mmol of copper nitrate hexahydrate were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 1 was formed. Subsequently, 0.6 g of trimesic acid and 4.0 g of polyvinylpyrrolidone were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 2 was formed. Finally, solution 1 was slowly added dropwise to solution 2 using a peristaltic pump (4 mL / min). After stirring continuously for 30 minutes, the mixed solution was transferred to a stainless steel container lined with polytetrafluoroethylene and then heated at 100°C for 10 hours. The solid material after the reaction was washed three times with ethanol and N,N-dimethylformamide, respectively, and then dried in a vacuum freeze dryer for 48 hours to obtain a solid powder after the reaction was completed. 0.1 g of solid powder was thoroughly ground and mixed with 0.15 g of melamine. The mixture was placed in a tube furnace and heated to 550°C (2°C / min) and kept at this temperature for 3 h under a flowing nitrogen atmosphere. The temperature was then raised to 650°C (3°C / min) and kept at this temperature for 3 h. The mixture was then naturally cooled to room temperature to obtain nitrogen-doped carbon-coated copper-cobalt alloy balls. (2) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion 2.5 mg of the above material was added to 1 mL of ultrapure water and ultrasonicated for 30 min to obtain a nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion.

[0019] Example 6. The nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion is prepared as follows: (1) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy balls 7.0 mmol of cobalt nitrate hexahydrate and 0.54 mmol of copper nitrate hexahydrate were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 1 was formed. Subsequently, 0.6 g of trimesic acid and 4.0 g of polyvinylpyrrolidone were dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 2 was formed. Finally, solution 1 was slowly added dropwise to solution 2 using a peristaltic pump (4 mL / min). After stirring continuously for 30 minutes, the mixed solution was transferred to a stainless steel container lined with polytetrafluoroethylene and then heated at 150°C for 10 hours. The solid material after the reaction was washed three times with ethanol and N,N-dimethylformamide, respectively, and then dried in a vacuum freeze dryer for 48 hours to obtain a solid powder after the reaction was completed. 0.1 g of solid powder was thoroughly ground and mixed with 0.25 g of melamine. The mixture was placed in a tube furnace and heated to 550°C (2°C / min) and kept at this temperature for 3 h under a flowing nitrogen atmosphere. The temperature was then raised to 650°C (3°C / min) and kept at this temperature for 3 h. The mixture was then naturally cooled to room temperature to obtain nitrogen-doped carbon-coated copper-cobalt alloy balls. (2) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion 5 mg of the above material was added to 1 mL of ultrapure water and ultrasonicated for 30 min to obtain a nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion.

[0020] Example 7. The preparation steps of the Europium-based Metal-organic Framework-labeled carbohydrate antigen 724 detection antibody solution are as follows: (1) Preparation of the Europium-based Metal-organic Framework Dissolve 0.40 mmol of dipicolinic acid and 1 mmol of triethylamine in 15 mL of ethanol, and 0.25 mmol of europium chloride hexahydrate in 5 mL of water. The solutions were then mixed and sealed in a 50 mL round-bottom flask and stirred continuously in a 40°C oil bath for 2 hours. The resulting white precipitate was collected and purified three times with water and ethanol, respectively, and then dried in a vacuum oven at 40°C overnight to obtain a white europium-based metal-organic framework. (2) Preparation of Europium-based Metal-Organic Framework-labeled Carbohydrate Antigen 724 Detection Antibody Solution 1 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1 mol / L N-hydroxysuccinimide and 1 mL of europium metal organic framework were evenly mixed and incubated at 4°C for 5 h. The above solution was centrifuged at 12000 rpm, 100 μL of carbohydrate antigen 724 detection antibody solution was added, and the mixture was shaken at room temperature for 10 min to obtain a europium metal organic framework-labeled carbohydrate antigen 724 detection antibody solution.

