Preparation method and application of immunosensor based on pyrenyl covalent organic framework

Abstract

The invention belongs to the technical field of novel functional materials and biosensing detection, and provides a preparation method and application of a pyrenyl-based covalent organic framework immunosensor. Specifically, a pyrenyl covalent organic framework is adopted as an electrochemiluminescence material, and free rotation of 1, 3, 6, 8-tetra (4-formylphenyl) pyrene is effectively inhibited through connection of formylhydrazine, so that non-radiative transition is weakened, and the electrochemiluminescence signal intensity is enhanced. The nickel-iron-carbon nano tube loaded with the gold nano particles is used as a substrate material, on one hand, the three-dimensional interconnected carbon nano tube can improve the electron transfer efficiency, and the large-area gold nano particles can immobilize more antibodies, and on the other hand, the uniformly dispersed iron-nickel active center can promote the generation of more tripropylamine free radicals with strong oxidizing property, so that the anti-tumor effect is improved. The luminous efficiency of the pyrenyl covalent organic framework is further improved. The carbohydrate antigen 242 is detected according to different electrochemical luminescence signal intensities of to-be-detected substances with different concentrations.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a pyrene-based covalent organic framework immunosensor. Specifically, a sandwich-type electrochemiluminescent immunosensor for detecting carbohydrate antigen 242 is constructed by using nickel-iron-carbon nanotubes loaded with gold nanoparticles as a base material, a pyrene-based covalent organic framework as a luminescent material to label a carbohydrate antigen 242 detection antibody, and tripropylamine as a co-reactant, belonging to the field of new functional materials and biosensor detection technology. Background Art

[0002] Pancreatic cancer is a highly malignant digestive system tumor. Due to the deep location of the pancreas in the human body, the clinical symptoms of patients are not obvious in the early stage of the disease. When symptoms appear, the disease has reached the middle and late stages and there is a lack of effective treatment, resulting in an extremely high mortality rate. Carbohydrate antigen 242 is a sialylated mucin-type carbohydrate antigen that is mainly present in pancreatic tumor cells. Carbohydrate antigen 242 is also present in a small amount in normal human tissues, with a normal value of <20IU / mL. Its content increases rapidly when pancreatic cancer occurs, and its expression is not affected by bile secretion, bile duct cell destruction, pancreatic duct stenosis or obstruction. Carbohydrate antigen 242 has been considered an ideal pancreatic cancer marker and has great reference value for the differential diagnosis and prognosis of pancreatic cancer. Therefore, rapid and accurate detection of carbohydrate antigen 242 is of great significance for the clinical diagnosis and treatment of related diseases.

[0003] The methods currently used to detect carbohydrate antigen 242 include enzyme-linked immunosorbent assay, radioimmunoassay, chemiluminescence immunoassay, etc. However, the above methods have the disadvantages of radioactive pollution, low sensitivity, long detection cycle, and complicated steps. Compared with traditional analytical methods, electrochemiluminescence sensors effectively integrate the characteristics of electrochemical analysis technology and spectroscopic analysis technology, and have the advantages of small background interference, simple operation, high sensitivity, fast analysis speed, good controllability, and wide linear range. They play an increasingly important role in environmental testing, food safety, biochemical analysis, clinical diagnosis and other fields. Therefore, the present invention prepares a luminescent self-enhanced pyrene-based covalent organic framework immunosensor to achieve the detection of carbohydrate antigen 242.

[0004] In the present invention, the pyrene-based covalent organic framework utilizes the connection of formoyl hydrazide to effectively reduce the aggregation quenching effect caused by the accumulation of luminescent molecules 1,3,6,8-tetrakis (4-formylphenyl) and inhibit the rotation of luminescent molecules to reduce non-radiative transitions, so that the electrochemiluminescence signal is enhanced. Using nickel-iron-carbon nanotubes loaded with gold nanoparticles as substrate materials, on the one hand, the three-dimensionally interconnected carbon nanotubes can improve the electron transfer efficiency, a large number of gold nanoparticles can immobilize more antibodies, and on the other hand, the uniformly dispersed iron-nickel active centers can promote the generation of more strongly oxidizing tripropylamine free radicals, accelerating the generation of excited states of the pyrene-based covalent organic framework. The present invention utilizes layer-by-layer self-assembly technology to prepare an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which has a wide linear range, a low detection limit and good analytical performance, and realizes sensitive and rapid detection of carbohydrate antigen 242. Summary of the invention

