阅读说明：本技术 基于hrp催化聚苯胺原位生成用于afb1的检测方法 (Detection method for AFB1 based on HRP catalysis polyaniline in-situ generation ) 是由 刘元建 袁伟 熊晓辉 于 2019-09-16 设计创作，主要内容包括：本发明公开了基于HRP催化聚苯胺原位生成用于AFB1的检测方法,包括以下步骤：DNA正四面体探针金电极的合成；将AFB1-HRP抗原分别与金电极孵育；然后将孵育后的金电极与苯胺聚合缓冲液反应检测聚苯胺的电化学信号获得标准曲线；将待测样品、AFB1-HRP与金电极孵育,然后与苯胺聚合缓冲液反应检测聚苯胺的电化学信号；通过聚苯胺的电化学信号对应于标准曲线得出AFB1的浓度。本发明利用DNA正四面体的刚性结构,将黄曲霉毒素B1鼠单抗有序组装在金电极表面,有效提高了抗原抗体专一性识别结合效率；相比传统的单链DNA或者11-巯基十一烷酸等小分子探针,电化学信号明显增强,方法灵敏度提升1~2个数量级。(the invention discloses a detection method for AFB1 based on HRP-catalyzed polyaniline in-situ generation, which comprises the following steps of synthesizing a DNA regular tetrahedron probe gold electrode, respectively incubating AFB1-HRP antigens with the gold electrode, reacting the incubated gold electrode with an aniline polymerization buffer solution to detect an electrochemical signal of polyaniline to obtain a standard curve, incubating a sample to be detected, AFB1-HRP and the gold electrode, reacting with the aniline polymerization buffer solution to detect the electrochemical signal of the polyaniline, and obtaining the concentration of AFB1 according to the standard curve corresponding to the electrochemical signal of the polyaniline.)

1. the detection method for AFB1 based on HRP catalytic polyaniline in-situ generation is characterized by comprising the following steps:

1) synthesizing a DNA regular tetrahedron probe gold electrode;

2) Respectively incubating known AFB1-HRP antigens with different concentrations with the DNA regular tetrahedron probe gold electrode obtained in the step 1);

3) Then reacting the DNA regular tetrahedron probe gold electrode incubated in the step 2) with an aniline polymerization buffer solution to detect an electrochemical signal of polyaniline to obtain a standard curve of AFB1-HRP antigen concentration and the electrochemical signal of the polyaniline;

4) Incubating a sample to be detected with unknown concentration, AFB1-HRP and the DNA regular tetrahedron probe gold electrode obtained in the step 1), and then reacting with an aniline polymerization buffer solution to detect an electrochemical signal of polyaniline;

5) The concentration of AFB1 was obtained by mapping the electrochemical signal of polyaniline to a standard curve.

2. the detection method for AFB1 based on HRP-catalyzed polyaniline in situ generation as claimed in claim 1, wherein the specific steps of step 1) are as follows: uniformly mixing 4 nucleic acid solutions, TCEP and TM buffer, heating in water bath, cooling to obtain a solution, dropwise adding the solution on the surface of a gold electrode, incubating at room temperature, soaking the electrode in a mixed solution of EDC and NHS for room temperature activation, washing the electrode with PBS, adding a monoclonal antibody, and continuously incubating to obtain the DNA regular tetrahedron probe gold electrode.

3. The detection method for AFB1 based on in-situ generation of HRP-catalyzed polyaniline according to claim 2, wherein the 4 nucleic acid solutions are Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH and Tetra-D-SH, respectively, and the nucleotide sequences of the Tetra-A-COOH are as follows:

COOH-TTTTTTTTTT-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA；

the nucleotide sequence of the Tetra-B-SH is as follows:

HS-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC；

The nucleotide sequence of the Tetra-C-SH is as follows:

HS-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC；

The nucleotide sequence of the Tetra-D-SH is as follows:

HS-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT。

4. the detection method for AFB1 based on in ~ situ generation of HRP ~ catalyzed polyaniline in the step 2) is characterized in that the concentration of AFB1 ~ HRP antigen in the step 2) is 0.1 ~ 200 ng/mL.

