Synthesis of gold nanocluster-loaded lysozyme nanoparticles for label-free ratiometric fluorescent pH sensing: applications to enzyme–substrate systems and cellular imaging

This study developed a simple strategy to prepare lysozyme nanoparticle-encapsulated gold nanoclusters (LysNP-AuNCs) as a dual emission probe for ratiometric sensing of pH variation in enzyme-substrate systems and live cells. The reduction of a gold ion precursor with lysozyme generated lysozyme-stabilized AuNCs (Lys-AuNCs); they were demonstrated to be self-assembled into nano-aggregates during the formation of AuNCs. The aggregated Lys-AuNCs were treated with glutaraldehyde, triggering the conversion of aggregated lysozymes into green-emitting lysozyme nanoparticles (LysNPs) via the formation of C?N bonds. As a result, the AuNCs were well-distributed inside the LysNPs, as demonstrated by transmission electron microscopy and size-exclusion chromatography. The as-prepared LysNP-AuNCs exhibited two separate emission bands, a large Stokes shift, excellent photostability, and salt stability. The pH-induced change in the fluorescence of LysNPs enabled LysNP-AuNCs to ratiometrically detect slight changes (0.2 pH unit) in the pH range from 7.5 to 9.5. Additionally, LysNP-AuNCs were well-suited to probing enzymatic reaction-induced pH changes, which was exemplified by urease-mediated hydrolysis of urea. Under a single excitation, LysNP-AuNCs were used to monitor dual fluorescent images of HeLa cells under different pH conditions.