CN110441528B - Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor - Google Patents

Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor Download PDF

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CN110441528B
CN110441528B CN201910706346.6A CN201910706346A CN110441528B CN 110441528 B CN110441528 B CN 110441528B CN 201910706346 A CN201910706346 A CN 201910706346A CN 110441528 B CN110441528 B CN 110441528B
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core
electrode
shell structure
cardiac troponin
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CN110441528A (en
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吴丹
王路晓
邢彬
王翰禹
胡丽华
王欢
魏琴
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University of Jinan
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue

Abstract

The invention relates to Mo based on a core-shell structure2The construction of a C @ C nanosphere cardiac troponin I immunosensor belongs to the technical field of novel sensor construction. Based on good specificity among antigen and antibody, the sensor utilizes multi-walled carbon nano-tube @ perfluorosulfonic acid-polytetrafluoroethylene copolymer MWCNTs-NH modified by amino2@ Nafion solution is used as a substrate material and loaded with polyethyleneimine PEI (PEI) core-shell structure Mo2The C @ C nanospheres are used as secondary antibody markers, the boron nitride quantum dots BNQDs are used as co-reactants, and the cardiac troponin I immunosensor is constructed through layer-by-layer self-assembly. The electrochemiluminescence immunosensor constructed by the invention has wider detection range, higher sensitivity and lower detection limit, and has important significance for detecting the cardiac troponin I.

Description

Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
Technical Field
The invention relates to a preparation method and application of a transition metal nano material electrochemiluminescence immunosensor, in particular to MWCNTs-NH adopted by the invention2@ Nafion solution is used as a substrate material and loaded with polyethyleneimine PEI (PEI) core-shell structure Mo2The C @ C nanospheres are secondary antibody markers, the BNQDs are co-reactants, the troponin I immunosensor constructed by layer-by-layer self-assembly can realize the specificity detection of the troponin I, and the method belongs to the technical field of novel sensor construction.
Background
It is very beneficial to treat myocardial damage accurately in an early stage, prevent irreversible damage to the heart, especially in acute myocardial infarction patients, and significantly improve survival rate. The cardiac troponin is a regulatory protein for cardiac muscle contraction and is located at a position of the cardiac muscle fibrillin double strands. When myocardial injury occurs, cardiac troponin I is rapidly released into the blood, wherein the extent of myocardial injury is proportional to the amount of cardiac troponin I released into the blood. Currently, cardiac troponin I has been recognized as a marker for diagnosing acute myocardial infarction and acute coronary syndrome. To date, a variety of analytical methods for detecting cardiac troponin I have been established, such as liquid chromatography, capillary electrophoresis, and surface plasmon resonance detection, but most of them require expensive instruments and equipment, take long time for sample preparation, and are not suitable for screening of large amounts of samples. Therefore, it is necessary to establish a method for measuring cardiac troponin I which has high sensitivity, fast response speed and easy operation. Electrochemiluminescence immunosensors, especially sandwich-type electrochemiluminescence immunosensors, have attracted a wide interest in bioanalysis due to their advantages of high sensitivity, low cost, fast response time, and the like.
The invention constructs a novel electrochemiluminescence immunosensor based on nano functional materials, and the electrochemiluminescence immunosensor is used for detecting cardiac troponin I. Using MWCNTs-NH2@ Nafion solution is used as a substrate material and loaded with polyethyleneimine PEI (PEI) core-shell structure Mo2The C @ C nanospheres are used as secondary antibody markers, and the boron nitride quantum dots BNQDs are used as co-reactants. The test results show that the electrochemiluminescence immunosensor has high sensitivity, good stability and low detection limit, and based on the findings, the inventors completed the present invention.
Disclosure of Invention
One of the objects of the present invention is MWCNTs-NH2The @ Nafion solution is used as a substrate material, and an electrochemiluminescence immunosensor with good selectivity, rapidness and ultrasensitiveness is constructed by utilizing the specific combination of antigen and antibody, so that the simple and sensitive detection of cardiac troponin I is realized.
The second purpose of the invention is to load the core-shell structure Mo of polyethyleneimine PEI2The C @ C nanosphere is a second antibody marker, and provides Mo based on a transition metal material core-shell structure2The preparation method of the C @ C nanosphere electrochemiluminescence immunosensor has the advantages that the sensor prepared by the method is good in stability, selectivity, sensitivity and repeatability.
