Disclosure of Invention
One of the purposes of the invention is to adopt the graphite-like phase carbon nitride accurately regulated and controlled by 2,4, 6-triaminopyrimidine as a substrate luminescent material, accurately replace nitrogen atoms in triazine ring structures with carbon atoms, endow the triazine ring structures with excellent electrochemical luminescence behavior, good biocompatibility, stable electrochemical signals and excellent anti-interference capability, enable the triazine ring structures to be connected with primary antibodies of neuron-specific enolase to form stable primary antibody markers, and simplify the construction process of the sensor.
The invention also aims to use Fe-ZIF loaded copper sulfide quantum dots as quenching materials, and because the ultraviolet absorption spectrum of the Fe-ZIF loaded copper sulfide quantum dots is greatly overlapped with the electrochemical luminescence emission spectrum of the modified graphite-like carbon nitride, the high-efficiency quenching can be realized based on resonance energy transfer, and a novel resonance energy transfer donor-acceptor pair is provided for constructing an electrochemical immunosensor.
The invention also aims to provide a preparation method of the novel sandwich type electrochemical immunosensor based on resonance energy transfer.
The fourth purpose of the invention is to realize the construction of the electrochemical immunosensor and sensitively detect the neuron-specific enolase, and the detection limit is 21.6 fg mL-1The application of the electrochemical luminescence sensor in detecting neuron-specific enolase is achieved.
Technical scheme of the invention
1. A preparation method of an electrochemical immunosensor based on 2,4, 6-triaminopyrimidine-regulated graphite-like-phase carbon nitride is characterized by comprising the following steps:
(1) preparation of graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine
Preparing graphite-like carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine by copolymerizing melamine and 2,4, 6-triaminopyrimidine, fully and uniformly mixing 1 g of melamine and 10-50 mg of 2,4, 6-triaminopyrimidine in an agate mortar for grinding, then placing the mixture in a crucible with a cover, and placing the crucible in a muffle furnace for 5 ℃ for min-1Raising the temperature rising rate to 550 ℃, keeping heating for 4 h, and obtaining the graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine, wherein other conditions are the same when the 2,4, 6-triaminopyrimidine is not added, and the original graphite-like phase carbon nitride is prepared;
(2) preparation of primary anti-marker of graphite-like phase carbon nitride combined neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine
5-20 microgram mL-1The primary antibody of the neuron-specific enolase is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine for 6 hours at 4 ℃ to obtain a primary antibody marker of the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine and combined with the neuron-specific enolase recognition antibody;
(3) preparation of Fe-ZIF
Firstly, dissolving 1-3 g of 2-methylimidazole in 30 mL of anhydrous methanol, and marking as a solution A; in addition, 0.1-0.5 g of ferrous sulfate heptahydrate and 0.6-1 g of zinc nitrate hexahydrate are mixed and dissolved in 30 mL of anhydrous methanol and marked as solution B; mixing the two solutions, stirring uniformly, continuously stirring at 35 ℃ for reacting for 4 hours, centrifuging the mixed solution after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using anhydrous methanol, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain Fe-ZIF powder;
(4) preparation of copper sulfide quantum dots
Dissolving 20-50 mg of sodium sulfate nonahydrate in 3 mL of deionized water to obtain a sodium sulfide solution for later use, dissolving 50-100 mg of copper chloride and 80-100 mg of trisodium citrate in 300 mL of ultrapure water, magnetically stirring to obtain a light blue transparent solution, heating the fully dissolved solution, reacting for 30 min at 90 ℃, slowly dropwise adding the sodium sulfide solution in the heating process, observing that the solution gradually becomes dark green and precipitates are generated in the process, centrifuging the suspension after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using the ultrapure water, dissolving the washed copper sulfide quantum dot suspension in 5 mL of deionized water, and storing for later use at 4 ℃;
(5) preparation of Fe-ZIF loaded copper sulfide quantum dot composite material