5. The detection method for AFB1 based on HRP-catalyzed polyaniline in situ generation as claimed in claim 1, wherein the aniline polymerization buffer solution in step 3) is prepared from acetic acid-sodium acetate solution, aniline and hydrogen peroxide.

6. The detection method for AFB1 based on HRP catalyzed polyaniline in situ generation as claimed in claim 1, wherein the volume ratio of the DNA regular tetrahedron probe gold electrode to the aniline polymerization buffer in step 3) is: 1:100-1:1000.

7. The detection method for AFB1 based on HRP-catalyzed polyaniline in-situ generation as claimed in claim 1, wherein the volume ratio of the sample to be detected in step 4), AFB1-HRP and the DNA regular tetrahedron probe gold electrode obtained in step 1) is 1:10-1: 30.

8. The detection method for AFB1 based on HRP-catalyzed polyaniline in situ generation as claimed in claim 1, wherein the sample to be detected is one or more of rice, wheat, corn, sorghum, barley and buckwheat.

9. the detection method for AFB1 based on HRP catalyzed polyaniline in situ generation as claimed in claim 1, wherein the diameter of the DNA regular tetrahedron probe gold electrode is 2 mm.

Technical Field

The invention belongs to the technical field of biosensing, and particularly relates to a detection method for AFB1 based on HRP catalysis polyaniline in-situ generation.

background

The mycotoxin refers to a secondary metabolite produced in the growth and reproduction process of fungi, and is various in variety, and more than 400 species are separated and identified at present and mainly comprise aflatoxin, zearalenone/alcohol and the like. Mycotoxins are generally substances of relatively small molecular mass that are not normally destroyed by the grain processing or cooking heat of the food product and are therefore somewhat more dangerous than other toxins such as bacterial toxins. The mycotoxin has great social harm, is mainly the most harmful aflatoxin AFB1, is also the main reason of harming the body health of children and teenagers in China, and has high carcinogenicity. At present, domestic cereal products have the problems of small scale, dispersion, unscientific airing method and the like, and AFB1 detects the defects of different products, which influences the self-checking effect of food production enterprises and the monitoring effect of a supervision mechanism. The AFB1 fast check product does not completely realize the localization, and the monopoly of foreign products is broken, but still occupies a large market. In order to prevent the food safety problem brought by AFB1 and reduce economic loss, it is especially important to develop a rapid and sensitive detection method for AFB 1.

At present, the detection methods of AFB1 adopted by most detection mechanisms in China are mainly divided into three types: one is a biological detection method, including a seed germination test, a vomiting test and the like, which is not favorable for quick detection and is rarely adopted; one is a physical and chemical detection method, Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). TLC is simple, but has high detection limit and poor sensitivity, and can only be semi-quantitative. Although the HPLC has high sensitivity, the sample processing steps are complicated, the operation is complex, the requirements on instruments and equipment are high, and the consumption of standard substances is high; another class is immunochemical assays such as ELISA and colloidal gold immunochromatographic assays. The ELISA method is particularly suitable for the centralized detection of a large number of samples, and the colloidal gold immunochromatography method is suitable for the instant detection of a single sample or a few samples on site, but false positive is easy to occur if the pretreatment of the samples is improper. False positive rate based on rapid detection of enzyme linked immunosorbent assay kit developed by Europroxima, Beacon, Beijing Holland, and Dian Biotechnology, Inc. of Hangzhou is generally higher.

therefore, the development of a new method for simply, quickly and accurately detecting the aflatoxin AFB1 in the grains has important significance for controlling the safety of the food generation!

