阅读说明：本技术 一种电化学免疫传感器及其制备方法与应用 (Electrochemical immunosensor and preparation method and application thereof ) 是由 赵文苹 孙霞 刘中合 郭业民 赵亚 于 2019-10-30 设计创作，主要内容包括：一种电化学免疫传感器及其制备方法与应用,属于电化学免疫传感器技术领域。为了解决当前对黄曲霉毒素B1检测专一性差,灵敏度低,检测周期长,不能现场在线检测及监测的问题,本发明提供一种电化学免疫传感器及其制备方法与应用,电化学免疫传感器的制备方法,包括以下步骤：(1)制备碳纤维,金纳米粒子修饰的金电极；(2)黄曲霉毒素抗体的固定；(3)BSA封闭非特异性结合位点,获得标准曲线,y＝0.1296+0.03774x,线性相关系数R<Sup>2</Sup>＝0.98858曲线线性检测范围为0.01～1000ng/mL。本发明所述的传感器可在0.11V左右的小范围电压下或确切电压下,进行黄曲霉毒素B1含量的测定。(An electrochemical immunosensor and a preparation method and application thereof, belonging to the technical field of electrochemical immunosensors. In order to solve the problems of poor specificity, low sensitivity, long detection period and incapability of on-site online detection and monitoring of aflatoxin B1 at present, the invention provides an electrochemical immunosensor and a preparation method and application thereof, wherein the preparation method of the electrochemical immunosensor comprises the following steps: (1) preparing a carbon fiber and gold nano particle modified gold electrode; (2) fixing the aflatoxin antibody; (3) blocking of non-specific binding sites with BSA gave a standard curve, y-0.1296 +0.03774x, linear correlation coefficient R 2 The linear detection range of the curve is 0.98858-0.01-1000 ng/mL. The sensor can carry out aflatoxin B under a small range voltage of about 0.11V or an exact voltageAnd (1) measuring the content.)

1. The electrochemical immunosensor is characterized in that a working electrode of the sensor is a gold electrode, and carbon fibers, nanogold, aflatoxin B1 antibody and BSA are modified on the gold electrode.

2. The preparation method of the electrochemical immunosensor, as claimed in claim 1, is characterized in that the carbon fiber and the nanogold are respectively dropped on the surface of the gold electrode, the modified gold electrode is obtained after incubation, then the aflatoxin B1 antibody is dropped on the surface of the electrode for incubation, and the obtained electrode resisting the aflatoxin B1 is incubated by BSA, so that the electrochemical immunosensor is obtained.

3. The method of claim 2, comprising the steps of:

(1) preparing carbon fiber and gold nano particle modified gold electrode: based on the area of the electrode, 0.5-1 mul of carbon fiber is dripped on the surface of the gold electrode per square millimeter, the incubation is carried out for 90-120min at the temperature of 23-27 ℃, then 0.5-1 mul of nano gold is dripped on the surface of the gold electrode per square millimeter, the incubation is carried out for 90-120min at the temperature of 23-27 ℃, and the AuNPs/CF/Au electrode is obtained;

(2) immobilization of aflatoxin B1 antibody: based on the electrode area, 0.5-1ul of aflatoxin B1 antibody with the mass concentration of 10-30 mug/mL is dripped on the surface of the AuNPs/CF/Au electrode of each square millimeter, and incubation is carried out for 60-90min at 23-27 ℃ to obtain an anti-AFB1/AuNPs/CF/Au electrode;

(3) blocking of BSA: and (3) dropwise adding 0.9-1.2 mu l of BSA with the mass concentration of 2.5% -3% on the surface of an anti-AFB1/AuNPs/CF/Au electrode of each square millimeter by taking the area of the electrode as a reference, and incubating for 60-90min at 23-27 ℃ to obtain the electrochemical immunosensor for detecting AFB 1.