Dissolving 50-100 mg of prepared Fe-ZIF powder in 300 mL of deionized water, performing magnetic stirring to fully disperse and dissolve the powder, then slowly dropwise adding the copper sulfide quantum dot suspension to fully load the copper sulfide quantum dots with Fe-ZIF, stirring for 8 hours, centrifuging the mixed solution, collecting the obtained product, washing and centrifuging for 3 times with deionized water, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain the Fe-ZIF loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of Fe-ZIF loaded copper sulfide quantum dot combined with neuron specific enolase recognition antibody
20-100 μ L of neuron-specific enolase secondary antibody (10 μ g mL)-1) Adding 1 mL to 1-3 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in an aqueous solution of the Fe-ZIF-loaded copper sulfide quantum dot composite material, centrifuging, and dispersing the obtained product in 1 mL of PBS to obtain a secondary antibody marker solution of the Fe-ZIF-loaded copper sulfide quantum dot combined with a neuron-specific enolase recognition antibody;
(8) preparation of electrochemical immunosensor
1) Throwing with aluminum oxidePolishing a glassy carbon electrode with the diameter of 4 mm by using polishing powder, cleaning with ultrapure water, and carrying out 6 mu L and 0.5-2.5 mg mL treatment on the polished glassy carbon electrode-1The primary-antibody marker solution of the graphite-like phase carbon nitride combined with neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-100 ng mL-1A series of neuron specific enolase antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding 6 muL of Fe-ZIF loaded copper sulfide quantum dots and a second antibody marker solution combined with the neuron specific enolase recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting the neuron specific enolase.
2. The detection method of the electrochemical immunosensor comprises the following steps:
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 500-800V, and the electrochemical luminescence sensor contains 20-100 mmoL L-1The test was performed in a PBS buffer solution of potassium persulfate;
(2) detecting the neuron specific enolase standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the neuron-specific enolase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the neuron-specific enolase, and drawing a working curve;
(4) and replacing the neuron-specific enolase antigen standard solution with the neuron-specific enolase antigen sample solution to be detected for detection.
The sensor is used for specific enolization of neuronsThe linear detection range of the enzyme antigen is 0.00005-100 ng mL-1The detection limit is 21.6 fg mL-1。
The chemicals required for the synthesis were all purchased at the local reagent store and were not reprocessed.
Advantageous results of the invention
(1) According to the invention, the graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine is used as a substrate luminescent material, and part of nitrogen atoms in the graphite-like phase carbon nitride triazine ring structure are accurately replaced by carbon atoms, so that excellent electrochemical luminescence behavior is given to the material, the overall electron distribution of the material is greatly improved, the defects that the original graphite-like phase carbon nitride electrochemical luminescence signal is weak and the luminescence signal is unstable are overcome, and the method has important significance for the extended application of the graphite-like phase carbon nitride.
(2) The invention provides a novel donor-acceptor pair for resonance energy transfer, which is constructed by using graphite-like carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine as a substrate luminescent material and using Fe-ZIF loaded copper sulfide quantum dots as a quenching material.
(3) The invention provides a novel method for constructing an electrochemical immunosensor. According to the method, the primary antibody and the secondary antibody of the substance to be detected are respectively marked by the substrate luminescent material and the quenching material, and then the antigen to be detected is identified, so that the preparation of the sensor is simpler and more convenient, and the prepared sensor has good stability, high sensitivity and good reproducibility.
(4) The electrochemical immunosensor prepared by the invention sensitively detects the neuron specific enolase, and the detection range is 0.00005-100 ng mL-1The detection limit is 21.6 fg mL-1And the detection with simplicity, convenience, rapidness, high sensitivity, high specificity and high stability can be realized.
