Particle-by-Particle In Situ Characterization of the Protein Corona via Real-Time 3D Single-Particle-Tracking Spectroscopy**

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, primarily using bovine serum albumin (BSA) as a model system. The fluorescence signals and 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 twice the number of proteins compared to the ex situ-measured hard protein corona.

Automated summary

A new method is introduced to track individual nanoparticles in real-time and measure their protein coronas, revealing that the number of proteins in the dynamic in situ protein corona is twice that of the ex situ measured hard protein corona.