Elucidating Silver Nanocluster-Protein Binding Mechanism: From Ensemble Measurements to Single-Particle Dynamics

With an aim to understand protein interactions of ultrasmall nanomaterials, this study investigates whether ultrasmall silver nanoclusters (AgNCs) interact with proteins differently from the classical protein corona observed for larger metal nanoparticles. To address this question, the interaction between glutathione-capped silver nanoclusters (GSH-AgNCs) and bovine serum albumin (BSA) is systematically investigated using a combination of ensemble-averaged and single-particle fluorescence techniques. AgNCs are synthesized and characterized, followed by investigation of their interaction with BSA using steady-state fluorescence spectroscopy, ζ-potential measurements, and isothermal titration calorimetry. These ensemble-averaged techniques have consistently revealed that the BSA-to-AgNCs binding stoichiometry remains below the threshold (BSA/AgNCs > 1) required for the formation of a stable protein corona, indicating a nanocluster-rich binding regime. Further insights are obtained from fluorescence correlation spectroscopy, which shows that the hydrodynamic radius of the BSA-AgNCs complex is significantly smaller than the thickness expected for a fully developed protein corona. This observation has provided direct physical evidence against complete protein shell formation. Additionally, circular dichroism and synchronous fluorescence measurements have further demonstrated that BSA retains its native secondary structure upon association with AgNCs. Overall, the results have depicted that ultrasmall AgNCs associate with proteins through complex formation instead of forming a protein corona. This mechanistic understanding is important for the rational design of AgNCs-based biosensing and biomedical applications.