CN108828042B - Preparation method of sandwich type photoelectrochemical sensor of cardiac troponin I - Google Patents

Preparation method of sandwich type photoelectrochemical sensor of cardiac troponin I Download PDF

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CN108828042B
CN108828042B CN201810492609.3A CN201810492609A CN108828042B CN 108828042 B CN108828042 B CN 108828042B CN 201810492609 A CN201810492609 A CN 201810492609A CN 108828042 B CN108828042 B CN 108828042B
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孔玲
陈杰
陈志伟
孙晓凯
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Shandong University of Technology
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Abstract

The invention relates to a preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I, belonging to the technical field of nano functional materials, immunoassay and photoelectrochemical biosensing. Nano square TiO sheet sensitized by carboxylated CdS quantum dots2001 crystal ofThe visible light absorption of the composite material is enhanced, and the composite material TiO with remarkably improved photoelectric activity is obtained2CdS, by layer-by-layer self-assembly method, cardiac troponin I antibody, bovine serum albumin and cardiac troponin I antigen are assembled to TiO2On a/CdS composite material, Ag @ Cu is utilized2O core-shell nano particle as second antibody marker and TiO2The excellent photoelectric activity of CdS and the specific combination between the cardiac troponin I antigen antibodies realize the ultra-sensitive detection of the cardiac troponin I, and have important significance for the analysis and detection application of the cardiac troponin I.

Description

Preparation method of sandwich type photoelectrochemical sensor of cardiac troponin I
Technical Field
The invention belongs to the technical field of nano functional materials, immunoassay and photoelectrochemical biosensing, and provides a preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I. In particular to a carboxylated CdS quantum dot modified nano square piece TiO2The 001 crystal face of the complex is used as an antibody capture substrate, and Ag @ Cu with double inhibition effects is used2The O nuclear shell nano particles are used as a second antibody marker to prepare a sandwich type photoelectrochemical sensor for detecting a cardiac troponin I specific antigen.
Background
Cardiovascular diseases are the first killers threatening human health in the world at present, and acute myocardial infarction has the characteristics of quick onset, high disability rate, high death rate and the like, so the research on the acute myocardial infarction is a hot spot. Meanwhile, cardiac troponin is the internationally accepted marker with the best sensitivity and specificity for diagnosing acute myocardial infarction, and human cardiac troponin I is one of subunits of a cardiac troponin compound, has higher myocardial specificity and sensitivity, and is called as clinical 'gold standard'. Therefore, early diagnosis of cardiac troponin I is of great significance.
Cardiovascular diseases are the first killers threatening human health in the world at present, and acute myocardial infarction has the characteristics of quick onset, high disability rate, high death rate and the like, so the research on the acute myocardial infarction is a hot spot. Meanwhile, cardiac troponin is the internationally accepted marker with the best sensitivity and specificity for diagnosing acute myocardial infarction, and human cardiac troponin I is one of subunits of a cardiac troponin compound, has higher myocardial specificity and sensitivity, and is called as clinical 'gold standard'. Therefore, early diagnosis of cardiac troponin I is of great significance.
At present, enzyme-linked immunosorbent assay, enzyme-linked fluorescence assay, magneto-optical biosensor, enzyme-labeled electrophoresis, electrochemical immunoassay and the like are reported as assay methods for detecting cardiac troponin I. Although the detection methods of the cardiac troponin I are many, the methods have the disadvantages of complex steps, long time consumption, low detection sensitivity and high requirements for detection personnel. In order to overcome the defects of the analysis method, the invention designs the photoelectrochemistry immunoassay method which has strong specificity, high sensitivity, good selectivity and quick and simple operation.
