Thermodynamics of multilayer protein adsorption on a gold nanoparticle surface

Abstract

We report the thermodynamics of protein adsorption on negatively charged colloidal gold nanoparticles (GNPs) of 16 nm to 69 nm at pH 7.0. Three biologically important proteins of varying size, namely tetrameric alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, bovine serum albumin (BSA), and insulin from the porcine pancreas were taken and their adsorption on the GNP surface was investigated by dynamic light scattering (DLS), absorption spectroscopy and zeta potential measurements. The hydrodynamic size of the GNPs was found to increase with protein addition. At very low protein concentrations, the adsorption of these proteins was earlier described by Langmuir-type adsorption isotherms. However, when the protein concentration is raised, the adsorption data are found to fit the BET (Brunauer, Emmett, and Teller) adsorption isotherm well, indicating multilayer formation on the GNP surface. The equilibrium binding constants: K_S (monolayer) ∼10⁸–10⁹ M⁻¹, K_L (subsequent layers) ∼10⁵–10⁶ M⁻¹, and monolayer thickness are obtained from our measurements. K_S is found to increase with the size of the GNPs, while K_L does not change significantly with either the size of the GNPs or that of the protein. K_L, which originates from protein–protein interaction, compares very well with the protein dimerization constant in solution, indicating that the interaction beyond the first layer is not significantly influenced by the nanoparticles. The first layer forms the soft corona on the GNP surface since this layer, although stable, is removable by centrifugation. But the subsequent layers are weakly bound and we call it an ultra-soft corona in order to distinguish it qualitatively from the strongly bound first layer. The modest magnitudes of Gibbs free energy changes of 43–55 kJ mol⁻¹ for the first layer and 31–35 kJ mol⁻¹ for the subsequent layers indicate that ADH, BSA, and insulin are all physiosorbed on the GNP surface. The temperature dependent DLS data point out that the adsorption of these proteins on GNPs is endothermic but is accompanied by a large increase in entropy. The nature of the BET fits to the adsorption data shows that multilayer adsorption starts even before the monolayer formation is complete, and it does not occur in a layer-by-layer sequence.