阅读说明：本技术 一种基于锌和钼共掺杂钒酸铋阵列的光电化学己烯雌酚传感器的制备方法 (Preparation method of photoelectrochemical diethylstilbestrol sensor based on zinc and molybdenum co-doped bismuth vanadate array ) 是由 钱彦荣 魏琴 鞠熀先 冯金慧 任祥 王雪莹 范大伟 于 2019-11-22 设计创作，主要内容包括：本发明涉及一种基于锌和钼共掺杂的钒酸铋阵列的光电化学己烯雌酚传感器的制备方法及应用。本发明具体是将锌和钼共掺杂的钒酸铋阵列作为基底材料电沉积在氧化铟锡导电玻璃上,锌和钼共掺杂的钒酸铋的蠕虫状多孔结构具有大的表面积可增加光的捕获和生物分子的负载,同时,锌和钼元素的掺杂可加入额外的电子,促进光生电子空穴的分离效率；制备的锰铁氰化合物是一种对过氧化氢具有催化功能的金属无机框架材料,将其作为竞争标记物；基于此,本体系以过氧化氢作为电子供体,构建了一种基于减弱型信号竞争策略的光电化学传感器,实现了对己烯雌酚的灵敏检测,该方法对早期诊断和监测自身免疫性疾病具有重要意义。(The invention relates to a preparation method and application of a photoelectrochemical diethylstilbestrol sensor based on a zinc and molybdenum co-doped bismuth vanadate array. The zinc and molybdenum co-doped bismuth vanadate array is used as a substrate material to be electrodeposited on indium tin oxide conductive glass, a worm-shaped porous structure of the zinc and molybdenum co-doped bismuth vanadate has a large surface area, so that light capture and biomolecule load can be increased, and additional electrons can be added in the doping of zinc and molybdenum elements to promote the separation efficiency of photo-generated electron holes; the prepared manganese ferricyanide compound is a metal inorganic framework material with a catalytic function on hydrogen peroxide, and is used as a competitive marker; based on the method, hydrogen peroxide is used as an electron donor, a photoelectrochemical sensor based on a weakening signal competition strategy is constructed, sensitive detection on diethylstilbestrol is realized, and the method has important significance for early diagnosis and monitoring of autoimmune diseases.)

1. A preparation method of a photoelectrochemical diethylstilbestrol sensor based on a zinc and molybdenum co-doped bismuth vanadate array is characterized by comprising the following steps:

ultrasonically cleaning indium tin oxide conductive glass of 2.5 cm multiplied by 0.8 cm by acetone, ethanol and ultrapure water for 30min in sequence, and drying by nitrogen; electrodepositing a zinc and molybdenum co-doped bismuth vanadate array on a conductive surface of clean indium tin oxide glass to be used as a working electrode; continuously dropwise adding 4 mu L of 3 mmol/L thioglycollic acid to the surface of the electrode to fix the diethylstilbestrol wrapped by the bovine serum albumin, airing in a refrigerator at 4 ℃, and washing with ultrapure water; continuously dropwise adding 3 mu L1X 10^-2mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 2X 10^-3Activating carboxyl by using a mixed solution of mol/L N-hydroxysuccinimide, airing in a refrigerator at 4 ℃, and washing with ultrapure water; continue to useDropwise adding 5 muL 1 mug/mL bovine serum albumin coated diethylstilbestrol on the surface of an electrode, drying in a refrigerator at 4 ℃, washing with a phosphate buffer solution with pH7.4, continuously dropwise adding a 5 muL bovine serum albumin solution with the mass fraction of 0.1 ~ 1.0.0% on the surface of a modified electrode to seal non-specific active sites which are not combined on the surface of the electrode, drying in the refrigerator at 4 ℃, washing with a phosphate with pH7.4, continuously dropwise adding a 5 muL 0.1 pg/m ~ 20ng/mL diethylstilbestrol standard solution and a mixed solution of a manganese iron cyanide compound and a gold nanoparticle composite labeled diethylstilbestrol antibody on the surface of the electrode, drying in the refrigerator at 4 ℃, washing with the phosphate buffer solution with pH7.4, and preparing a fully modified working electrode, namely the competitive photoelectrochemical immunosensor for detecting diethylstilbestrol.

