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Competitive photometric and visual ELISA for aflatoxin B1 based on the inhibition of the oxidation of ABTS

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Abstract

This work reports on a sensitive colorimetric immunoassay for aflatoxin B1 (AFB1) in foodstuff. The reagent L-ascorbic acid 2-phosphate (AAP) is added to the system, and alkaline phosphatase (ALP) hydrolyzes AAP under formation of ascorbic acid and phosphate. The ascorbic acid produced reduces chloroauric acid to form zero-valent gold (in the form of nanoparticles). Hence, Au(III) is no longer available to oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) to form a green product. Rather, the solution remains colorless. By using ALP-labelled monoclonal anti-AFB1 antibody, a competitive enzyme-label immunoassay was developed for AFB1 in a microplate coated with the AFB1-BSA conjugate. Under optimal conditions, the absorbance of the solution at 415 nm increases linearly with increasing AFB1 concentration in range from 10 pg·mL−1 to 100 ng·mL−1 (while the color gradually turns to green), and the detection limit is 7.8 pg·mL−1. The precision of the method (expressed as RSD) is ±9.7%. The accuracy was validated by analyzing both naturally contaminated and spiked food samples, and the results matched the results obtained by ELISA very well.

Schematic of a colorimetric aflatoxin B1 immunoassay. Antibody is labeled with alkaline phosphatase which catalyzes the hydrolysis of ascorbic acid 2-phosphate (AAP) to form ascorbic acid which reduces chloroauric acid to form gold nanoparticles. Hence, Au(III) is no longer available to oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) to form a green product.

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Acknowledgements

Support by the National Natural Science Foundation of China (21475025), the People’s Livelihood Science and Technology Innovation Project of Chongqing City (cstc2016shmszx20001), and the Advanced Natural Science Foundation of Chongqing City (cstc2015jcyjBX0126) is gratefully acknowledged.

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Authors and Affiliations

  1. Chongqing Key Laboratory of Environmental Materials & Remediation Technologies, Chongqing University of Arts and Sciences, Chongqing, 402160, People’s Republic of China

    Ying Tang, Jin Zhang & Dianping Tang

  2. Fujian Provincial Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, People’s Republic of China

    Wenqiang Lai

  3. MOE Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, 350116, People’s Republic of China

    Wenqiang Lai & Dianping Tang

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  1. Ying Tang
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  2. Wenqiang Lai
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  3. Jin Zhang
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  4. Dianping Tang
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Correspondence to Dianping Tang.

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Tang, Y., Lai, W., Zhang, J. et al. Competitive photometric and visual ELISA for aflatoxin B1 based on the inhibition of the oxidation of ABTS. Microchim Acta 184, 2387–2394 (2017). https://doi.org/10.1007/s00604-017-2268-2

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