In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy

CONSPECTUS: Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the protein corona, forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with negatively charged NP surfaces, and the observed protein layer thickness always matched the known molecular dimensions of the proteins binding in certain orientations. Temperature and NP surface functionalization have also been identified as important parameters controlling protein corona formation. Notably, while the corona formed from a single type of serum protein was reversible, protein adsorption from complex biological media such as blood serum was entirely irreversible. These quantitative in vitro studies are of great relevance to the bio-nano community and especially to researchers developing engineered nanomaterials for biological and biomedical applications. Future efforts will be directed toward elucidating kinetic aspects of protein corona formation and the detailed structure of the adsorbed proteins at the molecular level. To better appreciate the biological responses triggered by NP exposure, more efforts will be devoted to the exploration of the biomolecular corona as it forms on NPs in contact with living cells, tissues, and even entire model organisms. These studies are challenging when performed in a well-controlled and quantitative fashion and rely on the availability of sophisticated analytical tools, particularly, quantitative optical imaging techniques including FCS and related fluctuation methods.