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Method for detecting aflatoxin B1 based on visual BPE-ECL technology

A technology for aflatoxin and technical detection, applied in the field of detection of aflatoxin B1 based on visual BPE-ECL technology, can solve the problems of high risk, inaccurate quantification, complicated operation process, etc., and achieve highly sensitive ECL detection and realization Effects of ECL detection and easy arraying

Active Publication Date: 2019-12-03
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the biological identification method mainly includes seed germination test and vomiting test, but this method is not conducive to rapid detection, and can only verify the toxic site and mechanism of toxicity, and can only be used for qualitative analysis, which is rarely used; chemical analysis mainly includes TLC Chromatography, but the operation process of this method is relatively complicated, the sample pretreatment workload is large, and the standard product needs to be directly contacted during the detection process, which has high risk and poor accuracy; immunoassay methods mainly include ELISA method and colloidal gold immunochromatography method, but the reproducibility of this method is poor, it cannot be accurately quantified, the result is prone to false positives, and there are many experimental interference factors (temperature, reaction time); the instrumental analysis method mainly includes high performance liquid chromatography, and the pretreatment of this method is complicated. , the experimental equipment is expensive, and it is impossible to quickly screen a large number of samples

Method used

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  • Method for detecting aflatoxin B1 based on visual BPE-ECL technology
  • Method for detecting aflatoxin B1 based on visual BPE-ECL technology
  • Method for detecting aflatoxin B1 based on visual BPE-ECL technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The method for detecting AFB1 in agricultural products based on visual BPE-ECL technology, the detection steps are:

[0038] 1. Preparation of screen-printed bipolar electrodes: firstly, polyethylene terephthalate (PET) is selected as a cheap electrically inert material as the substrate, and then two working electrode leads are printed with silver ink at both ends of the substrate; then Dry the substrate, print the carbon electrode with carbon paste in the middle of the two working electrodes and dry it to obtain the anode and cathode of the bipolar electrode; then print with photo-curable insulating paste (UV curable insulating ink, etc. The electrode standard layer is cured with ultraviolet light; finally, the electrode insulating layer is printed with light-curable insulating paste and cured with ultraviolet light. The overall length of the screen-printed electrode is about 3 cm, the width is about 1 cm, and the length of the bipolar electrode wire is about 12 mm. A...

Embodiment 2

[0045] The establishment of embodiment 2 standard curve--determination of detection limit and detection range

[0046] Take 10 μL of different concentrations (0, 0.1, 0.5, 1, 5, 10, 20, 50, 100 ng mL -1 ) of AFB1 standard with 10 μL of 100ng mL -1 HRP-AFB1 was mixed and reacted with the monoclonal antibody on the functional sensing interface constructed in Example 1. After airtight incubation at room temperature for 2 hours, after washing with PBS, 20 μL of 0.1M acetic acid / sodium acetate buffer solution (200 mM aniline, 20 mM Hydrogen peroxide, 0.5 μM DNA, pH 4.3), incubate at room temperature for 2 h in the dark. Do not wash, and add 20 μL co-reactant (10mM Ru(bpy) 3 (Cl) 2 ·6H 2 (0, 50 mM TPA), and the bipolar electrode was connected to an electrochemiluminescence analyzer (ECL) with a connector for measurement. Experimental results such as Figure 6 As shown, when the concentration of AFB1 is 0.1-100ng mL -1 Between, the intensity of electrochemiluminescence has a g...

Embodiment 3

[0047] Embodiment 3 specific detection

[0048] Take 7 EP tubes and add HRP-AFB1 and different mycotoxin solutions to each tube to obtain a final concentration of 100ng mL -1 HRP-AFB1 antigen and 100ng mL -1 Mycotoxins, 7 tubes of 100ng mL each -1 HRP-AFB1 antigen and 10ngmL -1 BSA-AFB1, 100ng mL -1 HRP-AFB1 antigen and 10ng mL -1 Aflatoxin M1 (AFM1), 100ng mL -1 HRP-AFB1 antigen and 100ng mL -1 Zearalenone (ZEN), 100ng mL -1 HRP-AFB1 antigen and 100ng mL -1 Ochratoxin A (OTA), 100ng mL -1 HRP-AFB1 antigen and 100 ng mL -1 Deoxynivalenol (DON), 100ng mL -1 HRP-AFB1 antigen and 100ng mL -1 Patulin, 20 μL was added to the functional sensing interface (the cathode end of the bipolar electrode) constructed in Example 1, and after the competition reaction was carried out for 2 hours, after washing with PBS, 20 μL, 0.1M acetic acid / Sodium acetate buffer solution (200 mM aniline, 20 mM hydrogen peroxide, 0.5 μM DNA, pH 4.3), incubated at room temperature for 2 h in the ...

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Abstract

The invention discloses a method for detecting aflatoxin B1 based on the visual BPE-ECL technology. The method comprises the following steps: 1) preparing a screen-printed bipolar electrode; 2) constructing a function sensing interface; 3) mixing AFB1 with unknown concentration and HRP-AFB1 with fixed concentration, and then, reacting with an AFB1 monoclonal antibody on the function sensing interface; and 4) detecting an electrochemiluminescence signal on a signal acquisition interface by using a bipolar electrode working principle. An electrochemical signal of chemical reaction is converted into an electrochemiluminescence signal capable of being sensitively detected by utilizing BPE, so that the problem that Faraday current and charging current cannot be distinguished by electrochemistryis solved; the function sensing interface and the signal acquisition interface are physically isolated by utilizing the BPE, so that direct contact between photoactive molecules and a complex reaction system is avoided, the false positive phenomenon is effectively inhibited, the analysis and detection range is expanded, and the detection method is simplified; and the method has the advantages ofbeing simple and convenient and good in sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of biosensing technology, and relates to a method for detecting aflatoxin B1 based on visualized BPE-ECL technology, in particular to a monoclonal antibody functionalized modification onto the surface of gold nanoparticles at the cathode end of a screen-printed bipolar electrode, a competition method Recognizing and binding to AFB1 or HRP-AFB1 causes changes in the degree of aniline polymerization at the cathode end of the bipolar electrode, and changes in luminescence intensity and luminescence potential are observed on an electrochemiluminescence analyzer, thereby quantitatively detecting AFB1 in agricultural products. Background technique [0002] Mycotoxins refer to the toxic secondary metabolites produced during the growth and reproduction of Aspergillus, Fusarium or Penicillium. There are many kinds of them. More than 400 species have been isolated and identified, mainly including Mycotoxins / alcohols, etc., myco...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N27/327G01N27/48G01N33/543G01N33/577
CPCG01N21/76G01N27/3277G01N27/48G01N33/5438G01N33/577
Inventor 刘元建李亚飞熊晓辉
Owner NANJING UNIV OF TECH
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