In Situ Saxs Reveals q-Dependent Features And Thermodynamic Behavior Of The Protein Corona

Protein corona (PC) quantification often relies on separation methods that may disrupt the delicate biomolecular interface formed on nanoparticle surfaces. Here, we present a fractionation-free approach for quantifying PC formation on spherical silica nanoparticles (SiO₂) using synchrotron-based small-angle X-ray scattering (SAXS) at the B21 beamline (Diamond Light Source, UK). This in situ method allows direct, non-destructive analysis of nanoparticle–protein interactions under native conditions, without labeling or isolation steps. By performing controlled protein titrations and applying quantitative modeling to scattering profiles, i.e. analyzing intensity changes proportional to sample concentration, we identified distinct signatures of corona formation, markedly different from the arithmetic sum of individual components, and consistently above beamline fluctuation thresholds. The method achieves high reproducibility and signal-to-noise ratios, with scattering intensities remaining well above the beamline fluctuation threshold. To further validate the approach, zwitterionic-functionalized SiO₂ nanoparticles (SiO₂-ZW) were employed as a negative control. SiO₂-ZW exhibited minimal changes in low-q scattering up to 2.0 mg/mL BSA, confirming antifouling performance, while only slight adsorption was observed at 5.0 mg/mL, underscoring the utility of SAXS for detecting subtle protein–surface interactions. Size-exclusion chromatography SAXS (SEC-SAXS) measurements corroborated these findings by isolating elution fractions and enabling component-resolved scattering analysis. The method was also extended to complex biological fluids, including human serum and fetal bovine serum, demonstrating its translational potential for diverse proteomic environments. The experimental data were adjusted to adsorption isotherms using Hill, Langmuir and Fruendlich models to better describe adsorption process for each protein media. Finally, this study introduces a fractionation-free SAXS-based methodology for probing PC formation under biologically relevant conditions.