Colorimetric-fluorescent dual-mode nanosensor-powered enzyme immunoassay for ochratoxin A via alkaline phosphatase-mediated silver nanoparticle growth and fluorescence inner filter effect

Ochratoxin A (OTA) seriously threatens public health and agricultural ecosystems, necessitating sensitive detection methods. In view of that traditional immunoassays rely on animal-derived antibodies and toxic substrates, we developed a dual-mode nanosensor integrating colorimetric and fluorescent detection via alkaline phosphatase (ALP)-mediated silver nanoparticles (AgNPs) growth and inner filter effect (IFE). The Ag + -triggered aggregation-induced emission (AIE) enhancement of gold nanoclusters (AuNCs) was investigated for constructing a dual-mode nanosensor. The nanosensor employs an OTA-specific nanobody-ALP fusion (Nb-ALP) to dephosphorylate ascorbic acid 2-phosphate into ascorbic acid (AA), which reduces Ag⁺ to abundant AgNPs with NaBH 4 -reduced AgNPs seeds. AgNPs amplify plasmonic absorbance for colorimetric ALP detection, while quenching Ag⁺@AuNC fluorescence via IFE for fluorescent detection. The nanosensor was further combined with the Nb-ALP-based immunoassay to develop a dual-mode nanosensor-powered enzyme immunoassay (DMN-EIA). The DMN-EIA exhibited detection limits of 0.14 ng/mL (colorimetric) and 0.28 ng/mL (fluorescent) with high selectivity for OTA, with spike recovery rates of 93.5 %–108.7 % and relative standard deviations not exceeding 20 %. Furthermore, the colorimetric (R 2 = 0.96) and fluorescent DMN-EIA (R 2 = 0.93) correlated well with high-performance liquid chromatography in detection of ten real pepper samples. Therefore, the developed dual-mode nanosensor and DMN-EIA represent reliable and promising tools for detecting OTA.