[0005] One of the purposes of the present invention is to use a self-enhanced luminescent pyrene-based covalent organic framework as a luminescent body, and the luminescent material exhibits excellent electrochemiluminescence performance.

[0006] The second purpose of the present invention is to synthesize nickel-iron-carbon nanotubes loaded with gold nanoparticles as a base material, and the material exhibits good electrical conductivity and antibody fixation ability; at the same time, the material acts as a co-reaction promoter, which can promote the decomposition of tripropylamine into more strong oxidizing free radicals, thereby increasing the number of excited state luminophores and enhancing the electrochemiluminescence efficiency of the pyrene-based covalent organic framework.

[0007] The third purpose of the present invention is to use nickel-iron-carbon nanotubes loaded with gold nanoparticles as a substrate to fix antibodies, use pyrene-based covalent organic frameworks as antibody markers for detecting carbohydrate antigen 242, use carbohydrate antigen 242 as a target analyte, and utilize the specific immune reaction between antigen and antibody to construct a sensitive and rapid enhanced electrochemiluminescence immunosensor to detect carbohydrate antigen 242.

[0008] The technical solution of the present invention is as follows: 1. A method for preparing a pyrene-based covalent organic framework immunosensor, comprising the following steps: (1) A glassy carbon electrode with a diameter of 3.0 to 5.0 mm was polished into a mirror surface with aluminum oxide polishing powder and ultrasonically cleaned in anhydrous ethanol; (2) taking 3.0 μL of 0.5-3.5 mg / mL of a dispersion of nickel-iron-carbon nanotubes loaded with gold nanoparticles and dropping it onto the electrode surface, drying it at room temperature, rinsing the electrode surface with ultrapure water, and drying it at room temperature; (3) Continue to add 6.0 μL of 5.0-15.0 μg / mL carbohydrate antigen 242 capture antibody to the electrode surface and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 0.8-1.2 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer, and dry in a 4°C refrigerator; (5) Add 6.0 μL of a series of different concentrations of carbohydrate antigen 242 antigen solutions, rinse the electrode surface with pH = 7.4 phosphate buffer, and dry in a 4°C refrigerator; (6) Finally, 6 μL of a 0.25-3.0 mg / mL pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30-40 min, and rinsed with a pH = 7.4 phosphate buffer to obtain an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which was then stored in a 4°C refrigerator for future use.

[0009] 2. The nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles is prepared by the following steps: (1) Preparation of NiFe-CNTs 368 mg of zinc chloride, 12-36 mg of ferric chloride hexahydrate and 19 mg of nickel chloride hexahydrate were added to 25 mL of formamide solution in sequence under stirring, and stirred vigorously for 5-15 min until a uniformly dispersed mixed solution was formed. Subsequently, the mixed solution was transferred to a 50 mL stainless steel container lined with polytetrafluoroethylene, placed in an oven and heated to 60°C-220°C, and maintained for 12 h. The solution after the reaction was taken out and cooled to room temperature, the product was collected by centrifugation, and the product was washed 3 times with ultrapure water and ethanol respectively. The collected precipitate was transferred to a 60°C oven for 10 h drying. The dried precursor powder was transferred to a tubular furnace, heated to 600°C-900°C in an inert gas atmosphere and maintained for 1 h, with a heating rate of 5°C / min. After the reaction was completed, black nickel-iron-carbon nanotube powder was obtained. (2) Preparation of NiFe-CNT dispersion loaded with gold nanoparticles Add 1 mL of 1.0 wt% chloroauric acid aqueous solution to 99 mL of ultrapure water, stir the mixture evenly and heat it to 100°C. Quickly add 2 mL of 1.0 wt% sodium citrate solution to the mixed solution and continue stirring for 20 min to obtain a deep red gold nanoparticle solution. Add 25-105 mg of the prepared nickel-iron-carbon nanotube powder to 20 mL of the gold nanoparticle solution and stir at a constant speed for 1-5 h. Wash the above solution with ultrapure water and ethanol by centrifugation, and dry it in vacuum at 60°C for 12 h to obtain nickel-iron-carbon nanotube material loaded with gold nanoparticles. Take 0.35-5 mg of the above material and add it to 1 mL of ultrapure water, continue ultrasonication for 30 min to obtain a nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles.

