Real-time Optical Tracking of Protein Corona Formation on Single Nanoparticles in Serum

Significant advances in synthesis and functionalization have provided state-of-the-art technology in controlling the physico-chemical properties of nanomaterials. These are finding numerous applications including in the biomedical field whereby nanoparticles are injected in vivo for medical imaging, theranostics and biosensing. However, interactions with proteins contained in biological fluids lead to the formation of a shell on the surface of the nanoparticles called "protein corona" (PC). PC plays a detrimental role for the intended applications as it may modify the interface of the nanoparticle and thereby block functional groups needed for recognition. It is therefore essential to understand the mechanisms of formation of these PCs in order to control the surface chemistry of the nanoparticles in complex biological fluids. Current characterization techniques can identify and quantify the composition of PCs using mass spectroscopy and electrophoresis. However, most of them do not enable real-time measurement in complex media because they require washing steps to remove excess protein. Finally, most techniques provide ensemble averages and are unable to access inter-particle heterogeneity. Here, we demonstrate the use of single-particle scattering microscopy combined with a microfluidic system to study PC formation in real-time at the single-nanoparticle level. The method is label-free and operates in undiluted blood serum. We probe PC formation on both, metallic and dielectric nanoparticles with different surface chemistries. Analysis of protein adsorption revealed unexpectedly strong heterogeneity whereby the amount of accumulated protein varies by up to a factor of 10 between the particles. Furthermore, it is found that the surface roughness of the nanoparticles affects the kinetics of the PC formation. The results of this in-situ characterization are a powerful tool to optimize the surface chemistry in order to minimize the formation of PCs and thus increase the efficiency of nanoparticles for applications such as targeted drug delivery.