[0021] Example 8. The preparation steps of the Europium-based Metal-organic Framework-labeled carbohydrate antigen 724 detection antibody solution are as follows: (1) Preparation of the Europium-based Metal-organic Framework Dissolve 0.50 mmol of dipicolinic acid and 1 mmol of triethylamine in 15 mL of ethanol, and 0.25 mmol of europium chloride hexahydrate in 5 mL of water. The solutions were then mixed and sealed in a 50 mL round-bottom flask and stirred continuously in a 40°C oil bath for 2 hours. The resulting white precipitate was collected and purified three times with water and ethanol, respectively, and then dried in a vacuum oven at 40°C overnight to obtain a white europium-based metal-organic framework. (2) Preparation of Europium-based Metal-Organic Framework-labeled Carbohydrate Antigen 724 Detection Antibody Solution 2.5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.5 mol / L N-hydroxysuccinimide and 1 mL of europium metal organic framework were evenly mixed and incubated at 4°C for 7 h. The above solution was centrifuged at 12000 rpm, 250 μL of carbohydrate antigen 724 detection antibody solution was added, and the mixture was shaken at room temperature for 25 min to obtain a europium metal organic framework-labeled carbohydrate antigen 724 detection antibody solution.

[0022] Example 9. The preparation steps of the Europium-based Metal-organic Framework-labeled carbohydrate antigen 724 detection antibody solution are as follows: (1) Preparation of the Europium-based Metal-organic Framework Dissolve 0.50 mmol of dipicolinic acid and 1 mmol of triethylamine in 15 mL of ethanol, and 0.25 mmol of europium chloride hexahydrate in 5 mL of water. The solutions were then mixed and sealed in a 50 mL round-bottom flask and stirred continuously in a 40°C oil bath for 2 hours. The resulting white precipitate was collected and purified three times with water and ethanol, respectively, and then dried in a vacuum oven at 40°C overnight to obtain a white europium-based metal-organic framework. (2) Preparation of Europium-based Metal-Organic Framework-labeled Carbohydrate Antigen 724 Detection Antibody Solution 5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 1 mol / L N-hydroxysuccinimide and 1 mL of europium metal organic framework were evenly mixed and incubated at 4°C for 10 h. The above solution was centrifuged at 12000 rpm, 500 μL of carbohydrate antigen 724 detection antibody solution was added, and the mixture was shaken at room temperature for 40 min to obtain a europium metal organic framework-labeled carbohydrate antigen 724 detection antibody solution.

[0023] Example 10. For the detection of carbohydrate antigen 724, the steps are as follows: (1) The test was performed using an electrochemical workstation with a three-electrode system. A silver / silver chloride electrode was used as the reference electrode, a platinum wire electrode was used as the auxiliary electrode, and the prepared biosensor based on the self-luminescent europium-based metal-organic framework was used as the working electrode. The high voltage of the photomultiplier tube was set to 600 V, the scanning potential was 0-1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 150 mmol / L triethylamine at pH 7.38, the electrochemiluminescence signal intensity generated by the electrochemiluminescence system in response to different concentrations of the analyte antigen was detected, and a working curve was drawn; (3) replacing the carbohydrate antigen 724 antigen solution with the sample solution to be tested; (4) using the working curve method to determine the concentration of carbohydrate antigen 724 in the sample to be tested; (5) Based on the linear relationship between the obtained luminescence intensity and the concentration of carbohydrate antigen 724, a working curve was drawn. The linear range was 0.005-100 U / mL, and the detection limit was 0.0024 U / mL.

Claims

1. A method for preparing a self-luminescent europium-based metal-organic framework electrochemiluminescence sensor, characterized in that: The following steps are involved: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) 3.0-5.0 μL of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion was added dropwise to the electrode surface and stored at room temperature to dry; (3) Continue to add 6.0 μL, 5.0-10.0 μg / mL carbohydrate antigen 724 capture antibody dropwise to the electrode surface, store in a 4°C refrigerator to dry, and rinse with ultrapure water; (4) 3.0 μL of 1% 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 7.38 phosphate buffer and stored in a refrigerator at 4°C to dry. (5) Add 6.0 μL of a series of carbohydrate antigen 724 antigen solutions of different concentrations, rinse the electrode surface with pH = 7.38 phosphate buffer, and dry in a refrigerator at 4°C; (6) Finally, 6 μL of 0.3-3.0 mg / mL europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30-40 min, and rinsed with pH = 7.38 phosphate buffer to prepare an europium-based metal organic framework-based electrochemiluminescence sensor, which was then stored in a 4°C refrigerator for subsequent use.