The invention utilizes photoelectrochemical analysis method to sensitize TiO by carboxylated CdS quantum dots2The 001 crystal face of the TiO-based composite material enhances the visible light absorption of the TiO-based composite material to obtain the TiO-based composite material with remarkably improved photoelectric activity2CdS, by layer-by-layer self-assembly method, cardiac troponin I antibody, bovine serum albumin and cardiac troponin I antigen are assembled to TiO2on/CdS composite material, Ag @ Cu is used2O core-shell nano-particles are used as a secondary antibody marker, and Ag @ Cu is used2Plasma resonance transfer of visible light energy by checking Ag particles in O core-shell nanoparticles and p-type semiconductor Cu2The O-shell photo-generated electron-hole pair competes for an electron donor ascorbic acid, the steric hindrance of the O-shell photo-generated electron-hole pair and the large specific surface area increase the immobilization capacity of the antibody, and TiO2Excellent photoelectric activity of CdS and specific combination between cardiac troponin I antigen antibodies to construct a peptide based on TiO2Sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS. The sensor has excellent photoelectrochemical activity, has the advantages of high sensitivity, wide linear range, low detection limit, rapid detection, relatively simple preparation process and the like, realizes the ultra-sensitive analysis of the cardiac troponin I, and provides a new method for effectively detecting the cardiac troponin I at present.
Disclosure of Invention
The invention provides a preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I, which realizes the ultra-sensitive detection of the cardiac troponin I. The invention aims to provide a preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I. The other purpose of the invention is to realize the rapid and sensitive detection of the cardiac troponin I by using the prepared sandwich type photoelectrochemical sensor.
The technical scheme of the invention is as follows:
1. a preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I comprises the following steps:
(1) ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) taking 8-10 muL, 8-12 mg/mL of nano square TiO 22Dripping the suspension on the conductive surface of the conductive glass electrode, and naturally airing at room temperature;
(3) in TiO2Dropwise adding 4-5 mu L carboxylated cadmium sulfide CdS quantum dot solution to the modified electrode surface, airing at room temperature, and washing the electrode surface with ultrapure water to obtain TiO2a/CdS electrode;
(4) in TiO2Dropwise adding 4-5 muL and 0.1mol/L thioglycolic acid on the surface of the CdS electrode, airing at room temperature, continuously dropwise adding 4-5 muL 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide, reacting for 20-40 min, washing with ultrapure water, and naturally airing;
(5) continuously dropwise adding 4-5 muL of cardiac troponin I antibody solution with the concentration of 3-5 mug/mL on the surface of the modified electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) continuously dropwise adding 4-5 muL of BSA solution with the mass fraction of 1-1.5% to seal the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water after reacting for 20-40 min, and airing in a refrigerator at 4 ℃;
(7) continuously dropwise adding 3-5 mu L of cardiac troponin I antigen solution with the concentration of 0.00002-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) continuously dropwise adding 4-8 mu L of 0.3-1.0 mg/mL Ag @ Cu2The O nucleocapsid nanometer particle marked cardiac troponin I antigen antibody solution is specifically identified with antigen on the surface of an electrode, incubated for 1 h at room temperature and cleaned to prepare the TiO nucleocapsid nanometer particle marked cardiac troponin I antigen based antibody solution2A sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS is stored in a refrigerator at 4 deg.C for use.
The 1-ethyl-3- (3-dimethyl ammonia)Propyl) -carbodiimide/N-hydroxysuccinimide 1X 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3mol/L of N-hydroxysuccinimide.
2. TiO2The preparation of CdS comprises the following steps:
(1) adding 0.6-1.0 mL of hydrofluoric acid into 8-10 mL of tetrabutyl titanate, stirring for 15-30 min, transferring the obtained solution into a polytetrafluoroethylene lining high-pressure reaction kettle, reacting for 22-26 h at 180 ℃, cooling to room temperature, centrifuging, washing, and drying in vacuum to obtain TiO2；
(2) Dropwise adding 125 mu L mercaptoacetic acid to 25 mL of 0.01 mol/L CdCl2Introducing nitrogen into the aqueous solution for 20-40 min, adjusting the pH value to 11 by using 1mol/L sodium hydroxide, slowly injecting 2.5 mL of 0.1mol/L sodium sulfide solution into the solution, reacting the mixture for 4 h in the nitrogen environment to obtain carboxylated CdS quantum dots, and storing the CdS quantum dots in a refrigerator at 4 ℃ for later use;
(3) taking TiO of 8-10 muL and 8-12 mg/mL2Dropwise adding the suspension to the conductive surface of the conductive glass electrode, naturally drying at room temperature, continuously dropwise adding 4-5 mu L carboxylated CdS quantum dot solution, drying at room temperature, washing the surface of the electrode with ultrapure water, and preparing TiO2/CdS。
3. Ag@Cu2The preparation method of the O core-shell nano particle comprises the following steps:
(1) dissolving 30-40 mg of silver nitrate in 180-220 mL of ultrapure water, refluxing at 85 ℃, and stirring for 30 min;
(2) adding 4 mL of 1% trisodium citrate solution into the solution (1), and boiling for 30min to obtain a silver particle solution;
(3) under magnetic stirring, 10-12 g of PVP is added into 500 mL of 0.01 mol/L Cu (NO)3)2In the water solution, after the powder is completely dissolved, 40.0 mL of silver particle solution is dripped;
(4) 340 mu L N was added immediately at room temperature2H4-H2O (35%) solution, the mixture was stirred for 5 min, and the Ag @ Cu was washed with water and absolute ethanol2Dispersing the O core-shell nano particles by using absolute ethyl alcohol, and storing the O core-shell nano particles in a refrigerator at 4 ℃ for later use.
