Au/Ag Bimetallic Nanoclusters Stabilized by Glutathione and Lysozyme for Ratiometric Sensing of H₂O₂ and Hydroxyl Radicals

Metal nanoclusters, widely used as fluorescent probes in biosensing and bioimaging, still suffer from some barriers, such as low quantum yield, impurities, and poor sensitivity. Developing nanocluster-based fluorescent probes with good performance and a facile separation method for the synthesized nanoclusters to improve the sensing ability is of great importance. Herein, we reported a convenient approach to purify Au/Ag bimetallic nanoclusters stabilized by dual ligands of GSH and lysozyme (GSH-Lys@Ag-AuNCs) via isoelectric point precipitation, and the purified GSH-Lys@Ag-AuNCs exhibited an improved sensing sensitivity and linear range to the Fenton reagent (H₂O₂ + Fe²⁺). The fluorescence intensity ratio of F₄₄₀/F₆₄₀ was linear to the H₂O₂ concentration over the wide range of 0.2–500 μmol L^–1 (R² = 0.97), with a low detection limit of 0.05 μmol L^–1. Utilizing the catalytic oxidation reaction of the substrate under a specific oxidase by producing H₂O₂, the probe was further employed to glucose and uric acid assays with satisfactory results in human serum samples. Additionally, fluorescence confocal imaging experiments were performed to monitor the fluctuation of the ^•OH level in living cells, and the fluorescence intensity ratio of the blue channel to the red channel showed excellent ratiometric changes with the ^•OH contents.