The third purpose of the invention is to construct a novel Mo based on a core-shell structure by taking BNQDs as a coreactant2A C @ C nanosphere cardiac troponin I immunosensor.
Technical scheme of the invention
1. Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
(1) With Al2O3Polishing a glassy carbon electrode with the diameter of 4 mm by using polishing powder, respectively ultrasonically cleaning the glassy carbon electrode for 30 min by using absolute ethyl alcohol and ultrapure water in sequence, and then carrying out ultrasonic cleaning on the glassy carbon electrode by using 6 mu L and 0.1-1.0 mg mL-1The MWCNTs-NH is a multi-walled carbon nanotube @ perfluorosulfonic acid-polytetrafluoroethylene copolymer modified by amino2Dripping @ Nafion solution on the surface of the electrode, and airing at room temperature;
(2) 6 muL and 10 mmol L-1The terpyridyl ruthenium solution is dripped on the surface of the electrode and dried at room temperature;
(3) dropwise adding 3 mu L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide EDC/NHS solution to the surface of the electrode to activate MWCNTs-NH2@ amino group in Nafion, washing electrode with phosphate buffer solution PBS of pH 7.4, and drying at room temperature;
(4) 6 mu L and 5-15 mu g mL-1Cardiac troponin I capture antibody cTnI-Ab1Dropwise adding a bovine serum albumin BSA solution with the mass fraction of 0.5% -2.0% to the surface of the electrode, washing the electrode with PBS with the pH of 7.4, and airing at room temperature;
(5) dropwise adding 6 mu L and 10-4 ~ 50 ng mL-1A series of cardiac troponin I antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS with pH of 7.4, and dried at room temperature;
(6) dropwise adding 6 mu L core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Drying the solution at room temperature to obtain the final productMo based on core-shell structure2A C @ C nanosphere cardiac troponin I immunosensor.
2. MWCNTs-NH2Preparation of @ Nafion solution
Adding 0.4 g of dicycloethyl carbodiimide DCC and 20 mg of carboxylated carbon nano tube MWCNTs-COOH into 2.5 mL of ethylenediamine EDA solution, heating and reacting at 120 ℃ for 4 days, obtaining precipitates through centrifugal separation, and repeatedly washing the precipitates with ultrapure water and absolute ethyl alcohol respectively to obtain aminated carbon nano tube MWCNTs-NH2Drying at 35 deg.C under vacuum, and weighing 0.04-0.4 mg MWCNTs-NH2Dispersing a sample in 0.4 mL of perfluorosulfonic acid-polytetrafluoroethylene copolymer Nafion solution with the mass fraction of 0.5%, and obtaining uniform MWCNTs-NH after ultrasonic treatment for 20 min2@ Nafion solution.
3. Core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
(1) Core-shell structure Mo2Preparation of C @ C nanospheres
Dissolving 12-30 mg of ammonium molybdate and 500-1200 mg of glucose in 45 mL of ultrapure water in sequence, transferring the solution into a high-pressure reaction kettle, reacting the solution at 180 ℃ for 10 hours, centrifuging the product after the reaction is finished in a centrifuge tube, washing the product with absolute ethyl alcohol and the ultrapure water for three times respectively, then placing the product in a vacuum drying oven at 25 ℃ for 12 hours, grinding the obtained sample, and calcining the product in a tube furnace at 800 ℃ for 2 hours under argon to obtain Mo with a core-shell structure2C @ C nanospheres;
(2) core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
3.0 mL, 1.0 mg mL-1 Core-shell structure Mo2C @ C nanospheres dispersed in 12 mL solution containing 0.625 mol L-1Potassium chloride and 1.25 mg mL-1In the mixed solution of PEI, shaking for 12 h after 2 h of ultrasonic treatment to obtain a uniform black solution, centrifugally collecting a solid product, washing with ultrapure water for three times, and dispersing the solid product in 6 mL of ultrapure water to obtain Mo2C @ C/PEI solutionThen 1 mL of the solution is removed, and 200 muL of cardiac troponin I detection antibody cTnI-Ab is added2Shaking and incubating for 12-24 h at 4 ℃, centrifuging, and re-dispersing the solid in 1 mL of PBS with pH of 7.4 to obtain the polyethyleneimine PEI-loaded Mo with the core-shell structure2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2。
4. Detection of cardiac troponin I
(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and a modified working electrode in a cassette of a chemiluminescence detector, adding 10 mL of mixed solution of tri-n-propylamine solution containing NaCl and BNQDs into an electrolytic cell, wherein the volume ratio of the tri-n-propylamine solution to the mixed solution is 5:1, and the concentration of the tri-n-propylamine solution is 1-5 mmol L-1；
(2) Detecting the cardiac troponin I standard solutions with different concentrations by using a cyclic voltammetry method, wherein the high voltage of a photomultiplier is set to be 800V, and the scanning voltage is set to be 0-1.0V;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration logarithm of the cardiac troponin I.