4. The detection method of the cardiac troponin I specific antigen comprises the following steps:
(1) testing by using a three-electrode system of an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared cardiac troponin I specific antigen sandwich type photoelectrochemical sensor as a working electrode, and testing in a PBS (phosphate buffer solution) which is 10-15 mL, has a pH of 5.0-8.5 and contains 0.1mol/L ascorbic acid;
(2) detecting the specific antigen solution of the cardiac troponin I by adopting a time-current method, setting the voltage to be 0V, running the time to be 100 s, recording the current change and drawing a working curve, wherein an excitation light source is an LED lamp;
(3) replacing a cardiac troponin I specific antigen solution with a sample solution to be detected for detection, detecting according to the methods in the steps (2) and (3), and responding to the photocurrent intensityIAnd working curve to obtain the content of the troponin I in the sample to be detected. Experimental results prove that the time-current difference and the concentration of the specific antigen of the cardiac troponin I in the invention keep a good linear relation within the range of 0.02 pg/mL-50 ng/mL, and the detection limit reaches 6.7 fg/mL.
Advantageous results of the invention
(1) The synthesized nanometer square titanium dioxide TiO tablet of the invention2The 001 crystal face accounts for more than 80%, and the sensor has the advantages of good photoelectric activity, large surface area, low price, good stability and the like, and can increase the load on functional materials, improve the conductivity of the materials and enhance the sensitivity of the sensor.
(2) Sensitization of TiO by carboxylated CdS quantum dots2001 crystal face of (2) can reinforce TiO2The absorption capability to visible light is improved, and the TiO with obviously improved photoelectric activity is obtained2The composite material can accelerate the separation of electron hole pairs, increase the conductivity of the electron hole pairs and further improve the photoelectric activity of the sensor.
(3) By using Ag @ Cu2O core-shell nano-particles are used as a secondary antibody marker, and Ag @ Cu is used2Plasma resonance transfer of visible light energy by checking Ag particles in O core-shell nanoparticles and p-type semiconductor Cu2The O-shell photo-generated electron hole pair competes for an electron donor (ascorbic acid), the steric hindrance of the O-shell photo-generated electron hole pair and the large specific surface area increase the immobilization amount of the antibody, and the sensitivity of the sensor is improved, the linear range is widened, the detection limit of the sensor is effectively reduced and the ultrasensitive detection of the troponin I is realized according to the difference of the photoelectric signal intensity of the substance to be detected with different concentrations combined with the secondary antibody marker.
Detailed Description
The invention will now be further illustrated by, but not limited to, the following specific embodiments
Example 1 preparation method of Sandwich-type photoelectrochemical sensor for cardiac troponin I
(1) Ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) taking 8 mu L, 8 mg/mL nano square titanium dioxide TiO2Dripping the suspension on the conductive surface of the conductive glass electrode, and naturally airing at room temperature;
(3) in TiO2Dropwise adding 4 muL carboxylated CdS quantum dot solution on the modified electrode surface, airing at room temperature, rinsing the electrode surface with ultrapure water, and preparing TiO2a/CdS electrode;
(4) in TiO2Dripping 4 muL and 0.1mol/L thioglycollic acid on the surface of the CdS electrode, airing at room temperature, continuously dripping 4 muL of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide, reacting for 30min, washing with ultrapure water, and naturally airing;
(5) continuously dropwise adding a cardiac troponin I antibody solution with the concentration of 3 mug/mL on the surface of the modified electrode, washing the surface of the electrode with ultrapure water after reacting for 30min, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding 4 muL of BSA solution with the mass fraction of 1% to seal the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water after reacting for 30min, and airing in a refrigerator at 4 ℃;
(7) continuously dropwise adding 3 mu L of cardiac troponin I antigen solution with the concentration of 0.00002-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) continuously dropwise adding 4 mu L of 0.4 mg/mL Ag @ Cu2The O nucleocapsid nanometer particle marked cardiac troponin I antigen antibody solution is specifically identified with antigen on the surface of an electrode, incubated for 1 h at room temperature and cleaned to prepare the TiO nucleocapsid nanometer particle marked cardiac troponin I antigen based antibody solution2A sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS is stored in a refrigerator at 4 deg.C for use.
