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Rapid detection method for aflatoxin B1

A technology for the detection of aflatoxin and aflatoxin B1 is applied in the field of rapid detection of aflatoxin B1, which can solve the problems of cumbersome sample pretreatment procedures, expensive instruments, and low quantum yield, and is suitable for popularization and application. highly targeted effect

Active Publication Date: 2019-02-05
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of AFB1 mainly include: chromatography, enzyme-linked immunosorbent assay, inductively coupled plasma mass spectrometry and capillary electrophoresis, etc. Among them, chromatography is the most routine analysis for qualitative / quantitative detection of food chemical pollutant aflatoxin AFB1 technologies, including high performance liquid chromatography / mass spectrometry, liquid chromatography / mass spectrometry, reversed-phase high performance liquid chromatography, thin-layer chromatography, and gas chromatography coupled with mass spectrometry, etc., but cumbersome sample pretreatment procedures and expensive instruments are chromatography main problem
Methods such as enzyme-linked immunosorbent assay, inductively coupled plasma mass spectrometry and capillary electrophoresis have the problems of complex operation and time-consuming detection
Fluorescence method is used to detect food chemical pollutants, which has the advantages of simple operation, rapid automatic detection, etc. In recent years, fluorescent biosensors have been applied to the detection of aflatoxins. Although this method shows high sensitivity, it still has quenching. Difficult stripping of reagents, difficult preparation of single-layer nanomaterials, low quantum yield, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Preparation of DNA-CDs complex

[0054] (1) Synthesis of amino-functionalized aptamer DNA:

[0055] The amino functionalized aptamer DNA was synthesized by conventional methods, and its sequence is: NH 2 - AAA AAA AAG TTG GGCACG TGT TGT CTC TCT GTG TCT CGT GCC CTT CGC TAG GCC CAC AC (SEQ ID NO. 1).

[0056] (2) Preparation of carbon dots (CDs):

[0057] Citric acid (1.0507 g) and ethylenediamine (335 μL) were dissolved in 10 mL of deionized water and stirred for 30 minutes, then transferred to a hydrothermal reactor (23 mL) and heated at 200 °C for 5 hours, then cooled naturally, A brown solution was obtained. The brown solution was purified by a dialysis membrane (1000MWCO) for 24 hours to remove small molecular substances in the solution, and freeze-dried by a freeze dryer to obtain dry carbon dot CDs.

[0058] (3) DNA-CDs complex preparation:

[0059] DNA-CDs complexes were prepared by cross-linking CDs and amino-functionalized DNA through EDC / NHS (ED...

Embodiment 2

[0060] Example 2: Drawing of AFB1 Detection Linear Equation

[0061] (1) Dissolve the DNA-CDs complex (prepared in Example 1) in sodium chloride solution (200 mM), make the concentration of the DNA-CDs complex 0.07 mM, adjust the pH to 7 with phosphate buffer, and incubate for 30 min, Under the excitation of 360nm light, measure its fluorescence intensity F 1 Add humic acid to make the concentration of humic acid 0.16mg / ml, react for 3min, measure its fluorescence intensity F under the excitation of 360nm light 0 ;

[0062] (2) After the reaction, add a series of concentration gradients (0.1ng / ml, 0.2ng / ml, 0.3ng / ml, 0.4ng / ml, 0.5ng / ml, 0.6ng / ml, 0.7ng / ml, 0.8ng / ml ml) AFB1 standard substance, under the excitation of 360nm light, measure its fluorescence intensity F 2 ; AFB1 fluorescence quenching rate I% is calculated according to the formula:

[0063] I%=(F 2 –F 0 ) / (F 1 -F 0 ), where F 0 Indicates the fluorescence intensity under 360nm excitation light when humic a...

Embodiment 3

[0066] Embodiment 3: the detection of aflatoxin B1 in the sample

[0067] (1) Dissolve the DNA-CDs complex (prepared in Example 1) in sodium chloride solution (200 mM), make the concentration of the DNA-CDs complex 0.07 mM, adjust the pH to 7 with phosphate buffer, and incubate for 30 minutes , Measure its fluorescence intensity F under the excitation of 360nm light 1 Add humic acid to make the concentration of humic acid 0.16mg / ml, react for 3min, measure its fluorescence intensity F under the excitation of 360nm light 0 ;

[0068] (2) Add the peanut oil sample to be tested after the reaction, and measure its fluorescence intensity F under the excitation of 360nm light 2 ; AFB1 fluorescence quenching rate I% is calculated according to the formula:

[0069] I%=(F 2 –F 0 ) / (F 1 -F 0 ), where F 0 Indicates the fluorescence intensity under 360nm excitation light when humic acid is added. f 1 Indicates the fluorescence intensity under 360nm excitation light without addin...

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Abstract

The invention discloses a rapid detection method for aflatoxin AFB1. According to the rapid detection method for aflatoxin AFB1, a DNA-CDs composite is prepared in a simple and green synthesis method,a low-cost novel fluorescent detection system for the aflatoxin is constructed based on the [pi]-[pi] stacking interaction between the humic acid and the DNA-CDs, the DNA-CDs is taken as a fluorescent probe, and the fluorescence of the AFB1 is directly utilized, so that the efficient and sensitive detection for the aflatoxin is achieved. This is the first time to use the readily available humic acid as a fluorescent quenching material for toxin detection. The rapid detection method for aflatoxin AFB1 is simple and convenient to operate, high in targeting property and sensitivity, and low in cost.

Description

technical field [0001] The invention relates to the technical field of food detection, in particular to a rapid detection method for aflatoxin B1. Background technique [0002] Aflatoxin is a kind of derivative of dihydrofuranocoumarin produced by Aspergillus flavus and Aspergillus parasiticus. It is a common mycotoxin with teratogenic, mutagenic and carcinogenic effects. It is classified as I by the World Health Organization Carcinogens. Aflatoxins mainly include aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Among them, AFB1 is widely polluted and the most toxic, which seriously endangers people. health and food safety. Therefore, the development of highly sensitive AFB1 detection methods has become the focus of international attention. [0003] At present, the detection methods of AFB1 mainly include: chromatography, enzyme-linked immunosorbent assay, inductively coupled plasma mass spectrometry and capillary electrophoresis, etc...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 李向阳李宗益郭曼莉鲁金祥
Owner SHANDONG AGRICULTURAL UNIVERSITY
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