Correlating Synthesis Conditions with Structural Evolution and Near-Infrared Emission in Glutathione-Capped Gold Nanoclusters

Fluorescence imaging in the near-infrared (NIR) region has attracted increasing attention in biomedical research owing to its deep tissue penetration, reduced autofluorescence, and improved imaging contrast compared with visible-light imaging. Among NIR fluorescent probes, gold nanoclusters (AuNCs) are particularly attractive because of their size-dependent emission, excellent photostability, biocompatibility, and versatile surface chemistry. Here, we systematically investigate how reaction pH, HAuCl₄ concentration, and temperature influence the optical properties and structural evolution of glutathione-capped gold nanoclusters (GSH-AuNCs). The combined modulation of these parameters enables fine control over the emission characteristics, driving a transition from visible to NIR fluorescence. Near-neutral pH, elevated HAuCl₄ concentration, and higher reaction temperatures promoted the formation of NIR-emissive clusters. Under optimal conditions (95 °C, C _(Au) = 26 mM, and 80 μL of 1 M NaOH), GSH-AuNCs exhibited a single emission peak centered at 825 nm under 365 nm excitation, with a fluorescence intensity approximately 7.2-fold higher than that of conventional 640 nm-emissive GSH-AuNCs after absorption correction. Time-resolved fluorescence measurements revealed differences in excited-state dynamics among the differently emissive GSH-AuNCs, while circular dichroism spectroscopy showed systematic changes in ligand-metal coordination and Au-(I)-thiolate shell organization accompanying the emission red shift. Zeta potential and density measurements indicated precursor concentration-dependent changes in surface charge and structural compactness, suggesting that metal core growth and ligand-shell reorganization evolve in a partially decoupled manner. X-ray photoelectron spectroscopy confirmed similar elemental compositions and gold oxidation states across the differently emissive GSH-AuNCs, whereas increased Au/S ratios together with larger particle sizes determined by small-angle X-ray scattering evidenced progressive gold core growth with increasing precursor concentration. These complementary results show that the transition from red to NIR emission arises from the coupled evolution of gold core nuclearity, ligand-shell organization, and metal-ligand electronic interactions. To demonstrate biomedical applicability, GSH-AuNCs were functionalized with the SYL3C aptamer for selective detection of EpCAM-expressing PC3 cancer cells. Flow cytometry confirmed selective cellular recognition, highlighting the potential of these NIR nanoprobes for targeted cancer imaging. Overall, these findings provide a framework for engineering GSH-AuNCs with tailored structural and photophysical properties for biomedical imaging, biosensing, and image-guided therapeutic applications.