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a detection method for AFB1 in grains based on HRP catalysis polyaniline in-situ generation. The invention aims at the obvious change of electrochemical properties of aniline polymerized on the surface of a regular tetrahedron probe, so that a competition method is established for detecting AFB1, the principle is simple, high-efficiency quick detection can be achieved, and the miniaturization of a detection instrument is realized. The invention specifically recognizes AFB1-HRP antigen by forming a DNA regular tetrahedron probe based on a gold electrode surface functionalized modified monoclonal antibody, then catalyzes aniline in-situ deposition, and detects AFB1 by a competition method. The invention realizes the detection of AFB1 by a competition method by utilizing a DNA regular tetrahedron probe, aniline deposition property and antigen-antibody specific recognition property.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a detection method for AFB1 based on HRP catalysis polyaniline in-situ generation, which comprises the following steps:

1) Synthesizing a DNA regular tetrahedron probe gold electrode;

2) respectively incubating known AFB1-HRP antigens with different concentrations with the DNA regular tetrahedron probe gold electrode obtained in the step 1);

3) Then reacting the DNA regular tetrahedron probe gold electrode incubated in the step 2) with an aniline polymerization buffer solution to detect an electrochemical signal of polyaniline to obtain a standard curve of AFB1-HRP antigen concentration and the electrochemical signal of the polyaniline;

4) Incubating a sample to be detected with unknown concentration, AFB1-HRP and the DNA regular tetrahedron probe gold electrode obtained in the step 1), and then reacting with an aniline polymerization buffer solution to detect an electrochemical signal of polyaniline;

5) the concentration of AFB1 was obtained by mapping the electrochemical signal of polyaniline to a standard curve.

Before the synthesis of the DNA regular tetrahedron probe gold electrode, the first step is the selection of monoclonal antibody and fungomycin, and the monoclonal antibody of the invention is aflatoxin B1 mouse monoclonal antibody.

Furthermore, the invention comprises the selection of nucleic acid sequences, and the four modified nucleic acid sequences selected by the invention are Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH and Tetra-D-SH. The four nucleic acid sequences were artificially synthesized to obtain four nucleic acid solutions.

Wherein the specific steps of the step 1) are as follows: uniformly mixing 4 nucleic acid solutions, TCEP and TM buffer, heating in water bath, cooling to obtain a solution, then dropwise adding the solution on the surface of a gold electrode, incubating at room temperature, soaking the electrode in a mixed solution of EDC and NHS for room temperature activation, washing the electrode with PBS, adding aflatoxin B1 mouse monoclonal antibody, and continuing to incubate to obtain the DNA regular tetrahedron probe gold electrode.

wherein the 4 nucleic acid solutions are Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH and Tetra-D-SH respectively, and the nucleotide sequences of the Tetra-A-COOH are as follows:

COOH-TTTTTTTTTT-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA；

the nucleotide sequence of the Tetra-B-SH is as follows:

HS-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC；

The nucleotide sequence of the Tetra-C-SH is as follows:

HS-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC；

the nucleotide sequence of the Tetra-D-SH is as follows:

HS-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT。

Wherein, after four modified nucleic acid sequences (Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH and Tetra-D-SH) are mixed in equal proportion, three regular tetrahedron nucleic acid nano structures with Sulfydryl (SH) at the bottom and Carboxyl (COOH) at the top can be formed. SH can be connected with a gold electrode through an Au-S covalent bond to fix a regular tetrahedron, and carboxyl is coupled with a subsequent AFB1 monoclonal antibody to form a detection probe; common single nucleic acid strands such as SH-Tetra-T₁₀-COOH or small MUA although immobilization can be achievedthe antibody can not be arranged orderly like a regular tetrahedron due to only one chain;