4. The method according to claim 3, wherein the step (1) of preparing the carbon fiber, gold nanoparticle-modified gold electrode: and (3) dropwise adding 0.7 mu l of carbon fiber on the surface of the gold electrode per square millimeter by taking the area of the electrode as a reference, incubating for 90min at 25 ℃, dropwise adding 0.7ul of nanogold on the surface of the gold electrode per square millimeter, and incubating for 90min at 25 ℃ to obtain the AuNPs/CF/Au electrode.

5. The method for preparing the aflatoxin B1 antibody of the step (2), which is characterized in that the following steps are carried out: and (3) dropwise adding 0.7ul of aflatoxin B1 antibody with the mass concentration of 10 mu g/mL on the surface of the AuNPs/CF/Au electrode of each square millimeter by taking the area of the electrode as a reference, and incubating for 60min at 25 ℃ to obtain the anti-AFB1/AuNPs/CF/Au electrode.

6. The method of claim 3, wherein the blocking of BSA in step (3): and (3) based on the electrode area, dripping 1 mu l of BSA with the mass concentration of 2.5% on the surface of an anti-AFB1/AuNPs/CF/Au electrode of each square millimeter, and incubating for 60min at 25 ℃ to obtain the electrochemical immunosensor for detecting AFB 1.

7. Use of the electrochemical immunosensor of claim 1 to detect AFB 1.

8. Use according to claim 7, characterized in that it comprises the following steps:

(1) incubating the solution to be detected and the sensor for 30-50min at 36-38 ℃, and then washing the solution clean with water;

(2) containing 0.1M KCL and 5mM [ Fe (CN)6 at pH 7.4-7.6]^3-/4-In the PBS solution, detecting by cyclic voltammetry;

(3) converting the measured immunoreaction current response value under the voltage of 0.09-0.12V by a normalized current method to obtain a y value, substituting the y value into a standard curve y of 0.1296+0.03774x, and obtaining the x value, namely the logarithm value of the concentration of AFB1 to further obtain the concentration of AFB 1.

9. The use according to claim 8, wherein the normalized current method is converted from y ═ value (immunoreaction current response of test sample-immunoreaction current response of test sample without incubation)/immunoreaction current response of test sample without incubation, and the detection range of AFB1 is 0.01-1000 ng/ml.

10. The use of claim 8, wherein step (2) comprises 0.1M KCL and 5mM [ Fe (CN)6]^3-/4-The pH of the PBS solution of (1) was 7.4; the voltage in the step (3) is 0.10-0.12V.

Technical Field

The invention belongs to the technical field of electrochemical immunosensors, and particularly relates to an electrochemical immunosensor and a preparation method and application thereof.

Background

Aflatoxins are toxic, carcinogenic secondary metabolites produced by aspergillus flavus and parasitic bacteria, and are the most toxic and most toxic of mycotoxins. Since aflatoxins are often found in food and agricultural products, they pose serious health and economic problems in many countries. Among aflatoxins, aflatoxin B1(AFB1) is the most toxic and is classified as a carcinogen by the international cancer research institute. Therefore, health threats to humans, livestock and poultry are extremely large, and high importance is given to countries in the world, and the maximum amount is established as a mandatory standard for food and feed.

At present, a plurality of methods for detecting aflatoxin exist, and a chemical analysis method, an instrumental analysis method, a biological identification method, an immunoassay method and the like are summarized. The analysis methods mainly carry out qualitative and quantitative analysis on the aflatoxin by combining an instrument according to the chemical structure and the biological characteristics of the aflatoxin. For example, aflatoxin is fluorescent, and Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) can be performed, but the method has poor specificity and low sensitivity, and is generally only used as an evidence for chemical analysis; the immunoassay method utilizing the enzyme and biochemical technology has the advantages of strong specificity, high sensitivity, accuracy and rapidness, simple operation and easy standardization, but needs special equipment and safety protection, thereby preventing wider application.