Advantageous results of the invention
(1) The inventor of the invention uses MWCNTs-NH2@ Nafion solution as substrate material due to MWCNTs-NH2The @ Nafion has good conductivity and large specific surface area, and improves the sensitivity and stability of the sensor;
(2) the invention uses PEI-loaded core-shell structure Mo2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Applied to the preparation of electrochemiluminescence immunosensors, Mo is in a core-shell structure2The C @ C nanospheres have good conductivity and stable chemical properties, and the stability and the reproducibility of the sensor are improved;
(3) the invention adopts BNQDs as a coreactant to construct Mo based on a core-shell structure2The C @ C nanosphere cardiac troponin I immunosensor is used for effectively detecting cardiac troponin I, and the method is simple to operate;
(4) the electrochemiluminescence immunosensor prepared by the invention is used for detecting the cardiac troponin I, has high stability, good reproducibility, high sensitivity and wide linear range, and can realize simple, quick, high-sensitivity and specific detection.
Detailed Description
Embodiment 1 Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
(1) With Al2O3Polishing glassy carbon electrodes with the diameter of 4 mm by polishing powder, respectively ultrasonically cleaning for 30 min by using absolute ethyl alcohol and ultrapure water in sequence, and then carrying out ultrasonic cleaning on the glassy carbon electrodes with the diameter of 6 muL and 0.1 mg mL-1The MWCNTs-NH is a multi-walled carbon nanotube @ perfluorosulfonic acid-polytetrafluoroethylene copolymer modified by amino2Dripping the @ Nafion solution on the surface of the electrode, and airing at room temperature;
(2) 6 mu L and 10 mmol L-1The terpyridyl ruthenium solution is dripped on the surface of the electrode and dried at room temperature;
(3) 3 μ L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide EDC/NHS solution was applied dropwise to the electrode surface to activate MWCNTs-NH2@ amino group in Nafion, washing the electrode with PBS of pH 7.4, and air-drying at room temperature;
(4) 6 mu L and 5 mu g mL-1Cardiac troponin I capture antibody cTnI-Ab1Dropwise coating a bovine serum albumin BSA solution with the mass fraction of 0.5% and the volume of 3 muL on the surface of the electrode, washing the electrode by PBS with the pH of 7.4, and airing at room temperature;
(5) dropwise adding 6 mu L and 10-4 ~ 50 ng mL-1A series of cardiac troponin I antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS with pH of 7.4, and dried at room temperature;
(6) dropwise adding 6 mu L core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2The solution is dried at room temperature to prepare Mo based on the core-shell structure2A C @ C nanosphere cardiac troponin I immunosensor.
ExamplesMo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
(1) With Al2O3Polishing glassy carbon electrodes with the diameter of 4 mm by polishing powder, respectively ultrasonically cleaning for 30 min by using absolute ethyl alcohol and ultrapure water in sequence, and then carrying out ultrasonic cleaning on the glassy carbon electrodes with the diameter of 6 muL and 0.5 mg mL-1The MWCNTs-NH is a multi-walled carbon nanotube @ perfluorosulfonic acid-polytetrafluoroethylene copolymer modified by amino2Dripping the @ Nafion solution on the surface of the electrode, and airing at room temperature;
(2) 6 mu L and 10 mmol L-1The terpyridyl ruthenium solution is dripped on the surface of the electrode and dried at room temperature;
(3) 3 μ L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide EDC/NHS solution was applied dropwise to the electrode surface to activate MWCNTs-NH2@ amino group in Nafion, washing the electrode with PBS of pH 7.4, and air-drying at room temperature;
(4) 6 mu L and 10 mu g mL-1Cardiac troponin I capture antibody cTnI-Ab1Dropwise adding a bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode, washing the electrode with PBS with the pH of 7.4, and airing at room temperature;
(5) dropwise adding 6 mu L and 10-4 ~ 50 ng mL-1A series of cardiac troponin I antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS with pH of 7.4, and dried at room temperature;
(6) dropwise adding 6 mu L core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2The solution is dried at room temperature to prepare Mo based on the core-shell structure2A C @ C nanosphere cardiac troponin I immunosensor.
