阅读说明：本技术 一种同时检测黄曲霉毒素b1和赭曲霉毒素a的电化学方法 (Electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A ) 是由 邹小波 徐艺伟 胡雪桃 石吉勇 李艳肖 李亚惠 王鑫 于 2019-09-27 设计创作，主要内容包括：本发明属于生物传感器和电化学检测领域,涉及一种同时检测黄曲霉毒素B1和赭曲霉毒素A的电化学方法；步骤为：首先制备得到丝网印刷电极；然后制备得到AFB1适配体与OTA适配体修饰电极,再将适配体修饰电极依次浸入AFB1、OTA、Tris-HCl缓冲溶液配制的标准溶液和AFB1适配体互补链、OTA适配体互补链、Tris-HCl缓冲溶液的混合液；最后,根据二茂铁的峰电流值、亚甲基蓝的峰电流值与AFB1和OTA浓度的关系,分别建立标准曲线；通过检测待测液的二茂铁和亚甲基蓝的峰电流值,即可实现样品中AFB1和OTA的同时检测；本发明能够对两种目标物同时检测,操作简便,并具有高灵敏度和高选择性。(The invention belongs to the field of biosensors and electrochemical detection, and relates to an electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A; the method comprises the following steps: firstly, preparing a screen printing electrode; then preparing an AFB1 aptamer and an OTA aptamer modified electrode, and sequentially immersing the aptamer modified electrode into a standard solution prepared from AFB1, OTA and Tris-HCl buffer solution and a mixed solution of an AFB1 aptamer complementary chain, an OTA aptamer complementary chain and Tris-HCl buffer solution; finally, respectively establishing a standard curve according to the relationship between the peak current value of ferrocene and the peak current value of methylene blue and the concentration of AFB1 and OTA; the AFB1 and OTA in the sample can be simultaneously detected by detecting the peak current values of the ferrocene and the methylene blue of the liquid to be detected; the invention can simultaneously detect two targets, has simple and convenient operation, and has high sensitivity and high selectivity.)

1. An electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A, which is characterized by comprising the following steps:

(1) preparing a screen printing electrode, wherein the screen printing electrode comprises a counter electrode, a working electrode I, a working electrode II and a reference electrode;

(2) preparing an aptamer modified electrode;

s1, firstly, immersing the screen printing electrode prepared in the step (1) into a chloroauric acid solution, applying constant potentials on 2 working electrodes of the screen printing electrode, keeping for a period of time, taking out the screen printing electrode, placing the screen printing electrode in a sulfuric acid solution again, performing cyclic voltammetry scanning, and activating gold nanoparticles to obtain the gold nanoparticle modified screen printing electrode;

s2, mixing the tris (2-carboxyethyl) phosphine hydrochloride solution with AFB1 aptamer stock solution to react to obtain a mixed solution A; mixing the tris (2-carboxyethyl) phosphine hydrochloride solution with OTA aptamer stock solution to react to obtain a mixed solution B; respectively diluting the mixed solution A and the mixed solution B to certain concentrations by using a Tris-HCl buffer solution;

s3, dripping the diluted mixed solution A in the S2 on the surface of the first screen printing electrode modified by the gold nanoparticles obtained in the S1; dripping the diluted mixed solution B in the step S2 on the surface of a screen printing electrode working electrode II soaked in the step S1, standing for a period of time at a certain temperature, washing the surfaces of the working electrode I and the working electrode II by using a Tris-HCl buffer solution, dripping mercaptohexanol solutions on the surfaces of the working electrode I and the working electrode II respectively after washing, storing for a period of time at a constant temperature, washing the surfaces of the working electrode I and the working electrode II by using the Tris-HCl buffer solution, and obtaining an aptamer modified electrode after washing;

(3) adding AFB1 and OTA into a Tris-HCl buffer solution to prepare standard solutions with different concentrations; respectively soaking a plurality of aptamer modified electrodes prepared in the step (2) into standard solutions with different concentrations, and storing for a period of time under a constant temperature condition; then taking out the aptamer modified electrode, immersing the aptamer modified electrode into a mixed solution C consisting of the AFB1 aptamer complementary chain, the OTA aptamer complementary chain and the Tris-HCl buffer solution, and standing for a period of time under a certain temperature condition;

finally, taking out the aptamer modified electricity, placing the aptamer modified electricity in an electrolytic cell, taking a phosphoric acid buffer solution as an electrolyte, and recording electric signals of methylene blue modified by the OTA aptamer complementary chain and ferrocene modified by the AFB1 aptamer complementary chain through a differential pulse voltammetry method; respectively establishing a standard curve according to the relation between the peak current value of ferrocene in a voltammogram and the concentration of AFB1 and the relation between the peak current value of methylene blue and the concentration of OTA;

