Studying the protein corona on nanoparticles by FCS

Engineered nanoparticles (NPs) have found widespread application in technology and medicine. Whenever they come in contact with a living organism, interactions take place between the surfaces of the NPs and biomatter, in particular proteins, which are currently not well understood. We have introduced fluorescence correlation spectroscopy (FCS) and dual-focus FCS (2fFCS) to measure protein adsorption onto small NPs (~10-30 nm diameter). 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. Investigations of the adsorption of a number of important serum proteins onto negatively charged, carboxyl-functionalized NPs revealed a stepwise increase of the NP size due to protein binding, clearly indicating that a protein monolayer enshrouds the NP. Structure-based calculations of the protein surface potentials reveal positively charged patches through which the proteins interact electrostatically with the negatively charged NP surfaces; the observed protein layer thickness is correlated with the molecular dimensions of the proteins binding in suitable orientations.