2. The method for preparing a self-luminescent europium-based metal-organic framework electrochemiluminescence sensor according to claim 1, wherein the nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion is characterized in that: Here are the steps: (1) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy balls Dissolve 4.5–7.0 mmol of cobalt nitrate hexahydrate and 0.54 mmol of copper nitrate hexahydrate in 60 mL of N,N-dimethylformamide and stir continuously for 15 minutes until a uniformly dispersed mixed solution 1 is formed. Subsequently, 0.6 g of trimesic acid and 4.0 g of polyvinylpyrrolidone are dissolved in 60 mL of N,N-dimethylformamide and stirred continuously for 15 minutes until a uniformly dispersed mixed solution 2 is formed. Finally, solution 1 is slowly added dropwise to solution 2 using a peristaltic pump (4 mL / min). After stirring continuously for 30 minutes, the mixed solution is transferred to a stainless steel container lined with polytetrafluoroethylene and then heated at 60–150°C for 10 hours. The resulting solid material is washed three times with ethanol and N,N-dimethylformamide, respectively, and then dried in a vacuum freeze dryer for 48 hours to obtain a solid powder. 0.1 g of solid powder was thoroughly ground and mixed with 0.1-0.25 g of melamine. The mixture was placed in a tube furnace and heated to 550°C (2°C / min) and kept at this temperature for 3 h under a flowing nitrogen atmosphere. The temperature was then raised to 650°C (3°C / min) and kept at this temperature for 3 h. The mixture was then naturally cooled to room temperature to obtain nitrogen-doped carbon-coated copper-cobalt alloy balls. (2) Preparation of nitrogen-doped carbon-coated copper-cobalt alloy ball dispersion 0.35-5 mg of the above materials were added to 1 mL of ultrapure water and ultrasonicated for 30 min to obtain a nitrogen-doped carbon-coated copper-cobalt alloy sphere dispersion.

3. The method for preparing a self-luminescent europium-based metal organic framework electrochemiluminescence sensor according to claim 1, wherein the europium-based metal organic framework-labeled carbohydrate antigen 724 detection antibody solution is characterized in that: Here are the steps: (1) Preparation of Europium-based Metal-Organic Frameworks Dissolve 0.40-0.60 mmol of dipyridinecarboxylic acid and 1 mmol of triethylamine in 15 mL of ethanol, and dissolve 0.25 mmol of europium chloride hexahydrate in 5 mL of water. The solutions were then mixed and sealed in a 50 mL round-bottom flask and stirred continuously in a 40°C oil bath for 2 hours. The resulting white precipitate was collected and purified three times with water and ethanol, respectively, and then dried in a vacuum oven at 40°C overnight to obtain a white europium-based metal-organic framework. (2) Preparation of Europium-based Metal-Organic Framework-labeled Carbohydrate Antigen 724 Detection Antibody Solution 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1-1 mol / L N-hydroxysuccinimide and 1 mL of europium metal organic framework were evenly mixed and incubated at 4°C for 5-10 hours. The solution was centrifuged at 12000 rpm, 100-500 μL of carbohydrate antigen 724 detection antibody solution was added, and the mixture was shaken at room temperature for 10-40 minutes to obtain a europium metal organic framework-labeled carbohydrate antigen 724 detection antibody solution.

4. The method for preparing a self-luminescent europium-based metal-organic framework electrochemiluminescence sensor according to claim 1, which is used for the detection of carbohydrate antigen 724, characterized in that: Here are the steps: (1) The test was performed using an electrochemical workstation with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, the prepared biosensor based on the self-luminescent europium-based metal-organic framework was used as the working electrode, the high voltage of the photomultiplier tube was set to 600 V, the scanning potential was 0-1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 150 mmol / L triethylamine at pH 7.38, the electrochemiluminescence signal intensity generated by the electrochemiluminescence system for different concentrations of the analyte antigen was detected, and a working curve was drawn; (3) replacing the carbohydrate antigen 724 antigen solution with the sample solution to be tested; (4) Using the working curve method, the concentration of carbohydrate antigen 724 in the sample to be tested is calculated.