Bifunctional gold clusterzymes with surface-engineered catalytic triad design for selective dual-signal detection of p-nitrophenyl organophosphates for food safety

Selective detection of highly toxic p -nitrophenyl organophosphates (pNOPs) is of great importance for food safety and environmental risk assessment. Herein, peptide-coated bifunctional gold clusterzymes (AuCEs) that integrate hydrolase-like and peroxidase-like activities are developed via a surface-engineered catalytic triad strategy to achieve rapid, selective, and dual-mode colorimetric/fluorescent detection of pNOPs. The fluorescence signal is generated by the selective hydrolysis of pNOPs into p -nitrophenol (pNP), which interacts with AuCEs and quenches their fluorescence. For the colorimetric signal, pNOPs inhibit the multi-enzyme cascade system of acetylcholinesterase (AChE) and choline oxidase (ChOx) to reduce H 2 O 2 production and thereby affect the AuCEs-mediated oxidation of 3,3,5,5-tetramethyl-benzidine (TMB) into a blue product. This dual-signal detection method offers a linear fluorescence detection range from 6 to 540 ng/mL with a detection limit of 4 ng/mL (ppb), and a linear colorimetric detection range from 8 to 480 ng/mL with a detection limit of 1.08 ng/mL (ppb). Real food sample testing demonstrates that this dual-signal detection not only exhibits excellent selectivity for the p -nitrophenyl group, but also integrates self-calibration to enhance the accuracy of pNOPs detection.