Proximity Ligation Assay‐induced Structure‐switching Hairpin DNA toward Development of Electrochemical Immunosensor

Abstract

AbstractA novel electrochemical immunosensor was designed for sensitive detection of mycotoxins (aflatoxin B1, AFB1, used in this case) by coupling proximity ligation assay‐induced conformation switch of hairpin DNA with the antigen‐antibody reaction. The assay was carried out by anti‐AFB1 antibody‐conjugated DNA1 (mAb‐DNA1), AFB1‐BSA‐labeled DNA2 (AFB1‐DNA2) and hairpin DNA. Each hairpin included a stem of 6 base pairs and a 6 nucleotide (nt) loop with the labelled ferrocene tag at the 3′ end, which was immobilized on the electrode via self‐assembly of the terminal thiol moiety at the 5′ end. The proximity ligation assay was carried out via the specific antigen‐antibody reaction between mAb‐DNA1 and AFB1‐DNA2 to form an omega‐like DNA junction. Thereafter, the junction hybridized with hairpin DNA to open the probe, thus resulting in the ferrocene tag far away from the electrode for the decreasing of the redox current. Upon target AFB1 introduction, the competitive‐type immunoassay was executed between the analyte and AFB1‐DNA2 for mAb‐DNA1. Under the optimal conditions, the electrochemical signal was indirectly proportional to target AFB1 concentration, and allowed the detection of target AFB1 at a concentration as low as 3.2 pg mL−1. Our strategy also exhibited high selectivity, good reproducibility and precision. Importantly, the accuracy of this methodology was validated for analysis of spiked or naturally contaminated peanut samples, giving results matched well with those obtained from commercialized available AFB1 ELISA kit.