As a further preference, the synthesis steps of the DNA regular tetrahedron probe gold electrode are as follows: the nucleic acid sequences (Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH, Tetra-D-SH) were diluted to 50. mu.M with TE buffer (10mM Tris, pH8.0, 1mM EDTA); aflatoxin B1 murine monoclonal antibody was diluted to 100ng mL with PBS^-1(ii) a AFB1-HRP was diluted to 10ng mL with PBS^-1Freezing at-20 deg.C for use. Adding 1 μ L of nucleic acid solution (50 μ M Tetra-A-COOH, Tetra-B-SH, Tetra-C-SH and Tetra-D-SH) into an EP tube, 1 μ L of 500mM TCEP, 45 μ L of TM buffer (20mM Tris, pH8.0 and 50mM MgCl2), mixing, heating in water bath at 95 deg.C for 10min, cooling at 4 deg.C for 30min, adding 20 μ L of the above solution onto the surface of gold electrode, incubating at room temperature for 10-12 h, soaking the electrode in 1mL of mixed solution of 1.66 μ M EDC and 1mL4.15 μ M MNHS, activating at room temperature for 2h, washing the electrode with PBS for 3 times, and adding 20 μ L of 100ng mL^-1Incubating the aflatoxin B1 mouse monoclonal antibody for 10-12 h to obtain a regular tetrahedron probe gold electrode for detecting the probe DNA;

Wherein the concentration of the AFB1-HRP antigen in the step 2) is 0.1-200 ng/mL.

wherein, the aniline polymerization buffer solution in the step 3) is prepared by acetic acid-sodium acetate solution, aniline and hydrogen peroxide.

wherein, the volume ratio of the DNA regular tetrahedron probe gold electrode in the step 3) to the aniline polymerization buffer solution is as follows: 1:100-1: 1000.

Wherein the volume ratio of the sample to be detected in the step 4), AFB1-HRP and the DNA regular tetrahedron probe gold electrode obtained in the step 1) is 1:10-1: 30.

As a further preferred, the step of reacting the mixed solution of AFB1 with unknown concentration and AFB1-HRP with known fixed concentration in the step 4) with the detection probe comprises the following steps: 20 μ L of the actual sample treated according to the national standard and 100ng mL of the solution were added dropwise^-1AFB1-HRP mixed solution was applied to the detection probe and incubated at room temperature for 2 h.

as a further preferred, the HRP catalyzed aniline deposition step of step 4) is as follows: the gold electrode modified by the detection probe is soaked in 500 mu L aniline polymerization buffer solution (100mM acetic acid-sodium acetate, pH4.3, 200mM aniline, 20mM hydrogen peroxide, new configuration) and incubated for 2h at room temperature for differential pulse voltammetry detection.

Further preferably, the PBS buffer solution is 10mM PBS buffer solution with pH 7.2-7.4 containing NaCl and KCl, wherein the initial concentration of the NaCl is 136.89mM, and the initial concentration of the KCl is 2.67 mM.

Wherein the sample to be detected is one or more of rice, wheat, corn, sorghum, barley and buckwheat.

Wherein the diameter of the used DNA regular tetrahedron probe gold electrode is 2 mm.

The invention also comprises a comparison experiment of the traditional single-stranded DNA, 11-mercaptoundecanoic acid (MUA) and a DNA regular tetrahedron detection probe, which comprises the following steps: after the traditional single-stranded DNA probe and the MUA probe are respectively synthesized according to the step 1), the other steps are processed like the steps 2) to 5), differential pulse voltammetry detection is carried out, and the detection effects of the three probes are compared.

The principle of the invention is as follows: the invention marks Sulfydryl (SH) on three vertexes of a DNA regular tetrahedron, and modifies the DNA regular tetrahedron nano structure on the surface of a gold electrode through an Au-S covalent bond. And then activating carboxyl at the top end of a DNA regular tetrahedron by using 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS), coupling the activated carboxyl with a monoclonal antibody to form a detection probe, and promoting the AFB1 with unknown concentration in the sample to compete with the AFB1-HRP with known concentration for an active site by using an antigen-antibody specific recognition principle. The aniline is protonated in a buffer solution with pH4.3 and can be adsorbed to the phosphate skeleton of the DNA regular tetrahedron nano structure through electrostatic interaction. The HRP assembled on the gold electrode catalyzes aniline to generate polyaniline through in-situ polymerization on a DNA nano structure, and the current and potential changes of the electrode are observed through differential pulse voltammetry so as to detect aflatoxin AFB1 in the grains by the competition method.

