Homogeneous Quenching Immunoassay for Fumonisin B₁ Based on Gold Nanoparticles and an Epitope-Mimicking Yellow Fluorescent Protein

Abstract

Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B₁ (FB₁). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB₁ detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB₁ detection, with a dynamic range from 7.3 to 22.6 ng mL^-1, a detection limit of 1.1 ng mL^-1, and IC₅₀ value of 12.9 ng mL^-1, which was significantly lower than the IC₅₀ value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B₁ and B₂, as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB₁ 2000 μg kg^-1 and 103% for FB₁ 4000 μg kg^-1), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.