The electrochemical immunization method has strong selectivity, good specificity, simple operation and wide development prospect. Liu et al prepared a micro-comb electrode on the functional biorecognition surface of the gold nanoparticles using self-assembled horseradish peroxidase (HRP) and AFB1 antibody molecules, and used the bioelectrocatalytic reaction occurring on the micro-comb electrode to determine aflatoxin AFB1, with the lowest detection limit of the sensor being 0.1 ng/mL. Beheshti-Marnani et al studied the electrochemical characteristics of the reduced graphene modified electrode, and the aptamer sensor prepared by using the electrode was used for ultramicro detection of AFB1, and the detection result showed that the lowest detection limit of the sensor was 0.07 ng/mL. Gouda et al use an aptamer marked by methylene blue as a signal fragment, and propose an electrochemical aptamer biosensor based on a functionalized graphene oxide electrocatalytic amplification signal for aflatoxin B1 detection, wherein the minimum detection limit reaches 0.05 ng/mL. Yu et al propose an electrochemical impedance immunosensor constructed by MWCNTs/RTIL compound thin film modified electrode to rapidly and sensitively determine AFB1, the existence of MWCNTs ensures rapid electron transfer, ionic liquid provides a benign microenvironment for antibody, and the detection result shows: in the range of 0.1-10ng/mL, the sensor has good linear relation, and the lowest detection limit is 0.03 ng/mL. The detection signals of the methods are mostly obtained based on electrochemical analytical instruments in laboratories, but the agricultural production field environment is severe, the conditions are simple and crude, the detection result is required to be simple, convenient and easy to obtain, and the detection result can be seen in real time, and a detection method matched with a field rapid detection scene and a detection device in actual agricultural production is required to be invented.

Disclosure of Invention

The invention provides an electrochemical immunosensor and a preparation method and application thereof, and aims to solve the problems that aflatoxin B1 is poor in detection specificity, low in sensitivity, long in detection period and incapable of on-site on-line detection and monitoring.

The working electrode of the sensor is a gold electrode, and carbon fibers, nanogold, aflatoxin B1 antibody and BSA are modified on the gold electrode.

The preparation method of the electrochemical immunosensor comprises the steps of respectively dropping and coating carbon fiber and nanogold on the surface of a gold electrode, obtaining a modified gold electrode after incubation, then dropping and coating aflatoxin B1 antibody on the surface of the electrode for incubation, carrying out antibody fixation, finally carrying out BSA incubation on the prepared electrode, and sealing non-specific sites of the antibody fixed on the electrode, thus obtaining the electrochemical immunosensor.

The parameter optimization process in the sensor preparation process comprises the following steps:

(1) preparing carbon fiber and gold nano particle modified gold electrode: based on the area of the electrode, 0.5-1 mul of carbon fiber is dripped on the surface of the gold electrode per square millimeter, the incubation is carried out for 90-120min at the temperature of 23-27 ℃, then 0.5-1 mul of nano gold is dripped on the surface of the gold electrode per square millimeter, the incubation is carried out for 90-120min at the temperature of 23-27 ℃, and the AuNPs/CF/Au electrode is obtained;

(2) immobilization of aflatoxin B1 antibody: based on the electrode area, 0.5-1ul of aflatoxin B1 antibody with the mass concentration of 10-30 mug/mL is dripped on the surface of the AuNPs/CF/Au electrode of each square millimeter, and incubation is carried out for 60-90min at 23-27 ℃ to obtain an anti-AFB1/AuNPs/CF/Au electrode;

(3) blocking of BSA: and (3) dropwise adding 0.9-1.2 mu l of BSA with the mass concentration of 2.5% -3% on the surface of an anti-AFB1/AuNPs/CF/Au electrode of each square millimeter by taking the area of the electrode as a reference, and incubating for 60-90min at 23-27 ℃ to obtain the electrochemical immunosensor for detecting AFB 1.

