阅读说明：本技术 一种黄曲霉毒素电化学传感器的制备方法及应用 (Preparation method and application of aflatoxin electrochemical sensor ) 是由 李月云 禹晓东 李新进 徐振 张津津 霍之林 于 2020-06-17 设计创作，主要内容包括：本发明属于免疫分析和生物传感技术领域,提供了一种黄曲霉毒素电化学传感器的制备方法及应用。本发明使用AuPt纳米粒子作为基底材料,其优良的导电性可以有效减小背景信号,同时具有大的表面积可以负载足量的抗体；利用Au/Cu<Sub>x</Sub>O@CeO<Sub>2</Sub>与检测抗体孵化作为信号标记以进一步增强电化学传感器的催化性能、放大信号,实现了对黄曲霉毒素的定量检测,具有检测限低,灵敏度高,重复性、选择性和稳定性好等优势,对黄曲霉毒素的检测具有重要的科学意义和应用价值。(The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method and application of an aflatoxin electrochemical sensor. According to the invention, AuPt nanoparticles are used as a substrate material, the excellent conductivity of the AuPt nanoparticles can effectively reduce background signals, and meanwhile, the AuPt nanoparticles have large surface area and can load sufficient antibodies; using Au/Cu x O@CeO 2 Incubation with a detection antibody is used as a signal marker to further enhance the catalytic performance of the electrochemical sensor and amplify signals, so that quantitative detection of aflatoxin is realized, and the method has the advantages of low detection limit, high sensitivity, good repeatability, selectivity and stability and the like, and has important scientific significance and application value for detection of aflatoxin.)

1. A preparation method of an aflatoxin electrochemical sensor is characterized by comprising the following steps:

(1) al for glassy carbon electrode with diameter of 4 mm₂O₃Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;

(2) dripping a 6 mu L of 1.0-3.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;

(3) continuing to capture the aflatoxin capture antibody Ab of 6 muL and 8-12 mug/mL₁Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;

(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;

(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;

(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 muL and 1.5-3.5 mg/mL_x[email protected]₂-Ab₂The solution is put on the surface of an electrode and is put at 4 DEG CAnd (3) incubating in a refrigerator for 40 min, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.

2. The method for preparing the aflatoxin electrochemical sensor, according to claim 1, the preparation of the AuPt nanoparticle solution is characterized by comprising the following steps:

(1) preparation of AuPt nanoparticles

Sequentially adding 50-150 mg of poloxamer F127 and 20-40 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8mL, 10-30 mmol/L chloroplatinic acid, 2.5 mL and 10-30 mmol/L chloroauric acid, and uniformly stirring; adding 2.5 mL of ascorbic acid of 0.05-0.15 mol/L, and reacting for 15min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12h to prepare AuPt nanoparticles;

(2) preparation of AuPt nanoparticle solution

5-15 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water, and the solution is subjected to ultrasonic treatment for 10 min to prepare the AuPt nanoparticle solution.

3. The method for preparing an aflatoxin electrochemical sensor of claim 1, wherein the detection antibody hatches Au/Cu_x[email protected]₂-Ab₂The preparation of the solution is characterized by comprising the following steps:

（1）Au/Cu_x[email protected]₂preparation of

Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu₂An O cube;

adding 20-40 mg of Cu into 30 mL of ultrapure water₂Stirring the mixture evenly, adding 500-700 mu L of cerium nitrate and 0.1 mol/L of cerium nitrate, and heating the mixture to 60 ℃; adding 3.0-4.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 55-75 mu L of chloroauric acid of 20mmol/L, and reacting for 15 min; adding 65 μ L, 20mmol/L sodium citrate, and reacting for 30 min; centrifugal cleaning with ultrapure water for three timesVacuum drying at 60 ℃ for 8-12 h to prepare Au/Cu_x[email protected]₂；

The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;

(2) detection of antibody hatching Au/Cu_x[email protected]₂-Ab₂Preparation of the solution

1.0-3.0 mL of Au/Cu with the concentration of 2 mg/mL_x[email protected]₂Adding the solution into 0.5-1.5 mL of aflatoxin detection antibody solution Ab with the concentration of 10 mu g/mL₂Performing shake incubation for 12h in a constant-temperature oscillation box at 4 ℃, centrifuging and washing, adding 1.0-3.0 mL of phosphoric acid buffer solution with pH =7.38, and preparing the detection antibody incubation Au/Cu_x[email protected]₂-Ab₂The solution was stored at 4 ℃ until use.

