Particle-by-Particle in Situ Characterization of the Protein Corona via Real-Time 3D Single Particle Tracking Microscopy

Nanoparticles (NPs) adsorb proteins when exposed to biological fluids, forming a dynamic protein corona that affects their fate in biological environments. A comprehensive understanding of the protein corona is lacking due to the inability of current techniques to precisely measure the full corona in situ at the single particle level. Herein, we introduce a 3D real-time single-particle tracking spectroscopy to "lock-on" to single freely-diffusing polystyrene NPs and probe their individual protein coronas. The diffusive motions of the tracked NPs enable quantification of the "hard corona" using mean-squared displacement analysis. Critically, this method's particle-by-particle nature enabled a lock-in-type frequency filtering approach to extract the full protein corona, despite the typically confounding effect of high background signal from unbound proteins. From these results, the dynamic in situ full protein corona is observed to contain double the number of proteins than are observed in the ex situ measured "hard" protein corona.