(4) sample detection: firstly, crushing a sample to obtain sample powder, adding the sample powder into an ethanol solution, fully oscillating, centrifuging, collecting supernate, and mixing with a Tris-HCl buffer solution to obtain a sample extracting solution; and (3) replacing AFB1 and OTA for preparing a standard solution with a sample extracting solution to be detected according to the operation in the step (3) by using the aptamer modified electrode prepared in the step (2), obtaining a voltammetry curve of the sample extracting solution, obtaining peak current values of ferrocene and methylene blue in the voltammetry curve, and substituting the peak current values into the standard curve equation established in the step (3), so that the AFB1 and the OTA in the sample can be detected simultaneously.

2. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A according to claim 1, which is characterized in that the screen-printed electrode is prepared by the following steps: firstly, designing the shape of an electrode by using computer drawing software, and respectively preparing three screen printing plates for printing silver paste, carbon paste and insulating ink by a laser printing method; firstly, silver paste is printed, and after drying, three parallel and mutually independent silver paste patterns are obtained, wherein the silver paste pattern in the middle is Y-shaped, and the silver paste patterns on two sides of the Y shape are strip-shaped; then, printing carbon paste, and drying to obtain three independent carbon paste patterns, wherein one carbon paste pattern is arc-shaped and is connected with the tail end of one strip-shaped silver paste pattern, and the other two carbon paste patterns are oval-shaped and are respectively connected with the two tail ends of the Y-shaped silver paste pattern; finally, printing insulating ink and drying to obtain a rectangular insulating ink pattern, wherein the rectangular insulating ink covers the joint of the carbon paste pattern and the silver paste pattern and part of the silver paste pattern; the drying conditions are all 70-90 ℃, and the drying time is all 10-60 min.

3. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in step (2), in S1, the concentration of the chloroauric acid solution is 0.2-2 mmol/L; the constant potential applied to the working electrode is-3.0 to-0.3V, and the constant potential is kept for 1 to 10 min; the concentration of the sulfuric acid solution is 0.1-1.0 mol/L; the low potential of the cyclic voltammetry scanning is 0-0.2V, the high potential is 1.4-1.6V, the scanning speed is 10-100 mV/s, and the number of scanning cycles is 5-20 cycles.

4. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in step (2) S2, the concentration of the AFB1 aptamer stock solution is 100 μmmol/L, and the concentration of tris (2-carboxyethyl) phosphine hydrochloride is 100 mmol/L; the volume ratio of the AFB1 aptamer stock solution to tris (2-carboxyethyl) phosphine hydrochloride is 10:1, and the mixing reaction time is 30-60 min.

5. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in step (2) S2, the concentration of the OTA aptamer stock solution is 100 μmmol/L, the concentration of tris (2-carboxyethyl) phosphine hydrochloride is 100mmol/L, the volume ratio of the OTA aptamer stock solution to the tris (2-carboxyethyl) phosphine hydrochloride is 10:1, and the mixing reaction time is 30-60 min; the concentration of the Tris-HCl buffer solution is 10-100 mmol/L, and the pH value is 7.4; the concentration of the diluted mixed solution A is 10 mu mol/L; the concentration of the diluted mixed solution B was 10. mu. mol/L.

6. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in step (2) S3, the drop-coating dosage of the diluted mixed solution A is 4-8 μ L; the drop coating dosage of the diluted mixed solution B is 4-8 mu L; the certain temperature condition is 4 ℃, and the standing time is 1-15 h; the concentration of the mercaptohexanol solution is 1-10 mmol/L; the using amount of the mercapto-hexyl alcohol solution which is dripped on the surfaces of the first working electrode and the second working electrode is 4-8 mu L; the constant temperature is 4 ℃, and the preservation time is 0.5-2 h; the concentration of the Tris-HCl buffer solution for washing the surfaces of the first working electrode and the second working electrode is 10mmol/L, and the pH value is 7.4.

7. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in the step (3), the concentration of the Tris-HCl buffer solution is 10-100 mmol/L, and the pH value is 7.4; the concentration range of the standard solution is 1-1000 mug/L; the constant temperature is 37 ℃, and the preservation time is 0.5-2 h; the concentration of the mixed solution C is 10-100 mu mol/L; the certain temperature condition is 37 ℃, and the standing time is 0.5-2 h; the concentration of the phosphoric acid buffer solution is 0.1-0.5 mol/L, and the pH value is 6-9; the low potential of the differential pulse voltammetry is-0.6 to-0.4V, and the high potential is 0.4 to 0.6V.

8. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A in claim 1, wherein in the step (3), the standard curve equation is expressed as: y1 ═ ax1+ b and y2 ═ cx2+ d, where y1 is the concentration of AFB1, μ g/L; y2 is the concentration of OTA, μ g/L; x1 represents ferrocene peak current value, μ A; x2 represents the methylene blue peak current value, μ A; a and c are coefficients of the equation, and b and d are constant terms of the equation.

9. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in the step (4), the dosage ratio of the sample powder to the alcoholic solution is 1-5 g: 5-10 mL; the concentration of the ethanol solution is 80%; the oscillation time is 20-40 min; the volume ratio of the supernatant to the Tris-HCl buffer solution is 1: 8-9.

10. The electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A as claimed in claim 1, wherein in the step (4), the rotating speed of the centrifugation is 3000-6000 r/min, and the centrifugation time is 3-10 min; the concentration of the Tris-HCl buffer solution is 10-100 mmol/L, and the pH value is 7.4.

Technical Field

The invention belongs to the field of biosensors and electrochemical detection, and particularly relates to an electrochemical method for simultaneously detecting aflatoxin B1 and ochratoxin A.

Background

Mycotoxins are secondary metabolites produced by fungi during their growth and have toxic effects on humans and animals. The aflatoxin is mainly a secondary metabolite produced by aspergillus flavus and aspergillus parasiticus, and is a mycotoxin with extremely strong toxicity. Aflatoxins have a damaging effect on liver tissue and can cause liver cancer and even death in severe cases. Aflatoxin B1(Aflatoxin B1, AFB1) is the most common of the aflatoxins contaminating food products, and is also the most toxic and carcinogenic. Ochratoxins are compounds produced by a variety of aspergillus and penicillium species, and there are A, B, C, D types, among which Ochratoxin a (OTA) has the highest contamination rate, contamination level and toxicity.

The detection method of mycotoxin mainly comprises gas chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry combined method and the like. Although these methods can achieve detection of mycotoxins, they require expensive equipment, specialized operators, and complex detection procedures that are difficult to meet with the need for rapid detection in the field. Electrochemical techniques have the advantages of high sensitivity, easy operation, low cost, etc., and have been used for the detection of mycotoxins. At present, the electrochemical detection of mycotoxins mainly aims at one mycotoxin, and the electrochemical detection method for simultaneously detecting two mycotoxins is rarely reported. The invention develops an electrochemical method for simultaneously detecting AFB1 mycotoxins and OTA mycotoxins, and can realize the simultaneous detection of AFB1 mycotoxins and OTA mycotoxins.

Disclosure of Invention

The invention aims to provide an aptamer-based AFB1 and OTA electrochemical simultaneous detection method, which has the advantages of simple operation, high sensitivity, strong specificity and the like, and can be used for detecting mycotoxin in the field of food safety.

In order to achieve the purpose, the screen printing electrode with two working electrodes is prepared through a screen printing technology, gold nanoparticles are modified on the surfaces of the working electrodes by adopting an electrodeposition method, then nucleic acid aptamers of AFB1 and OTA are respectively modified on the surfaces of the two working electrodes in a self-assembly mode, the hybridization of a complementary chain and the aptamers is influenced by utilizing the specific binding of mycotoxin and the aptamers, and the simultaneous detection of AFB1 and OTA is realized according to voltammetric signals of markers at the tail ends of the complementary chain. The detection method provided by the invention takes the voltammetric signals of the markers with different oxidation-reduction potentials as quantitative standards, and simultaneously carries out quantitative determination on the two fungaltoxins.

The electrochemical simultaneous detection method of the AFB1 mycotoxin and the OTA mycotoxin based on the aptamer comprises the following specific steps:

(1) preparing a screen printing electrode, wherein the screen printing electrode comprises a counter electrode, a working electrode I, a working electrode II and a reference electrode;

the preparation method of the screen printing electrode comprises the following specific steps: firstly, designing the shape of an electrode by using computer drawing software, and respectively preparing three screen printing plates for printing silver paste, carbon paste and insulating ink by a laser printing method; firstly, silver paste is printed, and after drying, three parallel and mutually independent silver paste patterns are obtained, wherein the silver paste pattern in the middle is Y-shaped, and the silver paste patterns on two sides of the Y shape are strip-shaped; then, printing carbon paste, and drying to obtain three independent carbon paste patterns, wherein one carbon paste pattern is arc-shaped and is connected with the tail end of one strip-shaped silver paste pattern, and the other two carbon paste patterns are oval-shaped and are respectively connected with the two tail ends of the Y-shaped silver paste pattern; finally, printing insulating ink and drying to obtain a rectangular insulating ink pattern, wherein the rectangular insulating ink covers the joint of the carbon paste pattern and the silver paste pattern and part of the silver paste pattern; the drying conditions are all 70-90 ℃, and the drying time is all 10-60 min.

