Adsorption behavior of proteins and colloidal particles studied using atomic force microscopy

There is much interest in characterizing the adsorption of proteins and larger colloidal particles at solid-liquid interfaces, as the phenomenon is an important part of a variety of industrial and biological processes. Theoretical studies of the phenomenon often are based on mechanistic simulations of the behavior of individual particles (or protein molecules) interacting with a solid surface. However, direct observations of adsorbed species in the colloidal size range are extremely difficult due to the very small size of the particles involved, and so experimental studies are generally based on indirect, non-localized measurements of the extent of adsorption. The goal in this experimental study was to bridge this gap between theory and experiment by observing directly the adsorption of proteins and particles in the nanometer scale using the high resolution imaging capability of atomic force microscopy (AFM).; Charged polystyrene latex particles, 100 nm in size, adsorbed to substrates of opposite surface charge were studied as model systems for protein adsorption. AFM images revealed that the particles adsorb irreversibly and with sub-monolayer coverage to the substrates. The initial kinetics of the processes were found to be diffusion-limited and the asymptotic kinetics were found to be consistent with the approach to saturation of the random sequential adsorption (RSA) model. Localized ordering of the charged latex particles at the solid-liquid interface was analyzed by calculating two-dimensional radial distribution functions for the observed arrangements. This method revealed a high degree of short-range order among adsorbed particles at surface coverages near saturation. The extent of surface exclusion by adsorbed particles was found to depend on the magnitude of the electrostatic repulsion between the charged particles, and thus surface coverage and the length scale of the short-range ordering are controlled by double layer screening. The ionic strength dependence of the structure of the adsorbed layer was found to agree well with an RSA model for charged spheres.; The adsorption of proteins, including ferritin and lysozyme, was also studied using in situ AFM analysis. On certain charged substrates, ferritin was found to adsorb irreversibly with sub-monolayer coverage and without noticeable surface diffusion. The surface coverage of adsorbed ferritin was found to be sensitive to solution pH and to ionic strength due to the effect of these parameters on the electrostatic interaction between ferritin molecules. Radial distribution analysis revealed short-range ordering of adsorbed ferritin controlled by interparticle electrostatic repulsion. As with much larger colloidal particles, the adsorption behavior of ferritin was in good agreement with RSA models of charged spheres, indicating a much wider range of applicability of RSA theory than was previously confirmed. The arrangements of individual lysozyme molecules on various substrates were found to be qualitatively similar to adsorbed layers of ferritin.