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Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification

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Abstract

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuff. The system consisted of anti-AFB1 antibody labeled DNA1 (Ab-DNA1), AFB1–bovine serum albumin (BSA)-conjugated DNA2 (AFB1-DNA2), and methylene blue functionalized hairpin DNA. Owing to a specific antigen–antibody reaction between anti-AFB1 and AFB1–BSA, the immunocomplex formed assisted the proximity hybridization of DNA1 with DNA2, thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB1, the analyte competed with AFB1-DNA2 for the conjugated anti-AFB1 on the Ab-DNA1, subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB1 in a dynamic working range of 0.01–30 ng mL-1 with a detection limit of 4.8 pg mL-1. In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB1-spiked peanut samples, giving results that matched well with those obtained with a commercial AFB1 ELISA kit.

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Acknowledgments

Support by the National Natural Science Foundation of China (grant nos. 21505060, 41176079, and 21475025), the National Science Foundation of Fujian Province (grant no. 2014J07001), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT15R11) is gratefully acknowledged.

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Authors and Affiliations

  1. Key Laboratory of Functional Small Organic Molecule, Ministry of Education, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, China

    Juan Tang, Yapei Huang, Huiqiong Liu & Cengceng Zhang

  2. 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, China

    Dianping Tang

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  1. Juan Tang
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  2. Yapei Huang
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  3. Huiqiong Liu
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Correspondence to Juan Tang or Dianping Tang.

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Published in the topical collection Isothermal Nucleic Acid Amplification in Bioanalysis with guest editor Maria Jesus Lobo Castañón.

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Tang, J., Huang, Y., Liu, H. et al. Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification. Anal Bioanal Chem 408, 8593–8601 (2016). https://doi.org/10.1007/s00216-016-9343-0

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  • DOI: https://doi.org/10.1007/s00216-016-9343-0

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