The protein corona is an adsorbed protein layer enshrouding nanoparticles (NP) in contact with biological fluid, altering their surface identity and ultimate fate in vivo. Understanding this protein layer is critical to predicting the biological behavior of different NP formulations. However, current methods fail to reveal direct NP-protein interactions in the solution phase at the single-particle level. Herein, we introduce a minimally perturbative single-particle tracking lock-on protein corona spectroscopy. Individual NP-protein corona complexes are captured and probed using active feedback 3D single-particle tracking (SPT) in situ without surface tethering. The synchronously attained three-dimensional trajectory and fluorescence intensity traces of single NPs and the associated protein corona enable direct particle-by-particle measurement of the NP-corona complexes’ size via mean square displacement (MSD) analysis while simultaneously quantifying the number of tightly bound proteins in the “hard” corona layer, with NP aggregation excluded. Moreover, we applied a lock-in frequency algorithm to the protein intensity time traces to directly extract the full protein corona (i.e., “hard” plus highly dynamic “soft” corona) signal in the native protein solution, despite the overwhelming background signal from freely diffusing proteins at nearly 1% of signal-to-background ratio. When applied to polystyrene (PS) NPs in the presence of bovine serum albumin (BSA), our results show a sub-monolayer formation of PS NP-BSA full corona. This full corona was found to contain double the number of proteins found in the hard corona, meaning that half of the full corona consists of the dynamic soft corona proteins. This method, capable of extending to other nanomaterial-protein systems, reveals a direct interaction between freely diffusing NP and its protein corona, opening the possibility to study transient NP-protein interaction both in situ and in vivo.

Probing the protein corona in situ with active feedback single-particle tracking spectroscopy

ACS Fall 2021

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