4. The aflatoxin electrochemical sensor prepared by the preparation method of claim 1, which is used for detecting aflatoxin, and comprises the following steps:

(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of potassium ferricyanide solution containing 10 mmol/L;

(2) detecting aflatoxin by using a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;

(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH =7.38 every 50 s, and the change in current was recorded.

5. The aflatoxin of claims 1, 2, 3 and 4 which is selected from one of the following: aflatoxin B1, aflatoxin G1.

Technical Field

The invention belongs to the technical field of immunoassay, nano materials and biosensing, and provides a preparation method and application of an aflatoxin electrochemical sensor.

Background

Aflatoxin has strong biological toxicity and carcinogenic capacity, and aflatoxin can be generated when the aflatoxin is polluted by aspergillus flavus and parasitic aspergillus flavus, and further pollutes animal-derived foods such as milk, eggs and meat through a food chain, and indirectly enters a human body, so that serious consequences such as liver and kidney injury, reproductive disorder, immunosuppression, carcinogenic teratogenesis and the like are finally caused.

The existing detection methods of aflatoxin comprise thin layer chromatography, liquid chromatography, radioimmunoassay, enzyme-linked immunosorbent assay, affinity chromatography, microcolumn screening, gold-labeled test paper method and the like. These methods have more or less disadvantages, such as tedious and complicated operation process, long time consumption and large labor capacity; the instrument and equipment are expensive, large in size and complex in operation, and the rapid field analysis is difficult to realize; sensitivity and reproducibility were poor. The electrochemical immunoassay developed in recent years is widely applied to aflatoxin detection, and has the advantages of high selectivity, quick response, simplicity in operation, convenience in carrying, suitability for field detection and the like, so researchers are actively exploring and developing various novel electrochemical sensors for detecting aflatoxin.

The invention successfully constructs an aflatoxin electrochemical sensor, the substrate material of the sensor is AuPt nano particles, the bimetallic structure of the sensor has good conductivity, the electron transmission rate can be accelerated, the rugged surface morphology ensures that the sensor has larger specific surface area, and the antibody Ab can be better loaded₁. Marker Au/Cu_x[email protected]₂The multi-thorn core-shell structure is adopted, the surface appearance of the multi-thorn structure not only increases the specific surface area of the material, but also can better fix the antibody Ab₂Meanwhile, the composite material has excellent catalytic activity, and can realize amplification of detection signals, so that the sensitivity of the immunosensor is increased. The invention adopts AuPt nano particles as substrate material, Au/Cu_x[email protected]₂The electrochemical sensor constructed by the detection antibody marker realizes the detection of aflatoxin, has low detection limit, high sensitivity, acceptable repeatability, selectivity and stability, and has important scientific significance and application value for the detection of aflatoxin.

Disclosure of Invention

The invention provides a preparation method and application of an aflatoxin electrochemical sensor,

the aflatoxin sensitive detection is realized.

The invention aims to provide a preparation method of an aflatoxin electrochemical sensor.

The second purpose of the invention is to apply the prepared aflatoxin electrochemical sensor to the high-sensitivity and specific detection of aflatoxin.

The technical scheme of the invention comprises the following steps.

1. A preparation method of an aflatoxin electrochemical sensor comprises the following steps:

(1) al for glassy carbon electrode with diameter of 4 mm₂O₃Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;

(2) dripping a 6 mu L of 1.0-3.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;

(3) continuing to capture the aflatoxin capture antibody Ab of 6 muL and 8-12 mug/mL₁Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;

(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;

(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;

(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 muL and 1.5-3.5 mg/mL_x[email protected]₂-Ab₂And putting the solution on the surface of an electrode, incubating for 40 min in a refrigerator at 4 ℃, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.