Embodiment 3 Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
(1) With Al2O3Polishing glassy carbon electrodes with the diameter of 4 mm by polishing powder, respectively ultrasonically cleaning for 30 min by using absolute ethyl alcohol and ultrapure water in sequence, and then carrying out ultrasonic cleaning on the glassy carbon electrodes with the diameter of 6 muL and 1.0 mg mL-1Amino modified multi-walled carbon nanotube @ perfluorosulfonic acid-polyTetrafluoroethylene copolymer MWCNTs-NH2Dripping the @ Nafion solution on the surface of the electrode, and airing at room temperature;
(2) 6 mu L and 10 mmol L-1The terpyridyl ruthenium solution is dripped on the surface of the electrode and dried at room temperature;
(3) 3 μ L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide EDC/NHS solution was applied dropwise to the electrode surface to activate MWCNTs-NH2@ amino group in Nafion, washing the electrode with PBS of pH 7.4, and air-drying at room temperature;
(4) 6 muL and 15 mug mL-1Cardiac troponin I capture antibody cTnI-Ab1Dropwise adding a bovine serum albumin BSA solution with the mass fraction of 2% to the surface of the electrode, washing the electrode with PBS with the pH of 7.4, and airing at room temperature;
(5) dropwise adding 6 mu L and 10-4 ~ 50 ng mL-1A series of cardiac troponin I antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS with pH of 7.4, and dried at room temperature;
(6) dropwise adding 6 mu L core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2The solution is dried at room temperature to prepare Mo based on the core-shell structure2A C @ C nanosphere cardiac troponin I immunosensor.
Example 4 MWCNTs-NH2Preparation of @ Nafion solution
Adding 0.4 g of dicycloethyl carbodiimide DCC and 20 mg of carboxylated carbon nano tube MWCNTs-COOH into 2.5 mL of ethylenediamine EDA solution, heating and reacting at 120 ℃ for 4 days, obtaining precipitates through centrifugal separation, and repeatedly washing the precipitates with ultrapure water and absolute ethyl alcohol respectively to obtain aminated carbon nano tube MWCNTs-NH2It was dried under vacuum at 35 ℃ and then 0.04 mg of MWCNTs-NH was weighed2Dispersing a sample in 0.4 mL of perfluorosulfonic acid-polytetrafluoroethylene copolymer Nafion solution with the mass fraction of 0.5%, and obtaining uniform MWCNTs-NH after ultrasonic treatment for 20 min2@ Nafion solution.
Example 5 MWCNTs-NH2Of @ Nafion solutionPreparation of
Adding 0.4 g of dicycloethyl carbodiimide DCC and 20 mg of carboxylated carbon nano tube MWCNTs-COOH into 2.5 mL of ethylenediamine EDA solution, heating and reacting at 120 ℃ for 4 days, obtaining precipitates through centrifugal separation, and repeatedly washing the precipitates with ultrapure water and absolute ethyl alcohol respectively to obtain aminated carbon nano tube MWCNTs-NH2It was dried under vacuum at 35 ℃ and then 0.1 mg of MWCNTs-NH was weighed2Dispersing a sample in 0.4 mL of perfluorosulfonic acid-polytetrafluoroethylene copolymer Nafion solution with the mass fraction of 0.5%, and obtaining uniform MWCNTs-NH after ultrasonic treatment for 20 min2@ Nafion solution.