The 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3mol/L of N-hydroxysuccinimide.
Example 2 preparation method of Sandwich-type photoelectrochemical sensor for cardiac troponin I
(1) Ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) taking 9 mu L and 10 mg/mL of TiO in nano square sheet2Dripping the suspension on the conductive surface of the conductive glass electrode, and naturally airing at room temperature;
(3) in TiO2Dropwise adding 5 muL carboxylated CdS quantum dot solution to the modified electrode surface, airing at room temperature, and washing the electrode surface with ultrapure water to obtain TiO2a/CdS electrode;
(4) in TiO2Dripping 5 muL and 0.1mol/L thioglycollic acid on the surface of the CdS electrode, airing at room temperature, continuously dripping 5 muL of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide, reacting for 30min, washing with ultrapure water, and naturally airing;
(5) continuously dropwise adding a cardiac troponin I antibody solution with the concentration of 4 mug/mL on the surface of the modified electrode, washing the surface of the electrode with ultrapure water after reacting for 40min, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding 5 muL of BSA solution with the mass fraction of 1% to seal the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water after reacting for 30min, and airing in a refrigerator at 4 ℃;
(7) continuously dropwise adding 4 mu L of cardiac troponin I antigen solution with the concentration of 0.00002-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) 6 mu L of 0.6 mg/mL Ag @ Cu is dripped2The O nucleocapsid nanometer particle marked cardiac troponin I antigen antibody solution is specifically identified with antigen on the surface of an electrode, incubated for 1 h at room temperature and cleaned to prepare the TiO nucleocapsid nanometer particle marked cardiac troponin I antigen based antibody solution2A sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS is stored in a refrigerator at 4 deg.C for use.
The 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3mol/L of N-hydroxysuccinimide.
Example 3 preparation method of Sandwich-type photoelectrochemical sensor for cardiac troponin I
(1) Ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) taking 10 mu L and 12mg/mL nano square titanium dioxide TiO2Dripping the suspension on the conductive surface of the conductive glass electrode, and naturally airing at room temperature;
(3) in TiO2Dropwise adding 5 muL carboxylated CdS quantum dot solution to the modified electrode surface, airing at room temperature, and washing the electrode surface with ultrapure water to obtain TiO2a/CdS electrode;
(4) in TiO2Dripping 5 muL and 0.1mol/L thioglycollic acid on the surface of the CdS electrode, airing at room temperature, continuously dripping 5 muL of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide, reacting for 40min, washing with ultrapure water, and naturally airing;
(5) continuously dropwise adding a cardiac troponin I antibody solution with the concentration of 5 mug/mL on the surface of the modified electrode, washing the surface of the electrode with ultrapure water after reacting for 40min, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding 4 muL BSA solution with the mass fraction of 1.5% to seal the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water after reacting for 40min, and airing in a refrigerator at 4 ℃;
(7) continuously dropwise adding 5 mu L of cardiac troponin I antigen solution with the concentration of 0.00002-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) continuously dropwise adding 5 mu L of 0.8 mg/mL Ag @ Cu2The O nucleocapsid nanometer particle marked cardiac troponin I antigen antibody solution is specifically identified with antigen on the surface of an electrode, incubated for 1 h at room temperature and cleaned to prepare the TiO nucleocapsid nanometer particle marked cardiac troponin I antigen based antibody solution2A sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS is stored in a refrigerator at 4 deg.C for use.
The 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3mol/L of N-hydroxysuccinimide.
