A fluorescence signal amplification strategy for modification-free ratiometric determination of tyrosinase in situ based on the use of dual-templated copper nanoclusters

A fluorescence resonance energy transfer (FRET)-based in situ fluorescence signal amplification strategy is described for the determination of tyrosinase (TYR). In this assay, a dual-templated copper nanocluster (CuNCs) stabilized by bovine serum albumin (BSA) and glycylglycine (Gly-Gly) was used as an energy donor. Metyrosine was employed as a TYR substrate because its enzyme catalytic product (methyldopa) was able to function as a monomer molecule to form fluorescent polymethyldopa (PMeDP) with the assistance of BSA/Gly-Gly CuNCs. In this process, PMeDP can combine with BSA/Gly-Gly CuNCs without extra modification and then acts as an energy receptor, which leads to a remarkable FRET from BSA/Gly-Gly CuNCs to PMeDP. Interestingly, the fluorescence intensity of PMeDP was strengthened greatly in the FRET-based sensor compared to the separate excitation, which provided good sensitivity for TYR sensing. Illuminated under a UV light source, the fluorescence signal change is observed from dark violet to bright green. Therefore, the present sensing system affords a reliable ratiometric assay for TYR determination. Also, the ratio of fluorescence intensity between PMeDP ( λ em at 505 nm, F505) and BSA/Gly-Gly CuNCs ( λ em at 415 nm, F415) was used for quantitative determination of TYR. The sensing system was easily operated in aqueous media with an exciting detection limit of 44.0 U L −1 . This sensing strategy has been applied to the screening of inhibitors. Schematic representation of the strategy for the determination of tyrosinase.