Ratiometric Fluorescent Biosensing of Hydrogen Peroxide and Hydroxyl Radical in Living Cells with Lysozyme-Silver Nanoclusters: Lysozyme as Stabilizing Ligand and Fluorescence Signal Unit.

Construction of facile ratiometric fluorescent probes which possess sensitive and selective sensing ability for bioactive small molecules is highly desirable and challenging. Herein, silver nanoclusters capped with denatured lysozyme (dLys-AgNCs) were synthesized and proved to be dual emissive. The facility of the dLys-AgNCs ratiometric probe was attributed to the finding that the lysozyme acted not only as stabilizing ligand but also as fluorescence signal unit. In the presence of Fenton reagents, the emission of dLys-AgNCs at 640 nm was quenched by •OH, whereas the emission at 450 was enhanced due to •OH-induced oxidation of tyrosine in the lysozyme. This probe could be used for highly sensitive detection of H2O2. The fluorescence changes of F450/F640 had fantastic linearity to H2O2 concentrations in the range of 0.8-200 µmol/L (R2 = 0.9993), with a limit of detection (LOD) as low as 0.2 µmol/L. Additionally, this probe was also applied to H2O2-generated oxidase-based biosensing. As a proof-of-concept, glucose and acetylcholine chloride were detected with benefical LOD values of 0.6 µmol/L and 0.8 µmol/L, respectively. Furthermore, fluorescence confocal imaging demonstrated dLys-AgNCs had a sensitive response to fluctuation of •OH levels in living cells, which might have promising application in study of •OH-induced oxidative damage to proteins.