Ratiometric Fluorescence Sensing of Aflatoxin B1 Using a Cationic Perylene Probe and Rhodamine B

As one of the most toxic and prevalent mycotoxins, aflatoxin B₁ (AFB₁) poses a persistent threat to food safety and public health due to its widespread contamination of cereal commodities and its well-documented carcinogenic, genotoxic, and hepatotoxic effects. Although fluorescence sensing provides a rapid approach to AFB₁ screening, most existing fluorescence strategies rely on antibodies or aptamers, which are costly, laborious, and susceptible to environmental degradation; moreover, single-emission readouts remain vulnerable to variations in probe concentration, excitation conditions, and sample background. Herein, we developed a simple two-fluorophore, antibody- and aptamer-free ratiometric fluorescence system that pairs the AFB₁-responsive perylene derivative PTHA with Rhodamine B (RhB) as a reference fluorophore to afford a single-excitation, dual-emission readout. The system produced a concentration-dependent ratiometric signal over the linear range of 1-20 µg mL^-1, with an estimated statistical detection limit of 0.0165 µg mL^-1. It produced a substantially greater response to AFB₁ than to the tested mycotoxins and other potential interferents, indicating preliminary discrimination among the substances examined. The absorption changes and nearly unchanged fluorescence lifetime were consistent with predominantly static or static-like fluorescence attenuation involving ground-state association and possible redistribution of PTHA toward weakly emissive aggregated states. The performance of the method was evaluated in AFB₁-spiked rice, maize, and millet extracts, yielding recoveries of 91.5-109.0% and relative standard deviations of 1.2-3.6%. These results support the proof-of-concept use of the PTHA/RhB system for ratiometric determination of relatively high AFB₁ levels in pretreated cereal extracts.