Detailed Description
EXAMPLE 1 preparation of electrochemical immunosensor
(1) Preparation of graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine
Copolymerization of melamine and 2,4, 6-triaminopyrimidine to prepare 2,4, 6-trisThe graphite-like phase carbon nitride controlled by aminopyrimidine is prepared by mixing 1 g of melamine and 10 mg of 2,4, 6-triaminopyrimidine uniformly and thoroughly in an agate mortar for grinding, then placing the mixture in a crucible with a cover, and heating in a muffle furnace for 5 ℃ for min-1Raising the temperature rising rate to 550 ℃, keeping heating for 4 h, and obtaining the graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine, wherein other conditions are the same when the 2,4, 6-triaminopyrimidine is not added, and the original graphite-like phase carbon nitride is prepared;
(2) preparation of primary anti-marker of graphite-like phase carbon nitride combined neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine
5 microgram mL-1The primary antibody of the neuron-specific enolase is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine for 6 hours at 4 ℃ to obtain a primary antibody marker of the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine and combined with the neuron-specific enolase recognition antibody;
(3) preparation of Fe-ZIF
Firstly, dissolving 1 g of 2-methylimidazole in 30 mL of anhydrous methanol, and marking as a solution A; in addition, 0.1 g of ferrous sulfate heptahydrate and 0.6 g of zinc nitrate hexahydrate are mixed and dissolved in 30 mL of anhydrous methanol, and the solution B is marked; mixing the two solutions, stirring uniformly, continuously stirring at 35 ℃ for reacting for 4 hours, centrifuging the mixed solution after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using anhydrous methanol, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain Fe-ZIF powder;
(4) preparation of copper sulfide quantum dots
Dissolving 20 mg of sodium sulfate nonahydrate in 3 mL of deionized water to obtain a sodium sulfide solution for later use, dissolving 50 mg of copper chloride and 80 mg of trisodium citrate in 300 mL of ultrapure water, magnetically stirring to obtain a light blue transparent solution, heating the fully dissolved solution, reacting for 30 min at 90 ℃, slowly dropwise adding the sodium sulfide solution in the heating process, wherein the solution gradually becomes dark green and precipitates are generated in the process, centrifuging the suspension after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using the ultrapure water, dissolving the washed copper sulfide quantum dot suspension in 5 mL of deionized water, and storing for later use at 4 ℃;
(5) preparation of Fe-ZIF loaded copper sulfide quantum dot composite material
Dissolving the prepared 50 mg of Fe-ZIF powder in 300 mL of deionized water, carrying out magnetic stirring to fully dissolve and disperse the powder, then slowly dropwise adding the copper sulfide quantum dot suspension to fully load the copper sulfide quantum dots with Fe-ZIF, stirring for 8 h, centrifuging the mixed solution, collecting the obtained product, washing and centrifuging for 3 times with deionized water, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain the Fe-ZIF loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of Fe-ZIF loaded copper sulfide quantum dot combined with neuron specific enolase recognition antibody
mu.L of a secondary antibody to neuron-specific enolase (10. mu.g mL)-1) Added to 1 mL of 1 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in an aqueous solution of the Fe-ZIF-loaded copper sulfide quantum dot composite material, centrifuging, and dispersing the obtained product in 1 mL of PBS to obtain a secondary antibody marker solution of the Fe-ZIF-loaded copper sulfide quantum dot combined with a neuron-specific enolase recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 muL and 0.5 mg mL-12,4, 6-triaminopyrimidine-regulated graphite-like phase carbon nitride-binding neuron-specific enolizationDropwise adding a primary antibody marker solution of the enzyme recognition antibody to the surface of the electrode, and airing at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-100 ng mL-1A series of neuron specific enolase antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding 6 muL of Fe-ZIF loaded copper sulfide quantum dots and a second antibody marker solution combined with the neuron specific enolase recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting the neuron specific enolase.
