Protein Labeling Facilitates the Understanding of Protein Corona Formation via Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy.

Once nanoparticles enter into the biological milieu, nanoparticle-biomacromolecule complexes, especially the protein corona, swiftly form, which cause obvious effects on the physicochemical properties of both nanoparticles and proteins. Here, the thermodynamic parameters of the interactions between water-soluble GSH-CdSe/ZnS core/shell quantum dots (GSH-QDs) and human serum albumin (HSA) were investigated with the aid of labeling fluorescence of HSA. It was proved that the labeling fluorescence originating from a fluorophore (BDP-CN for instance) could be used to investigate the interactions between QDs and HSA. Gel electrophoresis displayed that the binding ratio between HSA and QDs was ∼2:1 by direct visualization. Fluorescence resonance energy transfer (FRET) results indicated that the distance between the QDs and the fluorophore BDP-CN in HSA was 7.2 nm, which indicated that the distance from the fluorophore to the surface of the QDs was ∼4.8 nm. Fluorescence correlation spectroscopy (FCS) results showed that HSA formed a monolayer of a protein corona with a thickness of 5.5 nm. According to the spatial structure of HSA, we could speculate that the binding site of QDs was located at the side edge (not the triangular plane) of HSA with an equilateral triangular prism. The elaboration of the thermodynamic parameters, binding ratio, and interaction orientation will highly improve the fundamental understanding of the formation of protein corona. This work has guiding significance for the exploration of the interactions between proteins and nanomaterials.