Binding-Induced DNA Dissociation Assay for Small Molecules: Sensing Aflatoxin B1

We describe a new fluorescence turn-on sensor for homogeneous detection of aflatoxin B1 (AFB1), a potent low molecular weight mycotoxin. A key innovation is the binding-induced intramolecular interaction involving the following two sets of probes: (1) a gold nanoparticle (AuNP) immobilized with hundreds of assistant oligonucleotides (AO) and dozens of anti-AFB1 monoclonal antibodies, and (2) the AFB1-BSA (BSA = bovine serum albumin) antigen conjugated with fluorophore-labeled signal oligonucleotides (SO) that contained a short sequence complementary to AO. Specific binding of AFB1-BSA to the antibody brought the fluorophore very close to the surface of the AuNP through a stable intramolecular hybridization between AO and SO, resulting in efficient quenching of fluorescence. The improved fluorescence quenching substantially reduced the background, due to the binding-induced intramolecular hybridization, and improved the signal-to-background ratio by 390%. In the presence of AFB1 in a sample, competitive binding of AFB1 in the sample to the antibodies immobilized on the AuNP caused the release of the fluorophore-labeled AFB1-BSA from the AuNP, turning on fluorescence. A detection limit of 2.3 nM was achieved, which meets the requirement for AFB1 detection at regulatory levels. Analyses of rice samples using this assay showed recoveries of 86–102%. Incorporating appropriate antibody probes could extend the assay to the detection of other small molecules.