Has the advantages that: compared with the prior art, the invention has the following characteristics and advantages:

1) According to the invention, the rigid structure of the DNA regular tetrahedron is utilized, and the monoclonal antibodies are orderly arranged on the surface of the gold electrode, so that the specific recognition and combination efficiency of the antigen and the antibody is effectively improved;

2) The DNA regular tetrahedron nanostructure is used as a template for aniline polymerization, is rich in phosphate double helix framework, can adsorb a large amount of aniline, and has the sensitivity as low as 0.05ng ml compared with the traditional single-stranded DNA or small molecular probes such as 11-mercaptoundecanoic acid (MUA) and the like^-1Whereas the conventional single-stranded DNA sensitivity is only 10ng ml^-1(ii) a The minimum detection limit is improved by 200 times, and the sensitivity of the method is improved by about 2 orders of magnitude.

3) the invention does not need to be detected by a precise instrument and has the advantages of low cost, rapidness, simplicity, convenience, sensitivity and good specificity.

Drawings

Fig. 1 shows a flow diagram of a detection method for AFB1 in grain based on HRP catalyzed polyaniline in situ generation;

FIG. 2A shows cyclic voltammetry; fig. 2B shows the nyquist diagram for the biosensor: a: bare gold electrode, b: DNA regular tetrahedron probe gold electrode, c: electrode modified by aflatoxin AFB1 antibody, d: AFB1-HRP modified electrode, e: an electrode after polymerization of aniline;

FIG. 3 shows the effect of scan rate on the sensor; FIG. 3A: cyclic voltammograms of the gold electrodes after polymerization at different scan rates (different rates (a)20, (b)50, (c)100, (d)150, (e)200, (f)250, (g)300, (h)350, (i)400, (j)450, (k)500mV s^-1) (ii) a FIG. 3B: the peak current is plotted against the square root of the scan rate; FIGS. 3C and 3D show scanning electron micrographs of gold electrodes before and after polymerization of aniline;

FIG. 4 shows the optimized validation graphs of different concentrations of AFB1-HRP, a-f: 0.1, 1, 10, 50, 100, 200 ng/mL;

FIG. 5 shows a graph of the electrochemical signal for detecting aflatoxin AFB1 specificity, from which aflatoxin AFB1 can be specifically recognized by DNA regular tetrahedron probes;

FIG. 6 comparison of the sensitivity of the DNA regular tetrahedron probe with the SH-T10-COOH probe;

FIG. 7 shows the MUA probe, SH-T₁₀-COOH probe (ssP) was compared with DNA regular tetrahedron probe (DTP).

Detailed Description

the present invention is further illustrated by the following specific examples and the accompanying drawings, and it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.

Reagents and instruments used in this experiment:

The nucleic acid sequences are shown in the table below and are obtained from Shanghai Biotechnology engineering (Shanghai) GmbH, aflatoxin B1-HRP (AFB1-HRP), aflatoxin B1-BSA (AFB1-BSA), aflatoxins (AFM1, AFFG1), aflatoxin B1 murine monoclonal antibody (AFB1 mAb), Patulin (Patulin), Ochratoxin (OTA), zeatin (F-2), vomitoxin (DON) are obtained from Shanghai Youlong Bio Inc, rice, wheat, corn, sorghum, barley, buckwheat are obtained from Suguo supermarket, sodium acetate (NaAc), tris (2-carboxy) phosphine hydrochloride (TCEP), PBS, 11-mercaptoundecanoic acid (MUA), potassium ferricyanide (K)₃[Fe(CN)₆]) Potassium ferrocyanide (K)₄[Fe(CN)₆]) Potassium chloride (KCl), Tris (hydroxymethyl) aminomethane (Tris), aniline (aniline), hydrogen peroxide (H)₂O₂) Ethylenediaminetetraacetic acid (EDTA), magnesium chloride (MgCl2), electrochemical workstation (CHI750E), scanning electron microscope (JEM-2100, JEOL, Japan), mixing vortexer (IKA German), centrifuge (Eppendorf German).

TABLE 1 nucleic acid sequences for use in the present invention