(2) Preparing an aptamer modified electrode:

s1, firstly, immersing the screen printing electrode prepared in the step (1) into a chloroauric acid solution, applying constant potentials on 2 working electrodes of the screen printing electrode, keeping for a period of time, taking out the screen printing electrode, placing the screen printing electrode in a sulfuric acid solution again, performing cyclic voltammetry scanning, and activating gold nanoparticles to obtain the gold nanoparticle modified screen printing electrode;

s2, mixing the tris (2-carboxyethyl) phosphine hydrochloride solution with AFB1 aptamer stock solution to react to obtain a mixed solution A; mixing the tris (2-carboxyethyl) phosphine hydrochloride solution with OTA aptamer stock solution to react to obtain a mixed solution B; respectively diluting the mixed solution A and the mixed solution B to certain concentrations by using a Tris-HCl buffer solution;

s3, dripping the diluted mixed solution A in the S2 on the surface of the first screen printing electrode modified by the gold nanoparticles obtained in the S1; dripping the diluted mixed solution B in the step S2 on the surface of a screen printing electrode working electrode II soaked in the step S1, standing for a period of time at a certain temperature, washing the surfaces of the working electrode I and the working electrode II by using a Tris-HCl buffer solution, dripping mercaptohexanol solutions on the surfaces of the working electrode I and the working electrode II respectively after washing, storing for a period of time at a constant temperature, washing the surfaces of the working electrode I and the working electrode II by using the Tris-HCl buffer solution, and obtaining an aptamer modified electrode after washing;

(3) electrochemical simultaneous detection of AFB1 and OTA: firstly, adding AFB1 and OTA into a Tris-HCl buffer solution to prepare standard solutions with different concentrations; respectively soaking a plurality of aptamer modified electrodes prepared in the step (2) into standard solutions with different concentrations, and storing for a period of time under a constant temperature condition; then, taking out the aptamer modified electrode, immersing the aptamer modified electrode into a mixed solution C consisting of the AFB1 aptamer complementary chain, the OTA aptamer complementary chain and the Tris-HCl buffer solution again, and standing for a period of time under a certain temperature condition;

finally, taking out the aptamer modified electricity, placing the aptamer modified electricity in an electrolytic cell, taking a phosphoric acid buffer solution as an electrolyte, and recording electric signals of methylene blue modified by the OTA aptamer complementary chain and ferrocene modified by the AFB1 aptamer complementary chain through a differential pulse voltammetry method; respectively establishing a standard curve according to the relation between the peak current value of ferrocene in a voltammogram and the concentration of AFB1 and the relation between the peak current value of methylene blue and the concentration of OTA;

(4) sample detection: firstly, crushing a sample to obtain sample powder, adding the sample powder into an ethanol solution, fully oscillating, centrifuging, collecting supernate, and mixing with a Tris-HCl buffer solution to obtain a sample extracting solution; and (3) using the aptamer modified electrode prepared in the step (2), replacing the standard solution coexisting with AFB1 and OTA with the sample extracting solution to be detected according to the detection program in the step (3), obtaining a voltammetry curve of the sample extracting solution, and substituting peak current values of ferrocene and methylene blue in the voltammetry curve into the standard curve equation established in the step (3), so that the simultaneous detection of AFB1 and OTA in the sample can be realized.

Preferably, in the step (2) S1, the concentration of the chloroauric acid solution is 0.2 to 2 mmol/L; the constant potential applied to the working electrode is-3.0 to-0.3V, and the constant potential is kept for 1 to 10 min; the concentration of the sulfuric acid solution is 0.1-1.0 mol/L.

Preferably, in step (2) S1, the low potential of the cyclic voltammetry scan is 0-0.2V, the high potential is 1.4-1.6V, the scanning speed is 10-100 mV/S, and the number of scanning cycles is 5-20 cycles.

