In situ characterization of nanoparticle protein corona using real-time 3D single-particle tracking
Biophysical Journal(2023)
Abstract
Nanoparticles (NPs) adsorb proteins when they are exposed to biological fluids, forming a dynamic protein corona that consists of tightly bound “hard” corona and rapidly exchanged “soft” corona. This protein layer alters the NPs’ surface identity and affects their fate in vivo. However, current methods fail to directly investigate single NP-protein interactions in the solution phase due to the fast NP diffusion and protein exchange dynamics. To address this gap, we introduce a real-time lock-on protein corona spectroscopy based on previously developed 3D single molecule active real-time tracking (3D-SMART), primarily using polystyrene NP (PSNP) and bovine serum albumin (BSA) as a model system. 3D-SMART uses real-time photon arrival information to estimate the PSNP's position within a rapid 3D laser scanning pattern. The position estimate is then used to move a piezoelectric stage to counteract the PSNP's diffusion, keeping the NP-protein complex locked in the center of detection volume for spectral interrogation. Consequently, the 3D trajectory of the NP-protein complex and the fluorescence traces of the associated proteins can be synchronously measured for size measurement and hard corona quantification. Moreover, we applied a lock-in filtering-based algorithm to the protein intensity trace to extract the full protein corona signal in situ at signal-to-background ratios of 1%. It was discovered that the full corona consists of a sub-monolayer of BSA on PSNP, which contains twice the quantity of proteins compared to hard corona measurements, indicating that half of the full protein corona can be classified as soft corona. To expand this method to smaller NP-protein systems, we have further implemented a Galvo mirror as control actuator with response time five times faster than the piezoelectric stage, opening the possibility of studying a wider range of transient NP-protein interaction in vivo.
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Key words
nanoparticle protein corona,real-time,single-particle
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