Fractionation‐Free Protein Corona Quantification Through Synchrotron‐Based Small‐Angle X‐ray Scattering

When nanoparticles (NPs) enter biological environments, they are rapidly coated by biomolecules, forming the protein corona (PC) that defines their biological identity and dictates how NPs are recognized, distributed, and processed by living systems. Capturing the authentic features of the PC demands experimental conditions that preserve its native state, which are difficult to achieve once NPs are removed from their biological milieu. Despite significant progress, current PC quantification methods still rely on separating the NP-PC complex from its native environment, thereby compromising the corona's integrity and preventing accurate evaluation of its physicochemical properties. Here, we introduce a fractionation-free approach based on synchrotron small-angle X-ray scattering (SAXS) to quantitatively determine the amount of protein adsorbed onto silica NPs under native conditions. By modeling the scattering contribution of free versus bound proteins, we directly extracted the adsorbed mass in both single-protein (serum albumin) and complex proteomic (human serum) systems. The resulting adsorption isotherms enabled the determination of thermodynamic parameters, distinguishing between simple monolayer-like and more complex adsorption regimes. Together, these findings establish SAXS as a non-invasive and quantitative technique for probing the PC in situ without perturbing equilibrium, advancing SAXS toward quantitative PC characterization.