2. The method for preparing the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum co-doped bismuth vanadate array according to claim 1, wherein the preparation method of the zinc and molybdenum co-doped bismuth vanadate array comprises the following steps:

the preparation method of the zinc and molybdenum codoped bismuth vanadate array comprises the following steps of firstly, electrodepositing bismuth oxyiodide nanosheets on indium tin oxide glass, specifically, dissolving 0.04 ~ 0.08 mol/L bismuth nitrate in 50mL of 0.4 ~ 0.8.8 mol/L potassium iodide solution, adjusting the pH of the solution to 1.4 ~ 1.7 by using concentrated nitric acid, then dropwise adding 20 mL of 0.23 mol/L p-benzoquinone solution into the solution, using the solution as an electrolyte for electrodeposition, performing electrodeposition in a three-electrode system with indium tin oxide glass as a working electrode, a platinum electrode as a counter electrode and a silver/silver chloride electrode as a reference electrode, setting cathode bias voltage to-0.1V, depositing for 3 ~ 5 min to obtain bismuth oxyiodide nanosheets, then, dropping 0.2 mol/L of a 0.5 ~ 3% molybdenum acetylacetonate and zinc acetylacetonate solution with molar ratios of 0.5% and 3 ~ 5% on a bismuth acetylacetonate zinc oxide nano-zinc oxide nanosheet, transferring the bismuth oxyiodide nanosheets on a bismuth vanadate nano-bismuth-oxide-bismuth-acetylacetonate nano-oxide nano-array, and a molybdenum-bismuth-vanadate nano-oxide nano-bismuth-oxide nano-bismuth-oxide nano-bismuth-oxide nano-bismuth-oxide nano.

3. The preparation method of the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum co-doped bismuth vanadate array as claimed in claim 1, wherein the preparation method of the manganese ferricyanide compound comprises the following steps:

0.3 ~ 0.8.8 g polyvinylpyrrolidone is dissolved in the mixed solution of 10mL absolute ethyl alcohol and 10mL distilled water, 0.04 ~ 0.08.08 g manganese acetate is dissolved in the solution and is called solution A, 0.06-0.1 g potassium ferricyanide is dissolved in the mixed solution of 10mL ethanol and 10mL distilled water and is called solution B, the solution B is slowly and dropwise added into the solution A, the mixed solution is stood for 24 h at room temperature, centrifuged, washed with distilled water and ethanol for a plurality of times and dried in vacuum at 60 ℃, and the prepared manganese ferricyanide compound is a hydrogen peroxide mimic enzyme, has better catalytic action on hydrogen peroxide and can consume hydrogen peroxide as an electron donor.

4. The method for preparing the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum co-doped bismuth vanadate array as claimed in claim 1, wherein the gold nanoparticles are prepared by the following steps:

50-55 mL of chloroauric acid solution with the mass fraction of 0.01%, boiling in an oil bath at 120 ℃, adding 2.5-5.0 mL of trisodium citrate solution with the mass fraction of 1% into the solution, and continuously boiling for 30min under stirring until the solution turns purple red to prepare gold nanoparticle solution; the prepared gold nanoparticles have uniform particle size and are beneficial to the connection of biomolecules.

5. The preparation method of the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum co-doped bismuth vanadate array as claimed in claim 1, wherein the preparation of the manganese hexacyanoferrate compound and the gold nanoparticle compound comprises the following steps:

firstly, carrying out amination on ferricyanide compound, dispersing 18 mg of ferromanganese ferricyanide compound in 10mL of ethanol solution containing 0.15 mL of 3-aminopropyltriethoxysilane, refluxing the suspension for 24 h at 90 ℃, cooling to room temperature, and then centrifugally washing the solution with ethanol to obtain aminated ferricyanide compound; and oscillating 10mL of gold nanoparticle solution and the amination ferricyanide compound overnight, centrifuging and washing to obtain the manganese ferricyanide compound and the gold nanoparticle compound.

6. The method for preparing the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum co-doped bismuth vanadate array according to claim 1, wherein a diethylstilbestrol antibody solution labeled by the manganese hexacyanoferrate compound and the gold nanoparticle composite is prepared by the following steps:

2 mL of 10 mug/mL diethylstilbestrol antibody solution, 10 μ L of 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10 μ L of 1 mg/mL of N-hydroxysuccinimide, oscillating for 30min at 4 ℃, then adding 3 mg of manganese ferricyanide compound and gold nanoparticle complex into the solution, oscillating for 6 h at 4 ℃, finally injecting 100 μ L of bovine serum albumin with the mass fraction of 0.1-1% into the solution, oscillating for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with pH of 7.4, and dispersing in 2 mLpH 7.4 phosphate buffer solution.