[0010] 3. The preparation steps of the pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution are as follows: (1) Preparation of pyrene-based covalent organic frameworks 15 mg of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, 5.5 mg of formic hydrazide, 0.2-1.5 mL of acetic acid, and 2-20 mL of o-dichlorobenzene / n-butanol were mixed in a hard glass tube. The mixture was ultrasonically treated for 5 min, degassed by three freeze-pump-thaw cycles, vacuum sealed, and heated continuously at 60°C to 150°C for 7 days. The reaction mixture was cooled to room temperature, and the light yellow precipitate was collected by centrifugation and washed three times with water, tetrahydrofuran, and chloroform mixed solution. Then, it was vacuum dried at 80°C overnight to obtain a light yellow pyrene-based covalent organic framework. (2) Preparation of pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1-1 mol / L N-hydroxysuccinimide and 1 mL of pyrene-based covalent organic framework were uniformly mixed and incubated at 4°C for 5-10 hours, then the solution was centrifuged at 12000 rpm, 100-500 μL of carbohydrate antigen 242 detection antibody solution was added, and the mixture was shaken at room temperature for 10-40 minutes to obtain a carbohydrate antigen 242 detection antibody solution labeled with a pyrene-based covalent organic framework.

[0011] 4. For the detection of carbohydrate antigen 242, the steps are as follows: (1) The test was carried out using an electrochemical workstation with a three-electrode system, with an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode, the prepared immunosensor based on the luminescent self-enhanced pyrene-based covalent organic framework as the working electrode, the high voltage of the photomultiplier tube set to 800 V, the scanning potential of 0-1.2 V, and the scanning rate of 0.1 V / s; (2) In 10 mL of phosphate buffer solution containing 150 mmol / L tripropylamine at pH = 7.4, the electrochemiluminescence signal intensity generated by the antigen at different concentrations is detected by an electrochemiluminescence system, and a working curve is drawn; (3) replacing the carbohydrate antigen 242 antigen solution with the test sample solution for testing; (4) Using the working curve method, the concentration of carbohydrate antigen 242 in the sample to be tested is calculated.

[0012] Beneficial results of the present invention (1) The present invention successfully synthesized a pyrene-based covalent organic framework with self-enhanced electrochemiluminescence. By introducing formyl hydrazide, the free rotation of 1,3,6,8-tetrakis(4-formylphenyl)pyrene luminescent molecules was effectively inhibited, thereby reducing non-radiative transitions. At the same time, the highly ordered rigid framework effectively improved the stacking state between luminescent molecules, exhibiting high-intensity luminescence efficiency, and solving the problem of low electrochemiluminescence signal.

[0013] (2) In the present invention, nickel-iron-carbon nanotubes loaded with gold nanoparticles are used as the base material. The three-dimensional interconnected carbon nanotubes greatly improve the electron transfer efficiency of the sensing interface, and the gold nanoparticles use stable gold-nitrogen bonds to fix a large number of antibodies. At the same time, the uniformly dispersed iron-nickel active centers can promote the coreactant tripropylamine to generate more strongly oxidizing tripropylamine free radicals, accelerate the generation of the excited state of the pyrene covalent organic framework, and thus enhance the electrochemiluminescence efficiency and improve the sensitivity of the immunosensor.