EXAMPLE 2 preparation of electrochemical immunosensor
(1) Preparation of graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine
2,4, 6-triaminopyrimidine-regulated graphite-like carbon nitride is prepared by copolymerizing melamine and 2,4, 6-triaminopyrimidine, 1 g of melamine and 30 mg of 2,4, 6-triaminopyrimidine are mixed sufficiently and uniformly in an agate mortar for grinding, then the mixture is placed in a crucible with a cover and is put in a muffle furnace for 5 ℃ for min-1Raising the temperature rising rate to 550 ℃, keeping heating for 4 h, and obtaining the graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine, wherein other conditions are the same when the 2,4, 6-triaminopyrimidine is not added, and the original graphite-like phase carbon nitride is prepared;
(2) preparation of primary anti-marker of graphite-like phase carbon nitride combined neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine
10 microgram mL-1The primary antibody of the neuron-specific enolase is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine for 6 hours at 4 ℃ to obtain the primary antibody of the graphite-like carbon nitride combined with the neuron-specific enolase recognition antibody regulated by the 2,4, 6-triaminopyrimidineAn agent;
(3) preparation of Fe-ZIF
Dissolving 2 g of 2-methylimidazole in 30 mL of anhydrous methanol, and marking as a solution A; in addition, 0.3 g of ferrous sulfate heptahydrate and 0.8 g of zinc nitrate hexahydrate are mixed and dissolved in 30 mL of anhydrous methanol and marked as solution B; mixing the two solutions, stirring uniformly, continuously stirring at 35 ℃ for reacting for 4 hours, centrifuging the mixed solution after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using anhydrous methanol, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain Fe-ZIF powder;
(4) preparation of copper sulfide quantum dots
Dissolving 30 mg of sodium sulfate nonahydrate in 3 mL of deionized water to obtain a sodium sulfide solution for later use, dissolving 80 mg of copper chloride and 90 mg of trisodium citrate in 300 mL of ultrapure water, magnetically stirring to obtain a light blue transparent solution, heating the fully dissolved solution, reacting for 30 min at 90 ℃, slowly dropwise adding the sodium sulfide solution in the heating process, wherein the solution gradually becomes dark green and precipitates are generated in the process, centrifuging the suspension after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using the ultrapure water, dissolving the washed copper sulfide quantum dot suspension in 5 mL of deionized water, and storing for later use at 4 ℃;
(5) preparation of Fe-ZIF loaded copper sulfide quantum dot composite material
Dissolving 80 mg of prepared Fe-ZIF powder in 300 mL of deionized water, carrying out magnetic stirring to fully dissolve and disperse the powder, then slowly dropwise adding the copper sulfide quantum dot suspension to fully load the copper sulfide quantum dots with the Fe-ZIF, stirring for 8 h, centrifuging the mixed solution, collecting the obtained product, washing and centrifuging for 3 times with the deionized water, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain the Fe-ZIF loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask and preparing into concentrated solutionDegree of 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of Fe-ZIF loaded copper sulfide quantum dot combined with neuron specific enolase recognition antibody
mu.L of neuron-specific enolase secondary antibody (10. mu.g mL)-1) Added to 1 mL of 2 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in an aqueous solution of the Fe-ZIF-loaded copper sulfide quantum dot composite material, centrifuging, and dispersing the obtained product in 1 mL of PBS to obtain a secondary antibody marker solution of the Fe-ZIF-loaded copper sulfide quantum dot combined with a neuron-specific enolase recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 1.5 mg mL-1The primary-antibody marker solution of the graphite-like phase carbon nitride combined with neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-100 ng mL-1A series of neuron specific enolase antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding 6 muL of Fe-ZIF loaded copper sulfide quantum dots and a second antibody marker solution combined with the neuron specific enolase recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting the neuron specific enolase.
EXAMPLE 3 preparation of electrochemical immunosensor
(1) Preparation of graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine
Preparation of 2,4, 6-triaminopyrimidine-regulated graphite-like phase nitriding through copolymerization of melamine and 2,4, 6-triaminopyrimidineCarbon, 1 g of melamine and 50 mg of 2,4, 6-triaminopyrimidine were mixed thoroughly and homogeneously in an agate mortar for grinding, and the mixture was placed in a crucible with a lid and heated in a muffle furnace at 5 ℃ for 5 min-1Raising the temperature rising rate to 550 ℃, keeping heating for 4 h, and obtaining the graphite-like phase carbon nitride regulated and controlled by 2,4, 6-triaminopyrimidine, wherein other conditions are the same when the 2,4, 6-triaminopyrimidine is not added, and the original graphite-like phase carbon nitride is prepared;
(2) preparation of primary anti-marker of graphite-like phase carbon nitride combined neuron-specific enolase recognition antibody regulated and controlled by 2,4, 6-triaminopyrimidine
20 microgram mL-1The primary antibody of the neuron-specific enolase is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine for 6 hours at 4 ℃ to obtain a primary antibody marker of the graphite-like carbon nitride regulated by 2,4, 6-triaminopyrimidine and combined with the neuron-specific enolase recognition antibody;