7. The preparation method of the photoelectrochemical diethylstilbestrol sensor based on the zinc and molybdenum codoped bismuth vanadate prepared by the preparation method of claim 1, which is used for detecting diethylstilbestrol, and comprises the following detection steps:

the method comprises the steps of using an electrochemical workstation to carry out testing by a three-electrode system, using an indium tin oxide modified electrode prepared according to claim 1 as a working electrode, using a saturated calomel electrode as a reference electrode, using a platinum wire electrode as an auxiliary electrode, carrying out testing in 10mL of phosphate solution with the pH value of 7.4 and containing 0.1 mol/L of hydrogen peroxide, detecting an analyte by using a time-current method, wherein the running time is 100 s, the wavelength of an irradiated LED lamp is 400 ~ 450nm, when the background current tends to be stable, continuously irradiating for 10 s every 10 s, recording the light current, drawing a working curve, and replacing diethylstilbestrol standard solution with diethylstilbestrol sample solution to be detected to carry out detection, wherein the result can be obtained by checking the working curve.

Technical Field

The invention belongs to the technical field of nano functional materials, immunoassay and biosensing, and particularly relates to a preparation method of a photoelectrochemical sensor for detecting diethylstilbestrol, which is constructed by taking zinc and molybdenum co-doped bismuth vanadate as a substrate material and a gold nanoparticle/manganese hexacyanoferrate compound as a competitive marker.

Background

Diethylstilbestrol is a synthetic estrogen compound and widely used in livestock production, aquaculture and clinical medicines for preventing pregnancy complications, and researches prove that the ordinary people exposed to diethylstilbestrol have increased risks of autoimmune diseases and cancers. It has been identified as an environmentally induced endocrine disrupting substance due to its potential toxicity and stability. At present, there are many methods for detecting diethylstilbestrol, such as enzyme-linked immunosorbent assay, electrochemical analysis, capillary electrophoresis technology, etc., but the methods have the disadvantages of complex operation, expensive instrument, time consumption, etc., so the invention develops a detection method with simple operation, low price and high sensitivity. The photoelectrochemical sensor constructed by the invention is a device for determining the concentration of an analyte by utilizing the photoelectric conversion property of a photoelectric active substance, and has the advantages of low background signal, high sensitivity, simple and convenient preparation and low cost because the detection signal is an electric signal and the excitation signal is a light source, thereby realizing the separation of the excitation signal and the detection signal, and having wide application in the fields of food analysis, environment detection, water quality analysis, biological analysis and the like.

Photoactive materials are key components of photoelectrochemical sensors. As a photoelectric conversion semiconductor material with excellent performance, bismuth vanadate is widely applied to the fields of photocatalysis, fuel cells and the like due to a narrow band gap (2.4 eV), but the application of the bismuth vanadate is limited by poor charge separation efficiency and charge transfer capacity. Therefore, in the invention, bismuth vanadate is prepared by electrodeposition and calcination methods and co-doped with zinc and molybdenum elements, so that the electronic structure of the bismuth vanadate can be regulated and controlled, and a local built-in electric field can be formed, thereby being beneficial to charge separation. The formed vermicular bismuth vanadate nano structure has a large surface area and is beneficial to loading biomolecules. In addition, the prepared peroxide mimic enzyme manganese ferricyanide compound can be used as a competitive marker to catalyze hydrogen peroxide, so that the sensor gets rid of the disadvantages of high environmental requirement, high cost and the like of the traditional enzyme sensor, and the sensitivity of the photoelectrochemical sensor is improved.

Disclosure of Invention

One of the purposes of the invention is to synthesize zinc and molybdenum element co-doped bismuth vanadate respectively, and form a built-in electric field in a bismuth vanadate array by doping elements, thereby improving the charge separation of bismuth vanadate, promoting the transfer of photo-generated electrons and improving the photoelectric conversion efficiency.

The other purpose of the invention is to synthesize a manganese ferricyanide compound and a gold nanoparticle compound, and a gold-ammonia bond is combined with a signal antibody to form a manganese ferricyanide compound and gold nanoparticle bioconjugate.