Example 4 TiO2The preparation of CdS comprises the following steps:
(1) adding 0.6 mL of hydrofluoric acid into 8 mL of tetrabutyl titanate, stirring for 15 min, transferring the obtained solution into a polytetrafluoroethylene lining high-pressure reaction kettle, reacting for 24 h at 180 ℃, cooling to room temperature, centrifuging, washing, and drying in vacuum to obtain TiO2；
(2) Dropwise adding 125 mu L mercaptoacetic acid to 25 mL of 0.01 mol/L CdCl2Introducing nitrogen into the aqueous solution for 20min, adjusting the pH value to 11 by using 1mol/L sodium hydroxide, slowly injecting 2.5 mL of 0.1mol/L sodium sulfide solution into the solution, reacting the mixture for 4 h in the nitrogen environment to obtain carboxylated CdS quantum dots, and storing the CdS quantum dots in a refrigerator at 4 ℃ for later use;
(3) TiO of 10 mu L and 8 mg/mL is taken2Dripping the suspension on the conductive surface of the conductive glass electrode, naturally drying at room temperature, continuously dripping 4 muL carboxylated CdS quantum dot solution, drying at room temperature, washing the surface of the electrode with ultrapure water, and preparing TiO2/CdS。
Example 5 TiO2Preparation of CdS by the following steps：
(1) Adding 0.8 mL of hydrofluoric acid into 9 mL of tetrabutyl titanate, stirring for 20min, transferring the obtained solution into a polytetrafluoroethylene-lined high-pressure reaction kettle, heating to react for 26 h at 180 ℃, cooling to room temperature, centrifuging, washing, and drying in vacuum to obtain TiO2；
(2) Dropwise adding 125 mu L mercaptoacetic acid to 25 mL of 0.01 mol/L CdCl2Introducing nitrogen into the aqueous solution for 30min, adjusting the pH value to 11 by using 1mol/L sodium hydroxide, slowly injecting 2.5 mL of 0.1mol/L sodium sulfide solution into the solution, reacting the mixture for 4 h in the nitrogen environment to obtain carboxylated CdS quantum dots, and storing the carboxylated CdS quantum dots in a refrigerator at 4 ℃ for later use;
(3) 10 mu L and 10 mg/mL TiO are taken2Dripping the suspension on the conductive surface of the conductive glass electrode, naturally drying at room temperature, continuously dripping 5 muL carboxylated CdS quantum dot solution, drying at room temperature, washing the surface of the electrode with ultrapure water, and preparing TiO2/CdS。
Example 6 TiO2The preparation of CdS comprises the following steps:
(1) adding 1.0 mL of hydrofluoric acid into 10 mL of tetrabutyl titanate, stirring for 30min, transferring the obtained solution into a polytetrafluoroethylene-lined high-pressure reaction kettle, heating to react for 24 h at 180 ℃, cooling to room temperature, centrifuging, washing, and drying in vacuum to obtain TiO2；
(2) Dropwise adding 125 mu L mercaptoacetic acid to 25 mL of 0.01 mol/L CdCl2Introducing nitrogen into the aqueous solution for 40min, adjusting the pH value to 11 by using 1mol/L sodium hydroxide, slowly injecting 2.5 mL of 0.1mol/L sodium sulfide solution into the solution, reacting the mixture for 4 h in the nitrogen environment to obtain carboxylated CdS quantum dots, and storing the carboxylated CdS quantum dots in a refrigerator at 4 ℃ for later use;
(3) taking 10 mu L and 12mg/mL TiO2Dripping the suspension on the conductive surface of the conductive glass electrode, naturally drying at room temperature, continuously dripping 4 muL carboxylated CdS quantum dot solution, drying at room temperature, washing the surface of the electrode with ultrapure water, and preparing TiO2/CdS。
Example 7 Ag @ Cu2The preparation method of the O core-shell nano particle comprises the following steps:
(1) dissolving 30 mg of silver nitrate in 180 mL of ultrapure water, refluxing at 85 ℃, and stirring for 30 min;
(2) adding 4 mL of 1% trisodium citrate solution into the solution (1), and boiling for 30min to obtain a silver particle solution;
(3) 10 g PVP was added to 500 mL of 0.01 mol/L Cu (NO) with magnetic stirring3)2In the aqueous solution, after the powder is completely dissolved, 40.0 mL of silver particle solution is dropwise added;
(4) 340 mu L N was added immediately at room temperature2H4-H2O (35%) solution, the mixture was stirred for 5 min, and the Ag @ Cu was washed with water and absolute ethanol2Dispersing the O core-shell nano particles by using absolute ethyl alcohol, and storing the O core-shell nano particles in a refrigerator at 4 ℃ for later use.
