Lysozyme-Functionalized 5-Methyl-2-thiouracil Gold/Silver Nanoclusters for Luminescence Assay of Alkaline Phosphatase

Herein, lysozyme-functionalized 5-methyl-2-thiouracil gold/silver nanoclusters (5-MTU/Lys Au/Ag NCs) with an orange-red fluorescence peak at 600 nm (quantum yield = 33.18%) were obtained through a simple blending route. Owing to the synergistic effect between Au and Ag and the formation of rigid host–guest assemblies between 5-MTU and the guanidine group of lysozymes, the chemical properties of 5-MTU/Lys Au/Ag NCs were significantly improved. Taking advantage of the exceptional optical properties and satisfactory stability of 5-MTU/Lys Au/Ag NCs, a fresh and sensitive fluorescent probe for monitoring alkaline phosphatase (ALP) was strategically constructed by the integration of 5-MTU/Lys Au/Ag NCs and the nanozyme with a graphene structure (Fe-G nanozyme) for the first time. Briefly, the fluorescence of 5-MTU/Lys Au/Ag NCs was initially quenched by indigo carmine (IC) through the inner filter effect (IFE) mechanism, but it was restored upon the introduction of H2O2 and Fe-G nanozyme by generating reactive oxygen species (ROS) (1O2/O2•–) and promoting the oxidative degradation of IC, whereas the fluorescence was quenched again in the presence of ALP via hydrolyzing l-ascorbic acid-2-phosphate (AAP). By monitoring the changes in signal intensity, the proposed fluorescent sensing platform presented a highly selective and excellent performance toward ALP, exhibiting a good linear relationship from 0.5 to 10 U L–1 and providing a low detection limit of 0.193 U L–1. Furthermore, the proposed biosensor showed satisfactory results for quantifying ALP in human serum.