The third purpose of the invention is to construct a competitive photoelectrochemical immunosensor by using a zinc and molybdenum element co-doped bismuth vanadate array as an optically active substrate material and using a manganese ferricyanide compound and a gold nanoparticle compound as signal markers, and the competitive photoelectrochemical immunosensor is used for rapid and sensitive detection of diethylstilbestrol.

The technical scheme of the invention is as follows:

1. a preparation method of a photoelectrochemical diethylstilbestrol sensor based on zinc and molybdenum co-doped bismuth vanadate comprises the following preparation steps:

the preparation method of the zinc and molybdenum codoped bismuth vanadate comprises the following steps of firstly, electrodepositing bismuth oxyiodide nanosheets on indium tin oxide glass, specifically, dissolving 0.04 ~ 0.08.08 mol/L bismuth nitrate in 50mL of 0.4 ~ 0.8.8 mol/L potassium iodide solution, adjusting the pH of the solution to 1.4 ~ 1.7.7 by concentrated nitric acid, then dropwise adding 20 mL0.23 mol/L p-benzoquinone solution into the solution, using the solution as an electrolyte for electrodeposition, performing electrodeposition in a three-electrode system with indium tin oxide glass as a working electrode, a platinum electrode as a counter electrode and a silver/silver chloride electrode as a three-electrode, setting a cathode bias voltage to-0.1V, depositing for 3 ~ 5 min to obtain bismuth oxyiodide nanosheets, setting a molar ratio of 0.5 ~ 3% and a molar ratio of 3 ~ 5% of molybdenum acetylacetonate and zinc acetylacetonate to 0.2 mol/0.2 min, and removing excess bismuth oxyiodide from the bismuth oxyiodide nanosheets, and simultaneously, and increasing the absorption efficiency of a doped nano-bismuth-oxide-bismuth-oxide nano-array, and removing the doped nano-bismuth-oxide nano-bismuth-oxide nano.

2. Preparing a manganese ferricyanide compound and gold nanoparticle compound detection antibody hatching substance solution; the preparation of gold nanoparticles is shown below: 50-55 mL of chloroauric acid solution with the mass fraction of 0.01%, boiling in an oil bath at 120 ℃, adding 2.5-5.0 mL of trisodium citrate solution with the mass fraction of 1% into the solution, and continuously boiling for 30min under stirring until the solution turns purple red to prepare gold nanoparticle solution; the prepared gold nanoparticles have uniform particle size and are beneficial to the connection of biomolecules.

3. The preparation process of manganese ferricyanide compound includes dissolving 0.3 ~ 0.8.8 g of polyvinylpyrrolidone in the mixed solution of 10mL of anhydrous alcohol and 10mL of distilled water, dissolving 0.04 ~ 0.08.08 g of manganese acetate in the solution, named solution A, dissolving 0.06-0.1 g of potassium ferricyanide in the mixed solution of 10mL of alcohol and 10mL of distilled water, named solution B, slowly and dropwise adding solution B into solution A, standing the mixed solution at room temperature for 24 hr, centrifuging, washing with distilled water and alcohol several times, vacuum drying at 60 deg.c, and preparing manganese ferricyanide compound as hydrogen peroxide enzyme with catalytic effect on hydrogen peroxide and consumption of hydrogen peroxide as electron donor;

firstly, carrying out amination on ferricyanide compound, dispersing 18 mg of ferromanganese ferricyanide compound in 10mL of ethanol solution containing 0.15 mL of 3-aminopropyltriethoxysilane, refluxing the suspension for 24 h at 90 ℃, cooling to room temperature, and then centrifugally washing the solution with ethanol to obtain aminated ferricyanide compound; oscillating 10mL of gold nanoparticle solution and an amination ferricyanide compound overnight, centrifuging and washing to obtain a manganese ferricyanide compound and a gold nanoparticle compound;

2 mL of 10 mug/mL diethylstilbestrol antibody solution, 10 μ L of 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10 μ L of 1 mg/mL of N-hydroxysuccinimide, oscillating for 30min at 4 ℃, then adding 3 mg of manganese ferricyanide compound and gold nanoparticle complex into the solution, oscillating for 6 h at 4 ℃, finally injecting 100 μ L of bovine serum albumin with the mass fraction of 0.1-1% into the solution, oscillating for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with the pH of 7.4, and dispersing in 2 mLpH 7.4 phosphate buffer solution.

