Inhibition of Serine Protease−Mediated Digestion of Gold Nanoclusters Enables Dual-Emission Fluorescence Detection of Aflatoxin B1

Abstract Given the significant health risks posed by aflatoxins in food and agricultural products, there is an urgent need for rapid and reliable detection methods. Conventional approaches often rely on antibody−antigen interactions and immunoaffinity columns, which can be expensive and limit widespread adoption in laboratories. To overcome these drawbacks, this study introduces a simple, dual-emission fluorescence biosensor for the detection of aflatoxin B1 (AFB1) without needing antibodies, aptamers and labels. The biosensor employs bovine serum albumin−stabilized gold nanoclusters (BSA−AuNCs) as fluorescent probes. Its operation relies on a serine protease that digests the BSA shell: proteolysis disassembles the BSA−AuNCs complex, and quenching the 650 nm red emission. Meanwhile, blue emission at 450 nm arises from the protease’s intrinsic fluorescence, primarily from tryptophan and tyrosine residues, enabling ratiometric detection. In the presence of AFB1, as a reversible competitive inhibitor, protease activity is inhibited, the BSA coating remains intact, and the AuNCs’ 650 nm fluorescence is recovered, while the enzyme’s emission remains unchanged. The resulting change in the F650/F450 emission ratio enables both visual and quantitative detection. The biosensor detects AFB1 over a linear range of 5.0−100.0 ng/mL, with a limit of detection of 2.0 ng/mL (S/N = 3), and delivers results within 20 min. This assay produces an immediate color shift, purple for samples without AFB1 and red for samples containing 100.0 ng/mL AFB1; lower AFB1 concentrations yield pink emission, enabling straightforward visual readout. The biosensor’s operation, based on the enzymatic digestion of BSA-AuNCs by serine protease and its inhibition by AFB1, was thoroughly characterized using UV−Vis spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, Fourier-transform infrared spectroscopy (FTIR), SDS-PAGE, and X-ray diffraction (XRD). Tested in oil-rich food matrices (e.g., corn, almonds, and sesame seeds), this approach offers a rapid, sensitive, and cost-effective alternative for AFB1 monitoring in food safety applications.