Fluorescein-5-isothiocyanate-conjugated protein-directed synthesis of gold nanoclusters for fluorescent ratiometric sensing of an enzyme-substrate system

This study describes the synthesis of a dual emission probe for the fluorescent ratiometric sensing of hydrogen peroxide (H2O2), enzyme activity, and environmental pH change. Green-emitting fluorescein-5-isothiocyanate (FITC) was conjugated to the amino groups of bovine serum albumin (BSA). This FITC-conjugated BSA acted as a template for the synthesis of red-emitting gold nanoclusters (AuNCs) under alkaline conditions. Under single wavelength excitation, FITC/BSA-stabilized AuNCs (FITC/BSA-AuNCs) emitted fluorescence at 525 and 670 nm, which are sensitive to changes in solution pH and H2O2 concentration, respectively. The effective fluorescence quenching of AuNCs by H2O2 enabled FITC/BSA-AuNCs to ratiometrically detect the H2O2 product-related enzyme system and its inhibition, including glucose oxidase-catalyzed oxidation of glucose, acetylcholinesterase/choline oxidase-mediated hydrolysis and oxidation of acetylcholine, and paraoxon-induced inhibition of acetylcholinesterase activity. When pH-insensitive AuNCs were used as an internal standard, FITC/BSA-AuNCs offered a sensitive and reversible ratiometric sensing of a 0.1-pH unit change in the pH range 5.0-8.5. The pH-induced change in FITC fluorescence enabled FITC/BSA-AuNCs to detect an ammonia product-related enzyme system. This was exemplified with the determination of urea in plasma by urease-mediated hydrolysis of urea.