Intrinsic fluorescence-enhancing bivalent aptamer-assisted ratiometric microsensor coupled smartphone imaging for label-free detection of aflatoxin B1 in food

Sensitive and reliable detection of aflatoxin B1 (AFB1) is critical for food safety monitoring. Herein, we developed an innovative amplification free, dual-mode microsensor integrated with smartphone imaging for visual quantitative detection of AFB1. We used a bivalent fluorescence-enhancing (BFE) aptamer and SYBR Green I (SG I) for a ratiometric fluorescence system. The rationally designed BFE aptamer featured two confined binding pockets that simultaneously achieved high-affinity recognition and intrinsic fluorescence amplification for AFB1. This unique design eliminated the need for complex probe labeling while significantly improving detection stability and sensitivity. SG I further complemented the system by generating a green emission upon binding to the BFE aptamer-AFB1 complex, enabling dual-color visualization and robust ratiometric quantification. To further expand the detection versatility, we constructed a smartphone-based digital platform, allowing on-site testing with favorable accuracy. The optimized method achieved wide detection ranges of 1–200 ng/mL (ratiometric fluorescence mode) and 1−100 ng/mL (visual mode), with detection limit of 0.69 ng/mL and 0.94 ng/mL, high selectivity and satisfied recoveries (94.0 %–108.3 %) in food sample analysis. The integration of target signal self-amplification with smartphone readout represents an advancement in portable food safety control, offering a practical approach for mycotoxin monitoring.