[0014] (3) The immunosensor based on the luminescent self-enhanced pyrene-based covalent organic framework prepared by the present invention has a linear range of 0.001 to 100 U / mL for carbohydrate antigen 242, a minimum detection limit of 0.00075 U / mL, good specificity, stability and reproducibility, simple operation, and can achieve sensitive and rapid detection of the target carbohydrate antigen 242. DETAILED DESCRIPTION (The present invention is now further described through specific embodiments, but is not limited thereto) Example 1. A method for preparing a pyrene-based covalent organic framework immunosensor, comprising the following steps: (1) A glassy carbon electrode with a diameter of 3.0 mm was polished into a mirror surface with alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) taking 3.0 μL of 0.5 mg / mL of the gold nanoparticle-loaded nickel iron-carbon nanotube dispersion and dropping it onto the electrode surface, drying it at room temperature, rinsing the electrode surface with ultrapure water, and drying it at room temperature; (3) Continue to add 6.0 μL, 5.0 μg / mL carbohydrate antigen 242 capture antibody to the electrode surface and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 0.8 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer, and dry in a 4°C refrigerator; (5) Add 6.0 μL of a series of different concentrations of carbohydrate antigen 242 antigen solutions, rinse the electrode surface with pH = 7.4 phosphate buffer, and dry in a 4°C refrigerator; (6) Finally, 6 μL of a 0.25 mg / mL pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30 min, and rinsed with a pH = 7.4 phosphate buffer to obtain an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which was then stored in a 4°C refrigerator for future use.

[0016] Example 2. A method for preparing a pyrene-based covalent organic framework immunosensor, comprising the following steps: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished into a mirror surface with alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) adding 3.0 μL of 2.5 mg / mL dispersion of nickel iron-carbon nanotubes loaded with gold nanoparticles to the electrode surface, drying at room temperature, rinsing the electrode surface with ultrapure water, and drying at room temperature; (3) Continue to add 6.0 μL, 10.0 μg / mL carbohydrate antigen 242 capture antibody to the electrode surface and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 1.0 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer, and dry in a 4°C refrigerator; (5) Add 6.0 μL of a series of different concentrations of carbohydrate antigen 242 antigen solutions, rinse the electrode surface with pH = 7.4 phosphate buffer, and dry in a 4°C refrigerator; (6) Finally, 6 μL of a 1.25 mg / mL pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 35 min, and rinsed with a pH = 7.4 phosphate buffer to obtain an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which was then stored in a 4°C refrigerator for future use.

[0017] Example 3. A method for preparing a pyrene-based covalent organic framework immunosensor, comprising the following steps: (1) A glassy carbon electrode with a diameter of 5.0 mm was polished into a mirror surface with alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) taking 3.0 μL of 3.5 mg / mL of the gold nanoparticle-loaded nickel iron-carbon nanotube dispersion and dropping it onto the electrode surface, drying it at room temperature, rinsing the electrode surface with ultrapure water, and drying it at room temperature; (3) Continue to add 6.0 μL, 15.0 μg / mL carbohydrate antigen 242 capture antibody to the electrode surface and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 1.2 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer, and dry in a 4°C refrigerator; (5) Add 6.0 μL of a series of different concentrations of carbohydrate antigen 242 antigen solutions, rinse the electrode surface with pH = 7.4 phosphate buffer, and dry in a 4°C refrigerator; (6) Finally, 6 μL of a 3.0 mg / mL pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 40 min, and rinsed with a pH = 7.4 phosphate buffer to obtain an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which was then stored in a 4°C refrigerator for future use.