Example 6 MWCNTs-NH2Preparation of the @ Nafion solution
Adding 0.4 g of Dicycloethylcarbodiimide (DCC) and 20 mg of carboxylated carbon nanotube MWCNTs-COOH into 2.5 mL of Ethylenediamine (EDA) solution, heating to react for 4 days at 120 ℃, obtaining precipitates by centrifugal separation, and repeatedly washing the precipitates with ultrapure water and absolute ethyl alcohol respectively to obtain aminated carbon nanotube MWCNTs-NH2It was dried under vacuum at 35 ℃ and then 0.4 mg of MWCNTs-NH was weighed2Dispersing a sample in 0.4 mL of perfluorosulfonic acid-polytetrafluoroethylene copolymer Nafion solution with the mass fraction of 0.5%, and obtaining uniform MWCNTs-NH after ultrasonic treatment for 20 min2@ Nafion solution.
Embodiment 7 core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
(1) Core-shell structure Mo2Preparation of C @ C nanospheres
Dissolving 12 mg of ammonium molybdate and 500 mg of glucose in 45 mL of ultrapure water in sequence, transferring the solution into a high-pressure reaction kettle, reacting the solution for 10 hours at 180 ℃, after the reaction is finished, placing the product into a centrifuge tube for centrifugation, washing the product with absolute ethyl alcohol and the ultrapure water for three times respectively, then placing the product into a vacuum drying chamber at 25 ℃ for 12 hours, grinding the obtained sample, and calcining the sample for 2 hours at 800 ℃ under argon by using a tube furnace to obtain Mo with a core-shell structure2C @ C nanospheres;
(2) loaded with polyethyleneCore-shell structure Mo of ethylenimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C @ C nanospheres dispersed in 12 mL of a dispersion containing 0.625 mol L-1Potassium chloride and 1.25 mg mL-1In the mixed solution of PEI, shaking for 12 h after 2 h of ultrasonic treatment to obtain a uniform black solution, centrifugally collecting a solid product, washing with ultrapure water for three times, and dispersing the solid product in 6 mL of ultrapure water to obtain Mo2C @ C/PEI solution, followed by 1 mL of the above solution and 200. mu.L of the cardiac troponin I detection antibody cTnI-Ab2Shaking and incubating at 4 ℃ for 12 h, centrifuging, and re-dispersing the solid in 1 mL of PBS with pH of 7.4 to obtain the polyethyleneimine PEI-loaded Mo with the core-shell structure2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2。
Embodiment 8 core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
(1) Core-shell structure Mo2Preparation of C @ C nanospheres
Dissolving 23 mg of ammonium molybdate and 1000 mg of glucose in 45 mL of ultrapure water in sequence, transferring the solution into a high-pressure reaction kettle, reacting the solution for 10 hours at 180 ℃, after the reaction is finished, placing the product into a centrifuge tube for centrifugation, washing the product with absolute ethyl alcohol and the ultrapure water for three times respectively, then placing the product into a vacuum drying chamber at 25 ℃ for 12 hours, grinding the obtained sample, and calcining the product for 2 hours at 800 ℃ under argon by using a tube furnace to obtain Mo with a core-shell structure2C @ C nanospheres;
(2) core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C @ C nanospheres dispersed in 12 mL of a dispersion containing 0.625 mol L-1Potassium chloride and 1.25 mg mL-1Shaking for 12 h after 2 h of ultrasonic treatment in the mixed solution of PEI to obtain a uniform black solution, centrifuging to collect a solid product, washing with ultrapure water for three times, anddispersing the Mo into 6 mL of ultrapure water to obtain Mo2C @ C/PEI solution, followed by 1 mL of the above solution and 200. mu.L of the cardiac troponin I detection antibody cTnI-Ab2Shaking and incubating at 4 ℃ for 18 h, centrifuging, and re-dispersing the solid in 1 mL of PBS with pH of 7.4 to obtain the polyethyleneimine PEI-loaded Mo with the core-shell structure2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2。