Preferably, in S2 of the step (2), the concentration of the AFB1 aptamer stock solution is 100 mu mmol/L, and the concentration of tris (2-carboxyethyl) phosphine hydrochloride is 100 mmol/L; the volume ratio of the AFB1 aptamer stock solution to tris (2-carboxyethyl) phosphine hydrochloride is 10:1, and the mixing reaction time is 30-60 min.

Preferably, in step (2) S2, the concentration of the OTA aptamer stock solution is 100 μmmol/L, the concentration of tris (2-carboxyethyl) phosphine hydrochloride is 100mmol/L, the volume ratio of the OTA aptamer stock solution to the tris (2-carboxyethyl) phosphine hydrochloride is 10:1, and the mixing reaction time is 30-60 min.

Preferably, in S2 of step (2), the concentration of the Tris-HCl buffer solution is 10 to 100mmol/L, and the pH is 7.4; the concentration of the diluted mixed solution A is 10 mu mol/L; the concentration of the diluted mixed solution B was 10. mu. mol/L.

Preferably, in step (2) S3, the amount of the diluted mixed solution a is 4 to 8 μ L; the drop coating dosage of the diluted mixed solution B is 4-8 mu L; the certain temperature condition is 4 ℃, and the standing time is 1-15 h.

Preferably, in the step (2) S3, the mercaptohexanol solution has a concentration of 1 to 10 mmol/L; the using amount of the mercapto-hexyl alcohol solution which is dripped on the surfaces of the first working electrode and the second working electrode is 4-8 mu L; the constant temperature is 4 ℃, and the preservation time is 0.5-2 h; the concentration of the Tris-HCl buffer solution for washing the surfaces of the first working electrode and the second working electrode is 10mmol/L, and the pH value is 7.4.

Preferably, in the step (3), the concentration of the Tris-HCl buffer solution is 10 to 100mmol/L, and the pH is 7.4; the concentration range of the standard solution is 1-1000 mug/L; the constant temperature is 37 ℃, and the preservation time is 0.5-2 h.

Preferably, in the step (3), the concentration of the mixed solution C is 10-100 [ mu ] mol/L; the certain temperature condition is 37 ℃, and the standing time is 0.5-2 h.

Preferably, in the step (3), the concentration of the phosphoric acid buffer solution is 0.1-0.5 mol/L, and the pH value is 6-9; the low potential of the differential pulse voltammetry is-0.6 to-0.4V, and the high potential is 0.4 to 0.6V.

Preferably, in step (3), the standard curve equation is expressed as: y1 ═ ax1+ b and y2 ═ cx2+ d, where y1 is the concentration of AFB1, μ g/L; y2 is the concentration of OTA, μ g/L; x1 represents ferrocene peak current value, μ A; x2 represents the methylene blue peak current value, μ A; a and c are coefficients of the equation, and b and d are constant terms of the equation.

Preferably, in the step (4), the dosage ratio of the sample powder to the alcoholic solution is 1-5 g: 5-10 mL; the concentration of the ethanol solution is 80%; the oscillation time is 20-40 min; the volume ratio of the supernatant to the Tris-HCl buffer solution is 1: 8-9.

Preferably, in the step (4), the rotating speed of the centrifugation is 3000-6000 r/min, and the centrifugation time is 3-10 min; the concentration of the Tris-HCl buffer solution is 10-100 mmol/L, and the pH value is 7.4.

The DNA sequences involved in the present invention are shown in Table 1:

TABLE 1 DNA sequences according to the invention

The invention has the advantages of

(1) The screen printing electrode with the two working electrodes is prepared, and the aptamer of AFB1 and OTA is respectively modified on the surfaces of the two working electrodes, so that the mutual interference of the two aptamers coexisting on the surface of the same working electrode is avoided.

(2) The detection method provided by the invention can be used for simultaneously detecting two targets and has high sensitivity and high selectivity compared with a single-signal electrochemical sensor by utilizing the specific binding of the aptamer with AFB1 and OTA.

(3) The invention takes the voltammetric signals of the markers with different oxidation-reduction potentials as the quantitative standard, not only can avoid the interference of background substances in a complex environment, but also can realize the simultaneous detection of two targets.

Drawings

FIG. 1 is a flow chart of a screen printing electrode preparation process, wherein 1 is a first working electrode, 2 is a second working electrode, 3 is a reference electrode, and 4 is a counter electrode.

FIG. 2 is a flow chart of the preparation of aptamer-modified electrodes and the simultaneous detection of both AFB1 and OTA mycotoxins; wherein the graph is a voltammogram obtained by electrochemical differential pulse voltammetry.

Detailed Description