[0018] Example 4. The nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles is prepared in the following steps: (1) Preparation of NiFe-CNTs 368 mg of zinc chloride, 12 mg of ferric chloride hexahydrate and 19 mg of nickel chloride hexahydrate were added to 25 mL of formamide solution in sequence under stirring and stirred vigorously for 5 min until a uniformly dispersed mixed solution was formed. Subsequently, the mixed solution was transferred to a 50 mL stainless steel container lined with polytetrafluoroethylene, placed in an oven and heated to 60 ° C, and kept for 12 h. The solution after the reaction was taken out and cooled to room temperature, the product was collected by centrifugation, and the product was washed 3 times with ultrapure water and ethanol respectively. The collected precipitate was transferred to a 60 ° C oven for 10 h drying. The dried precursor powder was transferred to a tubular furnace, heated to 600 ° C in an inert gas atmosphere and kept for 1 h, with a heating rate of 5 ° C / min. After the reaction was completed, black nickel iron-carbon nanotube powder was obtained. (2) Preparation of NiFe-CNT dispersion loaded with gold nanoparticles 1 mL of 1.0 wt% chloroauric acid aqueous solution was added dropwise to 99 mL of ultrapure water, and the mixture was stirred evenly and heated to 100°C. 2 mL of 1.0 wt% sodium citrate solution was quickly added to the mixed solution, and stirring was continued for 20 min to obtain a deep red gold nanoparticle solution. 25 mg of the prepared nickel iron-carbon nanotube powder was added to 20 mL of the gold nanoparticle solution and stirred at a constant speed for 1 h. After the above solution was centrifuged and washed with ultrapure water and ethanol respectively, it was vacuum dried at 60°C for 12 h to obtain a nickel iron-carbon nanotube material loaded with gold nanoparticles. 0.35 mg of the above material was added to 1 mL of ultrapure water, and ultrasonication was continued for 30 min to obtain a nickel iron-carbon nanotube dispersion loaded with gold nanoparticles.

[0019] Example 5. The nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles is prepared in the following steps: (1) Preparation of NiFe-CNTs 368 mg of zinc chloride, 25 mg of ferric chloride hexahydrate and 19 mg of nickel chloride hexahydrate were added to 25 mL of formamide solution in sequence under stirring and stirred vigorously for 10 min until a uniformly dispersed mixed solution was formed. Subsequently, the mixed solution was transferred to a 50 mL stainless steel container lined with polytetrafluoroethylene, placed in an oven and heated to 120 ° C, and kept for 12 h. The solution after the reaction was taken out and cooled to room temperature, the product was collected by centrifugation, and the product was washed 3 times with ultrapure water and ethanol respectively. The collected precipitate was transferred to a 60 ° C oven for 10 h drying. The dried precursor powder was transferred to a tubular furnace, heated to 750 ° C in an inert gas atmosphere and kept for 1 h, with a heating rate of 5 ° C / min, and black nickel iron-carbon nanotube powder was obtained after the reaction was completed. (2) Preparation of NiFe-CNT dispersion loaded with gold nanoparticles 1 mL of 1.0 wt% chloroauric acid aqueous solution was added dropwise to 99 mL of ultrapure water, and the mixture was stirred evenly and heated to 100°C. 2 mL of 1.0 wt% sodium citrate solution was quickly added to the mixed solution, and stirring was continued for 20 min to obtain a deep red gold nanoparticle solution. 75 mg of the prepared nickel iron-carbon nanotube powder was added to 20 mL of the gold nanoparticle solution and stirred at a constant speed for 2.5 h. After the above solution was centrifuged and washed with ultrapure water and ethanol respectively, it was vacuum dried at 60°C for 12 h to obtain a nickel iron-carbon nanotube material loaded with gold nanoparticles. 2.45 mg of the above material was added to 1 mL of ultrapure water, and ultrasonicated for 30 min to obtain a nickel iron-carbon nanotube dispersion loaded with gold nanoparticles.