4. Preparing a signal-weakening type photoelectrochemical immunosensor: ultrasonically cleaning indium tin oxide conductive glass of 2.5 cm multiplied by 0.8 cm by acetone, ethanol and ultrapure water for 30min in sequence, and drying by nitrogen; electrodepositing zinc and molybdenum co-doped bismuth vanadate on a conductive surface of clean indium tin oxide glass to serve as a working electrode; continuously dropwise adding 4 mu L of 3 mmol/L thioglycollic acid to the surface of the electrode to fix the diethylstilbestrol wrapped by the bovine serum albumin, airing in a refrigerator at 4 ℃, and washing with ultrapure water; continuously dropwise adding 3 mu L1 multiplied by 10^-2mol/L1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 2X 10^-3The method comprises the steps of activating carboxyl groups by a mixed solution of mol/L N-hydroxysuccinimide, airing in a refrigerator at 4 ℃, washing with ultrapure water, continuously dropwise adding 5 mu L of 1 mu g/mL bovine serum albumin coated diethylstilbestrol on the surface of an electrode, airing in the refrigerator at 4 ℃, washing with a phosphate buffer solution with pH of 7.4, continuously dropwise adding a 5 mu L of bovine serum albumin solution with the mass fraction of 0.1 ~ 1.0.0% on the surface of a modified electrode to seal non-specific active sites which are not combined on the surface of the electrode, airing in the refrigerator at 4 ℃, washing with phosphate with pH of 7.4, continuously dropwise adding a 5 mu L of 0.1 mu L/m ~ 20ng/mL of diethylstilbestrol standard solution and a diethylstilbestrol antibody mixed solution marked by a manganese hexacyanate compound and gold nanoparticle composite substance on the surface of the electrode, airing in the refrigerator at 4 ℃, washing with a phosphate buffer solution with pH of 7.4, and preparing a completely modified working electrode, namely a competitive photoelectrochemical immunosensor for detecting diethylstilbestrol.

5. The detection method of the competitive photoelectrochemical immunosensor for detecting diethylstilbestrol comprises the following steps of using an electrochemical workstation to carry out a test by a three-electrode system, using the indium tin oxide modified electrode prepared according to claim 1 as a working electrode, using a saturated calomel electrode as a reference electrode, using a platinum wire electrode as an auxiliary electrode, and carrying out a test in 10mL of a phosphate solution with the pH value of 7.4 and containing 0.1 mol/L of hydrogen peroxide, detecting an analyte by using a time-current method, wherein the running time is 100 s, and the wavelength of an irradiated LED lamp is 400 ~ 450 nm;

when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, recording the photocurrent, and drawing a working curve; and (3) replacing the diethylstilbestrol standard solution with the diethylstilbestrol sample solution to be detected for detection, and obtaining the result through a working curve.

Advantageous results of the invention

1. The zinc and molybdenum co-doped bismuth vanadate array synthesized by the method improves the photo-generated charge separation capacity and the carrier density, and meanwhile, the worm-shaped nano structure has a large specific surface area, so that the load capacity of biomolecules can be increased, the sensitivity of the sensor is remarkably improved, and the detection limit is reduced; meanwhile, the co-doping of the zinc and the molybdenum forms a built-in electric field in the bismuth vanadate array, so that the transfer of electrons can be promoted, and the photoelectrochemistry conversion efficiency is improved.

2. The method synthesizes the manganese ferricyanide compound and the gold nanoparticle compound, and constructs a signal-attenuated photoelectrochemical immunosensor; the ferromanganese ferricyanide compound is used as a metal organic framework material, has mimic enzyme with peroxidase activity, and can compete with a substrate material for hydrogen peroxide serving as an electron donor to construct a signal-attenuated photoelectrochemical competitive immunosensor.

3. According to the invention, the nano composite material manganese ferricyanide compound and gold nanoparticle compound are directly combined with the diethylstilbestrol antibody to construct an enzyme-free immunosensor, so that detection errors caused by enzyme inactivation or leakage are avoided; meanwhile, the manganese ferricyanide compound and the gold nanoparticle compound are utilized to weaken signals, so that the detection sensitivity of the photoelectrochemical sensor is greatly improved, and the method has important scientific significance and application value.

4. The signal attenuation type photoelectrochemical immunosensor prepared by the invention is used for detecting diethylstilbestrol, has short response time and good stability, and can realize simple, quick, high-sensitivity and specific detection of diethylstilbestrol.

Detailed Description

The invention will now be further illustrated by, but not limited to, the following specific embodiments