Preferably, the preparation of the carbon fiber and gold nanoparticle modified gold electrode in the step (1): and (3) dropwise adding 0.7 mu l of carbon fiber on the surface of the gold electrode per square millimeter by taking the area of the electrode as a reference, incubating for 90min at 25 ℃, dropwise adding 0.7ul of nanogold on the surface of the gold electrode per square millimeter, and incubating for 90min at 25 ℃ to obtain the AuNPs/CF/Au electrode.

Preferably, the aflatoxin B1 antibody in the step (2) is immobilized: and (3) dropwise adding 0.7ul of aflatoxin B1 antibody with the mass concentration of 10 mu g/mL on the surface of the AuNPs/CF/Au electrode of each square millimeter by taking the area of the electrode as a reference, and incubating for 60min at 25 ℃ to obtain the anti-AFB1/AuNPs/CF/Au electrode.

Preferably, blocking of BSA in step (3): and (3) based on the electrode area, dripping 1 mu l of BSA with the mass concentration of 2.5% on the surface of an anti-AFB1/AuNPs/CF/Au electrode of each square millimeter, and incubating for 60min at 25 ℃ to obtain the electrochemical immunosensor for detecting AFB 1.

The electrochemical immunosensor disclosed by the invention is applied to detection of AFB 1.

The method comprises the following steps:

(1) incubating the solution to be detected and the sensor for 30-50min at 36-38 ℃, and then washing the solution clean with water;

(2) containing 0.1M KCL and 5mM [ Fe (CN)6 at pH 7.4-7.6]^3-/4-In the PBS solution, detecting by cyclic voltammetry;

(3) converting the measured immunoreaction current response value under the voltage of 0.09-0.12V by a normalized current method to obtain a y value, substituting the y value into a standard curve y of 0.1296+0.03774x to obtain an x value, namely a logarithm value of the concentration of AFB1, and further obtaining the concentration of AFB1, wherein the normalized current method is used for converting y (immunoreaction current response value of a sample to be detected-immunoreaction current response value of a sample without incubation to be detected)/immunoreaction current response value without incubation to be detected, and the detection range of the AFB1 is 0.01-1000 ng/ml.

Preferably, the solution of step (2) contains 0.1M KCL and 5mM [ Fe (CN)6]^3-/4-The PBS solution of (3) had a pH of 7.4.

Preferably, the voltage in step (3) is 0.10-0.12V.

Advantageous effects

The carbon fiber is inorganic polymer fiber with carbon content higher than 90%, and is microcrystalline graphite material obtained by stacking organic fibers such as flaky graphite microcrystals along the axial direction of the fiber and performing carbonization and graphitization treatment. The carbon fiber has many excellent properties, high axial strength and modulus, low density, high specific performance, no creep, high temperature resistance in non-oxidation environment, good fatigue resistance, high conductivity, good X-ray permeability, insolubility and no expansion in organic solvent, acid and alkali, and corrosion resistance, and becomes an attractive material in electrochemistry for a remarkable year.

The nano gold (gold nano particles) refers to gold micro particles, the diameter of the nano gold (gold nano particles) is 1-100 nm, the nano gold (gold nano particles) has high electron density, dielectric property and catalytic action, can be combined with various biological macromolecules, and does not influence the biological activity of the nano gold (gold nano particles). The gold nanoparticles with different particle sizes can be conveniently prepared by the reduction method of chloroauric acid, and the color of the gold nanoparticles is red to purple according to the diameter. The nano gold can be used for a marking technology, and is essentially a coating process that high molecules such as protein and the like are adsorbed to the surface of nano gold particles. The adsorption mechanism is that negative charges on the surface of the gold nanoparticles are firmly combined with positive charge groups of protein due to electrostatic adsorption, and biomolecules are not denatured after adsorption. The spherical gold nanoparticles have a strong protein adsorption function, and can be non-covalently bound with immunoglobulin, toxin, enzyme, antibiotic, bovine serum albumin and the like, so that the spherical gold nanoparticles are very useful tools in basic research and experiments.

