Protein-encapsulated nanoclusters (NCs) are emerging as a versatile platform for in-vivo imaging and other biomedical applications due to their ultrasmall size and excitation in the near-infrared region. Encapsulation may however affect protein structure, size, charge, and its interaction with lipid membranes. In this study, bulk characterization methods along with surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy were employed to study the secondary structure of bovine serum albumin (BSA) with blue-emitting Au8NCs at the air/water and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) lipid/water interfaces. With this approach, the difference in the adsorption behavior between native BSA and BSA with an increasing number of blue-emitting NCs was investigated under different pH conditions. At pH 7, at which both BSA and the lipid are negatively charged, protein molecules are found to associate with the DPPG monolayer via hydrophobic interactions with no preferential orientation across the lipid monolayer. At pH 3, adsorption of BSA at the DPPG monolayer occurs mainly due to electrostatic interactions between the negatively charged lipid headgroups and the positively charged protein, resulting in a uniform orientation of the protein across the lipid monolayer. Complimentary bulk studies by circular dichroism and particle size measurements show that the encapsulation of Au8NCs is associated with the loss of BSA helicity, which makes BSA-encapsulated Au8NCs prone to oligomerization, especially at a high content of Au8NCs at one BSA protein. The results indicate that the hydrodynamic diameter of BSA with Au8NCs strongly depends on the molar fraction of gold, the pH, and the storage time. A prolonged storage of Au8NCs@BSA at pH 7 increases the rate of protein oligomerization.
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