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A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst

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Abstract

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B1 (AFB1). It is based on the just-in-time generation of an MnO2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO4) is converted into manganese dioxide (MnO2) which acts as an oxidase mimic that catalyzes the oxidation 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO4. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB1 and magnetic beads carrying bovine serum albumin conjugated to AFB1 are used for the determination of AFB1. In presence of AFB1, it will compete with the BSA-conjugated AFB1 (on the magnetic beads) for the labeled antibody against AFB1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB1 concentrations in the range from 0.1 to 100 ng mL−1, with a 0.1 ng mL−1 detection limit (at the 3Sblank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available.

Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO4)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B1 (AFB1) by coupling with hydrolytic reaction of AOx toward AA and the KMnO4-Mn(II)-TMB system [note: 3,3′,5,5′-tetramethylbenzidine: TMB].

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Acknowledgements

Support by the National Natural Science Foundation of China (Grants No. 21505060), the Outstanding Youth Science Foundation of Fujian Province (Year 2017), the Program for Excellent Talents of Minnan Normal University (Grant No. MJ1601), the Natural Science Foundation of Zhangzhou City, China (Grant No. ZZ2016J30), the National Science Foundation of Fujian Province (Grant No. 2014 J07001).

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Authors and Affiliations

  1. 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 & Maosheng Zhang

  2. Ji’an Vocational Polytechnic College, Ji’an, 343000, People’s Republic of China

    Qiao Zeng

  3. Ministry of Education Key Laboratory of Functional Small Organic Molecule, Department of Chemistry and chemical engineering, Jiangxi Normal University, Nanchang, 330022, People’s Republic of China

    Juan Tang

  4. Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education & Fujian Province), Institute of Nanomedicine and Nanobiosensing, Department of Chemistry, Fuzhou University, Fuzhou, 350108, People’s Republic of China

    Dianping Tang

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  1. Wenqiang Lai
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  2. Qiao Zeng
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  3. Juan Tang
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  4. Maosheng Zhang
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Correspondence to Wenqiang Lai.

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Lai, W., Zeng, Q., Tang, J. et al. A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst. Microchim Acta 185, 92 (2018). https://doi.org/10.1007/s00604-017-2651-z

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  • DOI: https://doi.org/10.1007/s00604-017-2651-z

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