Example 9 core-shell Structure Mo loaded with polyethyleneimine PEI2Second-antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
(1) Core-shell structure Mo2Preparation of C @ C nanospheres
Dissolving 30 mg of ammonium molybdate and 1200 mg of glucose in 45 mL of ultrapure water in sequence, transferring the solution into a high-pressure reaction kettle, reacting the solution at 180 ℃ for 10 hours, after the reaction is finished, placing the product into a centrifuge tube for centrifugation, washing the product with absolute ethyl alcohol and the ultrapure water for three times respectively, then placing the product into a vacuum drying chamber at 25 ℃ for 12 hours, grinding the obtained sample, and calcining the sample at 800 ℃ for 2 hours under argon by using a tube furnace to obtain Mo with a core-shell structure2C @ C nanospheres;
(2) core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C @ C nanospheres dispersed in 12 mL solution containing 0.625 mol L-1Potassium chloride and 1.25 mg mL-1In the mixed solution of PEI, shaking for 12 h after 2 h of ultrasonic treatment to obtain a uniform black solution, centrifugally collecting a solid product, washing with ultrapure water for three times, and dispersing the solid product in 6 mL of ultrapure water to obtain Mo2C @ C/PEI solution, followed by 1 mL of the above solution and 200. mu.L of the cardiac troponin I detection antibody cTnI-Ab2Shaking and incubating at 4 ℃ for 24 h, centrifuging, and re-dispersing the solid in 1 mL of PBS with pH of 7.4 to obtain the polyethyleneimine PEI-loaded Mo with the core-shell structure2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2。
Example 10 detection of cardiac troponin I
(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and a modified working electrode in a cassette of a chemiluminescence detector, adding 10 mL of mixed solution of tri-n-propylamine solution containing NaCl and BNQDs into an electrolytic cell, wherein the volume ratio of the tri-n-propylamine solution to the mixed solution is 5:1, and the concentration of the tri-n-propylamine solution is 1 mmol L-1；
(2) Detecting the cardiac troponin I standard solutions with different concentrations by using a cyclic voltammetry method, wherein the high voltage of a photomultiplier is set to be 800V, and the scanning voltage is set to be 0-1.0V;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration logarithm of the cardiac troponin I.
Example 11 detection of cardiac troponin I
(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and a modified working electrode in a cassette of a chemiluminescence detector, adding 10 mL of mixed solution of tri-n-propylamine solution containing NaCl and BNQDs into an electrolytic cell, wherein the volume ratio of the tri-n-propylamine solution to the mixed solution is 5:1, and the concentration of the tri-n-propylamine solution is 2 mmol L-1；
(2) Detecting the cardiac troponin I standard solutions with different concentrations by using a cyclic voltammetry method, wherein the high voltage of a photomultiplier is set to be 800V, and the scanning voltage is set to be 0-1.0V;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration logarithm of the cardiac troponin I.
Example 12 detection of cardiac troponin I
(1) Connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and a modified working electrode in a cassette of a chemiluminescence detector, adding 10 mL of mixed solution of tri-n-propylamine solution containing NaCl and BNQDs into an electrolytic cell, wherein the volume ratio of the tri-n-propylamine solution to the mixed solution is 5:1, and the concentration of the tri-n-propylamine solution is 5 mmol L-1；
(2) Detecting the cardiac troponin I standard solutions with different concentrations by using a cyclic voltammetry method, wherein the high voltage of a photomultiplier is set to be 800V, and the scanning voltage is set to be 0-1.0V;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration logarithm of the cardiac troponin I.