Example 8 Ag @ Cu2The preparation method of the O core-shell nano particle comprises the following steps:
(1) dissolving 36 mg of silver nitrate in 200 mL of ultrapure water, refluxing at 85 ℃, and stirring for 30 min;
(2) adding 4 mL of 1% trisodium citrate solution into the solution (1), and boiling for 30min to obtain a silver particle solution;
(3) 10.5 g PVP was added to 500 mL of 0.01 mol/L Cu (NO) with magnetic stirring3)2In the aqueous solution, after the powder is completely dissolved, 40.0 mL of silver particle solution is dropwise added;
(4) 340 mu L N was added immediately at room temperature2H4-H2O (35%) solution, the mixture was stirred for 5 min, and the Ag @ Cu was washed with water and absolute ethanol2Dispersing the O core-shell nano particles by using absolute ethyl alcohol, and storing the O core-shell nano particles in a refrigerator at 4 ℃ for later use.
Example 9 Ag @ Cu2The preparation method of the O core-shell nano particle comprises the following steps:
(1) dissolving 40mg of silver nitrate in 220 mL of ultrapure water, refluxing at 85 ℃, and stirring for 30 min;
(2) adding 4 mL of 1% trisodium citrate solution into the solution (1), and boiling for 30min to obtain a silver particle solution;
(3) 11 g PVP was added to 500 mL of 0.01 mol/L Cu (NO) with magnetic stirring3)2In the aqueous solution, after the powder is completely dissolved, 40.0 mL of silver particle solution is dropwise added;
(4) 340 mu L N was added immediately at room temperature2H4-H2O (35%) solution, the mixture was stirred for 5 min, and the Ag @ Cu was washed with water and absolute ethanol2Dispersing the O core-shell nano particles by using absolute ethyl alcohol, and storing the O core-shell nano particles in a refrigerator at 4 ℃ for later use.
Example 10 detection of cardiac troponin I-specific antigens
(1) Testing by using a three-electrode system of an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared cardiac troponin I specific antigen sandwich type photoelectrochemical sensor as a working electrode, and testing in a PBS buffer solution;
(2) detecting the specific antigen solution of the cardiac troponin I by adopting a time-current method, setting the voltage to be 0V, running the time to be 100 s, recording the current change and drawing a working curve, wherein an excitation light source is an LED lamp;
(3) replacing a cardiac troponin I specific antigen solution with a sample solution to be detected for detection, detecting according to the methods in the steps (2) and (3), and responding to the photocurrent intensityIAnd working curve to obtain the content of the troponin I in the sample to be detected.
The PBS buffer solution is 10-15 mL of phosphate buffer solution containing 0.1mol/L ascorbic acid and the pH value of the phosphate buffer solution is 5.0-8.5.

Claims (3)

1. A preparation method of a sandwich type photoelectrochemical sensor of cardiac troponin I is characterized by comprising the following steps:
(1) ultrasonically cleaning ITO conductive glass of 2.5 cm multiplied by 0.8 cm by using liquid detergent, acetone, absolute ethyl alcohol and ultrapure water in sequence, and drying by using nitrogen;
(2) taking 8-10 muL, 8-12 mg/mL of nano square TiO 22Dripping the suspension on the conductive surface of the conductive glass electrode, and naturally airing at room temperature;
(3) in TiO2Dropwise adding 4-5 mu L of carboxyl on the modified electrode surfaceDrying the cadmium sulfide CdS quantum dot solution at room temperature, and washing the surface of the electrode with ultrapure water to obtain TiO2a/CdS electrode;
(4) in TiO2Dropwise adding 4-5 muL and 0.1mol/L thioglycolic acid on the surface of the CdS electrode, airing at room temperature, continuously dropwise adding 4-5 muL 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide, reacting for 20-40 min, washing with ultrapure water, and naturally airing;
(5) continuously dropwise adding 4-5 muL of cardiac troponin I antibody solution with the concentration of 3-5 mug/mL on the surface of the modified electrode, reacting for 20-40 min, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) continuously dropwise adding 4-5 muL of BSA solution with the mass fraction of 1-1.5% to seal the non-specific active sites on the electrode surface, washing the electrode surface with ultrapure water after reacting for 20-40 min, and airing in a refrigerator at 4 ℃;