(3) preparation of Fe-ZIF
Dissolving 3 g of 2-methylimidazole in 30 mL of anhydrous methanol, and marking as a solution A; in addition, 0.5 g of ferrous sulfate heptahydrate and 1 g of zinc nitrate hexahydrate are mixed and dissolved in 30 mL of anhydrous methanol, and the solution is marked as solution B; mixing the two solutions, stirring uniformly, continuously stirring at 35 ℃ for reacting for 4 hours, centrifuging the mixed solution after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using anhydrous methanol, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain Fe-ZIF powder;
(4) preparation of copper sulfide quantum dots
Dissolving 50 mg of sodium sulfate nonahydrate in 3 mL of deionized water to obtain a sodium sulfide solution for later use, dissolving 100 mg of copper chloride and 100 mg of trisodium citrate in 300 mL of ultrapure water, magnetically stirring to obtain a light blue transparent solution, heating the fully dissolved solution, reacting for 30 min at 90 ℃, slowly dropwise adding the sodium sulfide solution in the heating process, wherein the solution gradually becomes dark green and precipitates are generated in the process, centrifuging the suspension after the reaction is finished, collecting the obtained product, washing and centrifuging for 3 times by using the ultrapure water, dissolving the washed copper sulfide quantum dot suspension in 5 mL of deionized water, and storing for later use at 4 ℃;
(5) preparation of Fe-ZIF loaded copper sulfide quantum dot composite material
Dissolving 100 mg of prepared Fe-ZIF powder in 300 mL of deionized water, carrying out magnetic stirring to fully dissolve and disperse the powder, then slowly dropwise adding the copper sulfide quantum dot suspension to fully load the copper sulfide quantum dots with Fe-ZIF, stirring for 8 h, centrifuging the mixed solution, collecting the obtained product, washing and centrifuging for 3 times with deionized water, and drying the washed product in a vacuum drying oven at 60 ℃ overnight to obtain the Fe-ZIF loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of Fe-ZIF loaded copper sulfide quantum dot combined with neuron specific enolase recognition antibody
mu.L of neuron-specific enolase secondary antibody (10. mu.g mL)-1) Added to 1 mL of 3 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in an aqueous solution of the Fe-ZIF-loaded copper sulfide quantum dot composite material, centrifuging, and dispersing the obtained product in 1 mL of PBS to obtain a secondary antibody marker solution of the Fe-ZIF-loaded copper sulfide quantum dot combined with a neuron-specific enolase recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 2.5 mg mL-1The primary-antibody marker solution of the 2,4, 6-triaminopyrimidine-regulated graphite-phase carbon nitride-combined neuron-specific enolase recognition antibodyDropwise adding the solution to the surface of an electrode, and airing at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-100 ng mL-1A series of neuron specific enolase antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding 6 muL of Fe-ZIF loaded copper sulfide quantum dots and a second antibody marker solution combined with the neuron specific enolase recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting the neuron specific enolase.
Example 4 detection of neuron-specific enolase
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 500V, and the electrochemical luminescence sensor contains 20 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting the neuron specific enolase standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) and observing the electrochemiluminescence intensity of the sensor before and after the addition of the neuron-specific enolase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the neuron-specific enolase, and drawing a working curve.
Example 5 detection of neuron-specific Enolase
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 600V, and the electrochemical luminescence sensor contains 50 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting the neuron specific enolase standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.5-0V;
(3) and observing the electrochemiluminescence intensity of the sensor before and after the addition of the neuron-specific enolase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the neuron-specific enolase, and drawing a working curve.
Example 6 detection of neuron-specific enolase
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 700V, and the electrochemical luminescence sensor contains 80 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting the neuron specific enolase standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.4-0V;
(3) and observing the electrochemiluminescence intensity of the sensor before and after the addition of the neuron-specific enolase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the neuron-specific enolase, and drawing a working curve.
Example 7 detection of neuron-specific enolase
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 800V, and the electrochemical luminescence sensor contains 100 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting the neuron specific enolase standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.3-0V;
(3) and observing the electrochemiluminescence intensity of the sensor before and after the addition of the neuron-specific enolase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the neuron-specific enolase, and drawing a working curve.