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

Abstract

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, which compromises the coronas integrity and prevents 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 (BSA) and complex proteomic (human serum) systems. The resulting adsorption isotherms enabled the determination of thermodynamic parameters such as binding constants and cooperativity, distinguishing between monolayer and multilayer adsorption regimes. Together, these findings establish SAXS as a robust, non-invasive, and quantitative technique for probing the protein corona in situ, without perturbing the native equilibrium. This methodology paves the way for the development of new in situ analytical frameworks across diverse nanomaterial and proteomic systems, advancing SAXS toward quantitative characterization of the protein corona. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@eea1f1org.highwire.dtl.DTLVardef@c9dcdforg.highwire.dtl.DTLVardef@c3dff6org.highwire.dtl.DTLVardef@1a49813_HPS_FORMAT_FIGEXP M_FIG C_FIG High-throughput synchrotron SAXS enables in situ, quantitative tracking of protein corona formation on nanoparticles.