[0020] Example 6. The nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles is prepared in the following steps: (1) Preparation of NiFe-CNTs 368 mg of zinc chloride, 36 mg of ferric chloride hexahydrate and 19 mg of nickel chloride hexahydrate were added to 25 mL of formamide solution in sequence under stirring and stirred vigorously for 15 min until a uniformly dispersed mixed solution was formed. Subsequently, the mixed solution was transferred to a 50 mL stainless steel container lined with polytetrafluoroethylene, placed in an oven and heated to 220 ° C, and kept for 12 h. The solution after the reaction was taken out and cooled to room temperature, the product was collected by centrifugation, and the product was washed 3 times with ultrapure water and ethanol respectively. The collected precipitate was transferred to a 60 ° C oven for 10 h drying. The dried precursor powder was transferred to a tubular furnace, heated to 900 ° C in an inert gas atmosphere and kept for 1 h, with a heating rate of 5 ° C / min, and black nickel iron-carbon nanotube powder was obtained after the reaction was completed. (2) Preparation of NiFe-CNT dispersion loaded with gold nanoparticles 1 mL of 1.0 wt% chloroauric acid aqueous solution was added dropwise to 99 mL of ultrapure water, and the mixture was stirred evenly and heated to 100°C. 2 mL of 1.0 wt% sodium citrate solution was quickly added to the mixed solution, and stirring was continued for 20 min to obtain a deep red gold nanoparticle solution. 105 mg of the prepared nickel-iron-carbon nanotube powder was added to 20 mL of the gold nanoparticle solution and stirred at a constant speed for 5 h. After the above solution was centrifuged and washed with ultrapure water and ethanol respectively, it was vacuum dried at 60°C for 12 h to obtain a nickel-iron-carbon nanotube material loaded with gold nanoparticles. 5 mg of the above material was added to 1 mL of ultrapure water, and ultrasonicated for 30 min to obtain a nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles.

[0021] Example 7. The preparation steps of the pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution are as follows: (1) Preparation of pyrene-based covalent organic frameworks 15 mg of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, 5.5 mg of formic hydrazide, 0.2 mL of acetic acid, and 2 mL of o-dichlorobenzene / n-butanol were mixed in a hard glass tube. The mixture was sonicated for 5 min, degassed by three freeze-pump-thaw cycles, vacuum sealed, and heated at 60 ° C for 7 days. The reaction mixture was cooled to room temperature, and the light yellow precipitate was collected by centrifugation and washed three times with water, tetrahydrofuran, and chloroform mixed solution. Then, it was vacuum dried at 80 ° C overnight to obtain a light yellow pyrene-based covalent organic framework. (2) Preparation of pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution 1 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1 mol / L N-hydroxysuccinimide and 1 mL of pyrene-based covalent organic framework were uniformly mixed and incubated at 4°C for 5 h. The solution was centrifuged at 12000 rpm, 100 μL of carbohydrate antigen 242 detection antibody solution was added, and the mixture was shaken at room temperature for 10 min to obtain a carbohydrate antigen 242 detection antibody solution labeled with a pyrene-based covalent organic framework.

[0022] Example 8. The preparation steps of the pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution are as follows: (1) Preparation of pyrene-based covalent organic frameworks 15 mg of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, 5.5 mg of formic hydrazide, 1.0 mL of acetic acid, and 10 mL of o-dichlorobenzene / n-butanol were mixed in a hard glass tube. The mixture was sonicated for 5 min, degassed by three freeze-pump-thaw cycles, vacuum sealed, and heated at 100 ° C for 7 days. The reaction mixture was cooled to room temperature, and the light yellow precipitate was collected by centrifugation and washed three times with water, tetrahydrofuran, and chloroform mixed solution. Then, it was vacuum dried at 80 ° C overnight to obtain a light yellow pyrene-based covalent organic framework. (2) Preparation of pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution 2.5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.5 mol / L N-hydroxysuccinimide and 1 mL of pyrene-based covalent organic framework were uniformly mixed and incubated at 4°C for 7.5 h, then the solution was centrifuged at 12000 rpm, 100-500 μL of carbohydrate antigen 242 detection antibody solution was added, and the mixture was shaken at room temperature for 25 min to obtain a carbohydrate antigen 242 detection antibody solution labeled with a pyrene-based covalent organic framework.

