Ratiometric fluorescence nanosensor-driven dual-mode enzyme immunoassay based on 9-anthraldehyde and mimotope peptide-alkaline phosphatase fusion for fluorescent and visual detection of ochratoxin A in pepper

Rapid and sensitive monitoring of ochratoxin A (OTA) in agricultural products can minimize its threats to public health. This study introduces a ratiometric fluorescence nanosensor-driven dual-mode enzyme immunoassay (RFN-DEIA) for fluorescent and visual detection of OTA. Herein, a bifunctional mimotope peptide-alkaline phosphatase fusion protein (MP-ALP) was utilized to compete for binding to the OTA-specific nanobody and dephosphorylate ascorbyl 2-phosphate. The connection between the MP-ALP-based immunoassay for OTA and the 9-anthraldehyde-based ratiometric fluorescence nanosensor for MP-ALP was facilitated using MnO 2 nanosheets. The optimized RFN-DEIA has a visual limit of detection (LOD) of 2.75 ng/mL and an instrumental LOD of 1.50 ng/mL. It exhibits high selectivity for OTA, and good recoveries of 80.66%–117.18% with relative standard deviations not exceeding 9.5% in recovery experiments. Furthermore, the RFN-DEIA was applied to detect OTA in real pepper samples and validated by high-performance liquid chromatography, demonstrating its effectiveness and reliability for OTA detection in pepper samples.