pH- and Ionic Strength-Controlled Switching between Protein Monolayer and Multilayer Adsorption on Gold Nanoparticles

The phenomenon of protein adsorption on nanoparticle surfaces in a biological milieu leads to the formation of a protein corona (PC) and its formation is governed by competing intermolecular forces. Among the different intermolecular forces, electrostatic interactions play a significant role, which can be tuned using pH and ionic strength. This study systematically investigates the effect of pH and ionic strength on the adsorption of three proteins: bovine serum albumin (BSA), myoglobin (Myo), and papain (Pap) onto citrate-capped gold nanoparticles (GNPs) of varying diameters (15-60 nm). Protein adsorption was examined at pH 5.2, 7.4, and 10.2, corresponding to conditions below, near, and above each protein's isoelectric point (pI). Protein adsorption was monitored in situ by dynamic light scattering (DLS), enabling direct tracking of hydrodynamic size evolution without separation artifacts. At pH 5.2, all proteins showed multilayer adsorption, quantified with BET fitting, revealing distinct first-layer binding constants (K₁) and subsequent-layer binding constants (K₂) with Gibbs free energies ΔG°₁ ≈ -49.5 to -53.7 kJ/mol and ΔG°₂ ≈ -41.9 to -44.8 kJ/mol. At pH 7.4 and 10.2, adsorption saturated at monolayer coverage, modeled by modified Langmuir isotherms (ΔG° ≈ -52.6 to -60.6 kJ/mol). Generally, multilayer adsorption is found at higher concentrations but here we use very low concentrations and use the pH and ionic strength of the medium to probe the role of electrostatics in multi and monolayer adsorption. Further, to delineate the contributions of electrostatic and nonelectrostatic forces, salt titration experiments were performed. Increasing ionic strength systematically reduced multilayer formation, indicating that electrostatic screening weakens interprotein interactions within the corona and goes back to monolayer. Quantitative BET analysis and polyelectrolyte theory yielded the electrostatic free energy component (ΔG°_elec) and salt-dependence parameter (SK). Smaller GNPs (15 nm) promoted stronger interprotein electrostatic coupling, reflected by ΔG°_elec compared to larger GNPs (60 nm).