I2/I?-mediated fluorescence quenching of an Ag+-doped gold nanocluster-based immunoassay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is a type of hazardous bacteria in the field of food safety. A sensitive and effective method is urgently needed to detect it, avoiding enormous harm for the human health. In this study, we synthesized stable Ag⁺-doped gold nanoclusters (Ag-AuNC) with a fluorescence intensity 4.8 times stronger than that of AuNC. It was further demonstrated that Ag⁰ existing in the AuNC core and a fraction of Ag⁺ anchored on the AuNC shell eliminated the surface defects and improved the luminescent properties of AuNC. A combination of I₂ and I^? was used to quench fluorescence-enhanced Ag-AuNC, which was first applied in ELISA for detecting E. coli O157:H7 to improve the sensitivity. In the presence of E. coli O157:H7, the biotinylated anti-E. coli O157:H7 mAb and streptavidin-alkaline phosphatase would be immobilized and catalyze l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to produce ascorbic acid. After addition of KIO₃, I₂/I^? were generated. The I₂ could trigger oxidative etching of Ag-AuNC and I^? could combine with Ag⁺ to decrease the Ag⁺ concentration of Ag-AuNC, which resulted in fluorescence quenching of Ag-AuNC. Under optimal conditions, the linear range of I₂/I^?-mediated fluorescence quenching of Ag-AuNC-based immunoassay for detecting E. coli O157:H7 was 3.3 × 10³ to 10⁶ cfu/mL, with a detection limit of 9.2 × 10² cfu/mL, 10.7-fold lower than that of the traditional ELISA. The proposed immunoassay exhibits excellent sensitivity, specificity, recovery, and accuracy, which is useful for quantitative detection of E. coli O157:H7 in food safety.