Decoding Protein Corona Through Synchrotron-Based Small-Angle X‑Ray Scattering

Nanoparticles (NPs) in biological environments rapidly become coated with a dynamic biomolecular layer known as the protein corona (PC), significantly influencing their biological identity and functionality. Traditional methods used to characterize the PC often disrupt its native state, limiting accurate insights into its true structural and compositional complexity. Synchrotron-based small-angle X-ray scattering (SAXS) provides a powerful alternative, enabling nondestructive, label-free, and in-solution analysis of the PC under physiologically relevant conditions. This minireview critically examines recent advancements in applying SAXS to decode the PC, highlighting methodological developments and exemplary studies demonstrating SAXS's unique ability to resolve interactions at the nano-bio interface. By discussing novel analytical frameworks, such as integrating SAXS with complementary techniques like small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM), we provide a comprehensive overview on the structural and thermodynamic features of the PC. Furthermore, we outline future opportunities including time-resolved SAXS to elucidate the kinetics of corona formation and the establishment of standardized protocols to enhance reproducibility and reliability. Ultimately, this review positions SAXS as an indispensable tool for advancing our understanding of nanoparticle-protein interactions and fostering innovation in nanomedicine.