2. The preparation method of the AuPt nano particle solution comprises the following steps:

(1) preparation of AuPt nanoparticles

Sequentially adding 50-150 mg of poloxamer F127 and 20-40 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8mL, 10-30 mmol/L chloroplatinic acid, 2.5 mL and 10-30 mmol/L chloroauric acid, and uniformly stirring; adding 2.5 mL of ascorbic acid of 0.05-0.15 mol/L, and reacting for 15min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12h to prepare AuPt nanoparticles;

(2) preparation of AuPt nanoparticle solution

5-15 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water, and the solution is subjected to ultrasonic treatment for 10 min to prepare the AuPt nanoparticle solution.

3. Detection of antibody hatching Au/Cu_x[email protected]₂-Ab₂The solution was prepared by the following steps:

（1）Au/Cu_x[email protected]₂preparation of

Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu₂An O cube;

adding 20-40 mg of Cu into 30 mL of ultrapure water₂Stirring the mixture evenly, adding 500-700 mu L of cerium nitrate and 0.1 mol/L of cerium nitrate, and heating the mixture to 60 ℃; adding 3.0-4.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 55-75 mu L of chloroauric acid of 20mmol/L, and reacting for 15 min; adding 65 μ L, 20mmol/L sodium citrate, and reacting for 30 min; centrifugally cleaning the solution for three times by ultrapure water, and drying the solution for 8 to 12 hours in vacuum at the temperature of 60 ℃ to obtain Au/Cu_x[email protected]₂；

The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;

(2) detection of antibody hatching Au/Cu_x[email protected]₂-Ab₂Preparation of the solution

1.0-3.0 mL of Au/Cu with the concentration of 2 mg/mL_x[email protected]₂Adding the solution into 0.5-1.5 mL of aflatoxin detection antibody solution Ab with the concentration of 10 mu g/mL₂Shaking and incubating in a constant temperature oscillating box at the temperature of middle and 4 DEG CDissolving for 12h, centrifuging, washing, adding into 1.0-3.0 mL of phosphoric acid buffer solution with pH =7.38, and preparing the detection antibody hatching substance Au/Cu_x[email protected]₂-Ab₂The solution was stored at 4 ℃ until use.

4. Detecting aflatoxin by the following steps:

(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of potassium ferricyanide solution containing 10 mmol/L;

(2) detecting aflatoxin by using a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;

(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH =7.38 every 50 s, and the change in current was recorded.

The aflatoxin is selected from one of the following: aflatoxin B1, aflatoxin G1.

The raw materials used in the present invention are all available from chemical agents companies or biopharmaceutical companies.

Advantageous results of the invention

(1) The substrate material used by the method is AuPt nanoparticles, and has the advantages of simplicity in synthesis, good stability and the like, and the gully-shaped surface appearance enables the substrate material to have a larger surface area and can load a large amount of antibody Ab₁Meanwhile, the bimetal structure also has good conductivity, and high-efficiency signal conduction can be realized. Au/Cu_x[email protected]₂As a detection antibody marker, the sensitivity of the electrochemical sensor can be increased, the catalytic activity of the electrochemical sensor can be improved, and the amplification of a detection signal can be realized. Au/Cu_x[email protected]₂Is a multi-spine core-shell structure, Cu₂The O and CuO have good catalytic activity under the synergistic catalytic action, can realize the amplification of detection signals, and simultaneously endow the detection signals with larger specific surface area due to the multi-thorn morphology, and can effectively load an antibody Ab₂. The invention adopts AuPt nano particles as substrate material, Au/Cu_x[email protected]₂The electrochemical sensor constructed by the detection antibody marker realizes sensitive and rapid detection of aflatoxin.

(2) The aflatoxin electrochemical sensor realizes detection of aflatoxin, wherein the linear range of detection of aflatoxin B1 is 10 pg-60 ng/mL, the detection limit is 3.33pg/mL, the linear range of detection of aflatoxin G1 is 10 pg-60 ng/mL, and the detection limit is 3.33pg/mL, which shows that the aflatoxin electrochemical sensor based on Au/Cu is based on Au/Cu_x[email protected]₂The electrochemical sensor can achieve the aim of accurate determination.

Detailed Description

The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.