The AuNPs and CF signal amplification material prepared by the invention has good electrochemistryThe activity and the detection capability of the prepared electrochemical immunosensor are wide in linear range and low in detection limit, and the AFB1 can be successfully and sensitively detected. Specifically recognizing a series of AFB1 antigen and antibody at different concentrations, incubating at 37 deg.C for 30min, washing with ultrapure water, and washing with a solution containing 0.1MKCl and 5mM [ Fe (CN)₆]^3-/4-The detection method comprises the steps of detecting AFB1 with different concentrations in PBS solution by using cyclic voltammetry, processing detection current values on a sensor before and after incubation of aflatoxin B1 by using a normalization method to obtain a standard curve taking logarithm of AFB1 concentration as an abscissa and a current difference value at a voltage of 0.11V corresponding to the maximum immunoreaction current response current normalization value as an ordinate, wherein the linear detection range of the curve is 0.01-1000 ng/mL, the detection limit is 0.01ng/mL, the linear regression equation is y 0.1296+0.03774x, and the linear correlation coefficient R is²＝0.98858。

The detection signal of the electrochemical immunosensor is weak generally and is easy to be influenced by various external interference factors, a normalized current method (NSPV) is used for processing the electrochemical signal detected by the sensor, the voltage value at the position with the maximum change rate of the sensor sensing signal is determined, a linear regression model extracted by taking the current response difference value before and after the reaction at the voltage value as the calculation basis is used, the detection sensitivity, the accuracy and the stability of the linear regression model are greatly improved, the detection effect of the sensor is practical and reliable, and the linear regression model has good development potential in the aspect of detecting the aflatoxin B1.

When the aflatoxin B1 content in a sample to be detected is actually measured, the electrochemical workstation can be applied under the condition of a laboratory to apply a certain range of voltage for measurement, and the aflatoxin B1 content can be measured under a small range of voltage of about 0.11V or under a definite voltage, so that the limitation of the method on experimental instruments in actual use is greatly reduced, the applicability of the method and an aflatoxin on-site rapid detection device is improved, the matching problem of the on-site rapid detection scene and the detection device in actual agricultural production is met, other biological identification elements (other antibodies) are replaced by the method, the detection technology and the application can be popularized in a larger range, and the benchmarking contribution is provided for the development of the on-site rapid technology.

Drawings

FIG. 1. different material modification sequences respond to different currents on the surface of a gold electrode (a) bare gold electrode Au; (b) nano gold/carbon fiber/gold electrode AuNPs/CF/Au; (c) carbon fiber/nano gold/gold electrode CF/AuNPs/Au;

FIG. 2. different material modification sequences respond to different currents on the glassy carbon electrode surface (a) bare glassy carbon electrode GCE; (b) nano-gold/carbon fiber/glassy carbon electrodes, AuNPs/CF/GCE; (c) carbon fiber/nanogold/glassy carbon electrode, CF/AuNPs/GCE;

FIG. 3 is a comparison of the effect of carbon fiber and nanogold composite films on the modification of gold and glassy carbon electrodes (a) AuNPs/CF/Au; (b) AuNPs/CF/GCE;

FIG. 4 is a graph of normalized values of sensor response current at different voltages incubated with different concentrations of aflatoxin;

FIG. 5 is a linear relationship graph of the concentration of aflatoxin at different concentrations and the current difference at 0.11V of the immunosensor after incubation with aflatoxin.

Detailed Description

The gold nano preparation method of the invention is consistent with the records 1311317 of Doron A, Katz E, Willner I.organic of Au colloidal films to ITO glass substrates of application of the metallic colloidal films to substrate redox-active monolayers [ J ] Langmuir,1995,11(4): 3-;

the carbon fiber of the present invention is purchased from German island gold technologies, Inc., Beijing.

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.