[0023] Example 9. The preparation steps of the pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution are as follows: (1) Preparation of pyrene-based covalent organic frameworks 15 mg of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, 5.5 mg of formic hydrazide, 1.5 mL of acetic acid, and 20 mL of o-dichlorobenzene / n-butanol were mixed in a hard glass tube. The mixture was sonicated for 5 min, degassed by three freeze-pump-thaw cycles, vacuum sealed, and heated at 150 ° C for 7 days. The reaction mixture was cooled to room temperature, and the light yellow precipitate was collected by centrifugation and washed three times with water, tetrahydrofuran, and chloroform mixed solution. Then, it was vacuum dried at 80 ° C overnight to obtain a light yellow pyrene-based covalent organic framework. (2) Preparation of pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution 5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 1 mol / L N-hydroxysuccinimide and 1 mL of pyrene-based covalent organic framework were uniformly mixed and incubated at 4°C for 10 h. The solution was centrifuged at 12000 rpm, 500 μL of carbohydrate antigen 242 detection antibody solution was added, and the mixture was shaken at room temperature for 40 min to obtain a carbohydrate antigen 242 detection antibody solution labeled with a pyrene-based covalent organic framework.

[0024] Example 10. For the detection of carbohydrate antigen 242, the steps are as follows: (1) The test was carried out using an electrochemical workstation with a three-electrode system, with an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode, the prepared immunosensor based on the luminescent self-enhanced pyrene-based covalent organic framework as the working electrode, the high voltage of the photomultiplier tube set to 800 V, the scanning potential of 0-1.2 V, and the scanning rate of 0.1 V / s; (2) In 10 mL of phosphate buffer solution containing 150 mmol / L tripropylamine at pH = 7.4, the electrochemiluminescence signal intensity generated by the antigen at different concentrations is detected by an electrochemiluminescence system, and a working curve is drawn; (3) replacing the carbohydrate antigen 242 antigen solution with the test sample solution for testing; (4) Using the working curve method, the concentration of carbohydrate antigen 242 in the sample to be tested is calculated. (5) Based on the linear relationship between the obtained luminescence intensity and the concentration of carbohydrate antigen 242, a working curve was drawn with a linear range of 0.001 to 100 U / mL and a detection limit of 0.00075 U / mL.

Claims

1. A method for preparing a pyrene-based covalent organic framework immunosensor, characterized in that: The following steps are involved: (1) A glassy carbon electrode with a diameter of 3.0 to 5.0 mm was polished into a mirror surface with aluminum oxide polishing powder and ultrasonically cleaned in anhydrous ethanol; (2) taking 3.0 μL of 0.5-3.5 mg / mL of a dispersion of nickel-iron-carbon nanotubes loaded with gold nanoparticles and dropping it onto the electrode surface, drying it at room temperature, rinsing the electrode surface with ultrapure water, and drying it at room temperature; (3) Continue to add 6.0 μL of 5.0-15.0 μg / mL carbohydrate antigen 242 capture antibody to the electrode surface and dry in a 4°C refrigerator; (4) Continue to add 3.0 μL of 0.8-1.2 mg / mL bovine serum albumin solution to the electrode surface to block nonspecific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer, and dry in a 4°C refrigerator; (5) Add 6.0 μL of a series of different concentrations of carbohydrate antigen 242 antigen solutions, rinse the electrode surface with pH = 7.4 phosphate buffer, and dry in a 4°C refrigerator; (6) Finally, 6 μL of a 0.25-3.0 mg / mL pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution was dropped onto the electrode, placed in a 4°C refrigerator for 30-40 min, and rinsed with a pH = 7.4 phosphate buffer to obtain an immunosensor based on a luminescent self-enhanced pyrene-based covalent organic framework, which was then stored in a 4°C refrigerator for future use.

