Gold nanoclusters-mediated enzyme cascade-amplified immunoassay for dual-mode sensing of aflatoxins

Aflatoxins still pose severe risks to public health due to their high toxicity and carcinogenicity. Herein, we present a novel dual-mode enzyme cascade-amplified immunoassay (ECAIA) for the ultrasensitive and reliable detection of aflatoxins. In this strategy, a nanobody-alkaline phosphatase fusion protein (VHH2-5-AP) was expressed and served as antigen surrogate to compete binding with aflatoxins towards antibody. Bovine serum albumin-gold nanoclusters (BSA-Au NCs) was synthesized and applied as a peroxidase mimic, catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) into its oxidized form (ox TMB), leading to a reduction in fluorescence due to the inner filter effect. VHH2-5-AP could hydrolyzes L-ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), thus preventing TMB oxidation and restoring BSA-Au NCs' fluorescence. In the presence of aflatoxins, less VHH2-5-AP would be captured, leading to oxTMB accumulation and fluorescence quenching. The proposed ECAIA achieved a limit of detection (LOD) of 0.04 ng/mL for colorimetric detection and 0.24 ng/mL for fluorescence detection with high selectivity for aflatoxins. Moreover, the assay exhibited excellent performance in complex food matrices, achieving recoveries between 84% and 116%, which underscores its considerable potential for practical applications in food safety monitoring.