Claims (3)

1. Mo based on core-shell structure2The method for constructing the C @ C nanosphere cardiac troponin I immunosensor is characterized by comprising the following steps of:
(1) with Al2O3Polishing a glassy carbon electrode with the diameter of 4 mm by using polishing powder, respectively ultrasonically cleaning the glassy carbon electrode for 30 min by using absolute ethyl alcohol and ultrapure water in sequence, and then carrying out ultrasonic cleaning on the glassy carbon electrode by using 6 mu L and 0.1-1.0 mg mL-1The MWCNTs-NH is a multi-walled carbon nanotube @ perfluorosulfonic acid-polytetrafluoroethylene copolymer modified by amino2Dripping @ Nafion solution on the surface of the electrode, and airing at room temperature;
(2) 6 mu L and 10 mmol L-1The terpyridyl ruthenium solution is dripped on the surface of the electrode and dried at room temperature;
(3) 3 μ L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide EDC/NHS solution was applied dropwise to the electrode surface to activate MWCNTs-NH2@ amino group in Nafion, washing electrode with phosphate buffer solution PBS of pH 7.4, and drying at room temperature;
(4) 6 mu L and 5-15 mu g mL-1Cardiac troponin I capture antibody cTnI-Ab1Dropwise adding a bovine serum albumin BSA solution with the mass fraction of 0.5% -2% to the surface of the electrode, washing the electrode with PBS with the pH of 7.4, and airing at room temperature;
(5) dropwise adding 6 mu L and 10-4 ~ 50 ng mL-1A series of cardiac troponin I antigens with different concentrations are applied to the surface of the electrode, incubated for 2 hours, washed by PBS with pH of 7.4, and dried at room temperature;
(6) dropwise adding 6 mu L core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2The solution is dried at room temperature to prepare Mo based on the core-shell structure2A C @ C nanosphere cardiac troponin I immunosensor;
core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2The solution preparation steps are as follows:
(1) core-shell structure Mo2Preparation of C @ C nanospheres
Dissolving 12-30 mg of ammonium molybdate and 500-1200 mg of glucose in 45 mL of ultrapure water in sequence, transferring the solution into a high-pressure reaction kettle, reacting the solution at 180 ℃ for 10 hours, after the reaction is finished, placing the product into a centrifuge tube for centrifugation, washing the product with absolute ethyl alcohol and the ultrapure water for three times respectively, then placing the product into a 25 ℃ vacuum drying chamber for 12 hours, grinding the obtained sample, and calcining the product at 800 ℃ for 2 hours under argon by using a tube furnace to obtain Mo with a core-shell structure2C @ C nanospheres;
(2) core-shell structure Mo loaded with polyethyleneimine PEI2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2Preparation of solutions
3.0 mL, 1.0 mg mL-1Core-shell structure Mo2C @ C nanospheres dispersed in 12 mL solution containing 0.625 mol L-1 Potassium chloride and 1.25 mg mL-1In the mixed solution of PEI, shaking for 12 h after 2 h of ultrasonic treatment to obtain a uniform black solution, centrifugally collecting a solid product, washing with ultrapure water for three times, and dispersing the solid product in 6 mL of ultrapure water to obtain Mo2C @ C/PEI solution, then 1 mL of the solution is removed, and 200 muL of cardiac troponin I detection antibody cTnI-Ab is added2Shaking and incubating for 12-24 h at 4 ℃, centrifuging, and re-dispersing the solid in 1 mL of PBS with pH of 7.4 to obtain the polyethyleneimine PEI-loaded Mo with the core-shell structure2Secondary antibody marker Mo of C @ C nanosphere2C@C/PEI-Ab2。
2. The Mo core-shell structure-based Mo core-shell structure of claim 12The method for constructing the troponin I immunosensor of the C @ C nanosphere is characterized in that the MWCNTs-NH2The preparation steps of the @ Nafion solution are as follows:
0.4 g of dicycloethylcarbodiimide DCC and 20 mg of carboxylated carbon nanotube MWCNTs-COOH were added to 2.5 mL of ethylenediamine EDA solutionHeating at 120 deg.C for 4 days, centrifuging to obtain precipitate, and repeatedly washing the precipitate with ultrapure water and anhydrous ethanol to obtain aminated carbon nanotube MWCNTs-NH2Drying at 35 deg.C under vacuum, and weighing 0.04-0.4 mg MWCNTs-NH2Dispersing a sample in 0.4 mL of perfluorosulfonic acid-polytetrafluoroethylene copolymer Nafion solution with the mass fraction of 0.5%, and obtaining uniform MWCNTs-NH after ultrasonic treatment for 20 min2@ Nafion solution.
3. The Mo core-shell structure-based Mo core-shell structure of claim 12The method for constructing the C @ C nanosphere cardiac troponin I immunosensor is characterized by being used for detecting the cardiac troponin I and comprising the following preparation steps:
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and a modified working electrode in a cassette of a chemiluminescence detector, adding 10 mL of mixed solution of tri-n-propylamine solution containing NaCl and boron nitride quantum dots BNQDs into an electrolytic cell, wherein the volume ratio of the tri-n-propylamine solution to the mixed solution is 5:1, and the concentration of the tri-n-propylamine solution is 1-5 mmol L-1；
(2) Detecting the cardiac troponin I standard solutions with different concentrations by using a cyclic voltammetry method, wherein the high voltage of a photomultiplier tube is set to be 800V, and the scanning voltage is set to be 0-1.0V;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration logarithm of the cardiac troponin I.
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