(7) continuously dropwise adding 3-5 mu L of cardiac troponin I antigen solution with the concentration of 0.00002-50 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(8) continuously dropwise adding 4-8 mu L of 0.3-1.0 mg/mL Ag @ Cu2The O nucleocapsid nanometer particle marked cardiac troponin I antigen antibody solution is specifically identified with antigen on the surface of an electrode, incubated for 1 h at room temperature and cleaned to prepare the TiO nucleocapsid nanometer particle marked cardiac troponin I antigen based antibody solution2A sandwich-type photoelectrochemical sensor of cardiac troponin I of CdS stored in a refrigerator at 4 ℃ for later use;
the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide content is 1 x 10-2mol/L of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 2X 10-3mol/L of N-hydroxysuccinimide;
the Ag @ Cu2The preparation steps of the O core-shell nano particles are as follows:
(1) dissolving 30-40 mg of silver nitrate in 180-220 mL of ultrapure water, refluxing at 85 ℃, and stirring for 30 min;
(2) adding 4 mL of 1% trisodium citrate solution into the solution in the step (1), and boiling for 30min to obtain a silver particle solution;
(3) under magnetic stirring, 10-12 g of PVP is added into 500 mL of 0.01 mol/L Cu (NO)3)2In the water solution, after the powder is completely dissolved, 40.0 mL of silver particle solution is dripped;
(4) adding 340 mu L35% hydrazine hydrate solution at room temperature immediately, stirring the mixture for 5 min, and cleaning Ag @ Cu with water and absolute ethyl alcohol2Dispersing the O core-shell nano particles by using absolute ethyl alcohol, and storing the O core-shell nano particles in a refrigerator at 4 ℃ for later use.
2. The method for preparing the sandwich-type photoelectrochemical sensor of cardiac troponin I according to claim 1, wherein said TiO is selected from the group consisting of2The preparation of the/CdS is characterized by comprising the following steps:
(1) adding 0.6-1.0 mL of hydrofluoric acid into 8-10 mL of tetrabutyl titanate, stirring for 15-30 min, transferring the obtained solution into a polytetrafluoroethylene lining high-pressure reaction kettle, reacting for 22-26 h at 180 ℃, cooling to room temperature, centrifuging, washing, and drying in vacuum to obtain TiO2；
(2) Dropwise adding 125 mu L mercaptoacetic acid to 25 mL of 0.01 mol/L CdCl2Introducing nitrogen into the aqueous solution for 20-40 min, adjusting the pH value to 11 by using 1mol/L sodium hydroxide, slowly injecting 2.5 mL of 0.1mol/L sodium sulfide solution into the solution, reacting the mixture for 4 h in the nitrogen environment to obtain carboxylated CdS quantum dots, and storing the CdS quantum dots in a refrigerator at 4 ℃ for later use;
(3) taking TiO of 8-10 muL and 8-12 mg/mL2Dropwise adding the suspension to the conductive surface of the conductive glass electrode, naturally drying at room temperature, continuously dropwise adding 4-5 mu L carboxylated CdS quantum dot solution, drying at room temperature, washing the surface of the electrode with ultrapure water, and preparing TiO2/CdS。
3. The method for preparing the sandwich-type photoelectrochemical sensor for cardiac troponin I according to claim 1, which is used for the detection of an antigen specific to cardiac troponin I and comprises the following steps:
(1) testing by using a three-electrode system of an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared cardiac troponin I specific antigen sandwich type photoelectrochemical sensor as a working electrode, and testing in a PBS (phosphate buffer solution) which is 10-15 mL, has a pH of 5.0-8.5 and contains 0.1mol/L ascorbic acid;
(2) detecting the specific antigen solution of the cardiac troponin I by adopting a time-current method, setting the voltage to be 0V, running the time to be 100 s, recording the current change and drawing a working curve, wherein an excitation light source is an LED lamp;
(3) replacing a cardiac troponin I specific antigen solution with a sample solution to be detected for detection, detecting according to the methods in the steps (2) and (3), and responding to the photocurrent intensityIAnd working curve to obtain the content of the troponin I in the sample to be detected.
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