2. The method for preparing a pyrene-based covalent organic framework immunosensor according to claim 1, wherein the nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles is characterized in that: Here are the steps: (1) Preparation of NiFe-CNTs 368 mg of zinc chloride, 12-36 mg of ferric chloride hexahydrate and 19 mg of nickel chloride hexahydrate were added to 25 mL of formamide solution in sequence under stirring, and stirred vigorously for 5-15 min until a uniformly dispersed mixed solution was formed. Subsequently, the mixed solution was transferred to a 50 mL stainless steel container lined with polytetrafluoroethylene, placed in an oven and heated to 60°C-220°C, and maintained for 12 h. The solution after the reaction was taken out and cooled to room temperature, the product was collected by centrifugation, and the product was washed 3 times with ultrapure water and ethanol respectively. The collected precipitate was transferred to a 60°C oven for 10 h drying. The dried precursor powder was transferred to a tubular furnace, heated to 600°C-900°C in an inert gas atmosphere and maintained for 1 h, with a heating rate of 5°C / min. After the reaction was completed, black nickel-iron-carbon nanotube powder was obtained. (2) Preparation of NiFe-CNT dispersion loaded with gold nanoparticles Add 1 mL of 1.0 wt% chloroauric acid aqueous solution to 99 mL of ultrapure water, stir the mixture evenly and heat it to 100°C. Quickly add 2 mL of 1.0 wt% sodium citrate solution to the mixed solution and continue stirring for 20 min to obtain a deep red gold nanoparticle solution. Add 25-105 mg of the prepared nickel-iron-carbon nanotube powder to 20 mL of the gold nanoparticle solution and stir at a constant speed for 1-5 h. Wash the above solution with ultrapure water and ethanol by centrifugation, and dry it in vacuum at 60°C for 12 h to obtain nickel-iron-carbon nanotube material loaded with gold nanoparticles. Take 0.35-5 mg of the above material and add it to 1 mL of ultrapure water, continue ultrasonication for 30 min to obtain a nickel-iron-carbon nanotube dispersion loaded with gold nanoparticles.

3. The method for preparing a pyrene-based covalent organic framework immunosensor according to claim 1, wherein the pyrene-based covalent organic framework-labeled carbohydrate antigen 242 detection antibody solution is characterized in that: Here are the steps: (1) Preparation of pyrene-based covalent organic frameworks 15 mg of 1,3,6,8-tetrakis(4-formylphenyl)pyrene, 5.5 mg of formic hydrazide, 0.2-1.5 mL of acetic acid, and 2-20 mL of o-dichlorobenzene / n-butanol were mixed in a hard glass tube. The mixture was ultrasonically treated for 5 min, degassed by three freeze-pump-thaw cycles, vacuum sealed, and heated continuously at 60°C to 150°C for 7 days. The reaction mixture was cooled to room temperature, and the light yellow precipitate was collected by centrifugation and washed three times with water, tetrahydrofuran, and chloroform mixed solution. Then, it was vacuum dried at 80°C overnight to obtain a light yellow pyrene-based covalent organic framework. (2) Preparation of pyrene-based covalent organic framework labeled carbohydrate antigen 242 detection antibody solution 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide, 0.1-1 mol / L N-hydroxysuccinimide and 1 mL of pyrene-based covalent organic framework were uniformly mixed and incubated at 4°C for 5-10 hours, then the solution was centrifuged at 12000 rpm, 100-500 μL of carbohydrate antigen 242 detection antibody solution was added, and the mixture was shaken at room temperature for 10-40 minutes to obtain a carbohydrate antigen 242 detection antibody solution labeled with a pyrene-based covalent organic framework.

4. A method for preparing a pyrene-based covalent organic framework immunosensor as claimed in claim 1, for detecting carbohydrate antigen 242, characterized in that: Here are the steps: (1) The test was carried out using an electrochemical workstation with a three-electrode system, with an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode, the prepared immunosensor based on the luminescent self-enhanced pyrene-based covalent organic framework as the working electrode, the high voltage of the photomultiplier tube set to 800 V, the scanning potential of 0-1.2 V, and the scanning rate of 0.1 V / s; (2) In 10 mL of phosphate buffer solution containing 150 mmol / L tripropylamine at pH = 7.4, the electrochemiluminescence signal intensity generated by the antigen at different concentrations is detected by an electrochemiluminescence system, and a working curve is drawn; (3) replacing the carbohydrate antigen 242 antigen solution with the test sample solution for testing; (4) Using the working curve method, the concentration of carbohydrate antigen 242 in the sample to be tested is calculated.