Thiamine-regulated fluorescence based on the oxidase-mimicking activity of BSA-MnO2 nanosheets for the detection of β-galactosidase and Escherichia coli

Specific-Species enzyme detection serves as a crucial method in foodborne pathogen surveillance. Particularly, endogenous β-galactosidase (β-Gal) enzyme has been widely applied to determine the concentration of Escherichia coli ( E. coli ). The main challenges include reducing analysis time and improving detection sensitivity. A novel strategy for biosensing β-Gal activity and E. coli are developed based on BSA‑manganese dioxide nanosheets (BSA-MnO 2 NS) with the oxidase-mimicking activity to regulate the fluorescence of thiamine. BSA-MnO 2 NS can catalyze the oxidation of non-fluorescent thiamine to generate strong fluorescent dehydrothiamine. When β-Gal enzymes is introduced, p -aminophenyl-β-D-galactopyranoside (PAPG) could be hydrolyzed to form para-aminophenol (PAG), which efficiently reduced BSA-MnO 2 NS into Mn 2+ , resulting in the loss of their oxidase-like activity and the decrease of dehydrothiamine fluorescence. Since endogenous β-Gal in E. coli cells can directly hydrolyze PAPG without destroying bacteria, which greatly reduced the analysis time. The reduction product PAG leads to MnO 2 NS decomposition and thus reduced dehydrothiamine fluorescence. According to this mechanism, fluorescence biosensor for β-Gal activity and E. coli are constructed utilizing thiamine luminescence modulated by BSA-MnO 2 NS, without requiring an additional oxidizer. β-Gal and E. coli showed good linearity in the range of 0.01–2.0 μg/mL and 1.0 × 10 2 –1.0 × 10 4  CFU/mL, and the detection limits reached 8.55 ng/mL and 89.45 CFU/mL, respectively. The proposed enzyme-nanozyme cascade reaction significantly improved the sensitivity. It is highly specific and can be used to distinguish E. coli from other pathogenic bacteria. The strategy has also been successfully applied to the detection of E. coli in real samples.