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

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.