Investigation of adhesion mechanisms of proteins on nanostructured surfaces by a combined local approach based on Atomic Force Microscopy and Fluorescence Microscopy

Protein adsorption is the triggering event for a variety of complex phenomena occurring at the nanoscale and determining cell adhesion, proliferation and differentiation. Biomaterials surfaces are rapidly coated by proteins that mediate the cell/substrate interactions, regulating the cell behavior through complex signaling pathways. A quantitative characterization of surface nanoscale properties and interactions influencing protein adsorption is therefore strategic for the understanding of the overall biocompatibility of surfaces. We present the results of an experimental study of the local morphological and physico-chemical properties of cluster-assembled nanostructured (ns) biocompatible TiO2 films, and of their role in protein (BSA) adsorption. In particular, we highlight the peculiar role of nanoscale surface morphology and surface charge distribution (in particular the IsoElectric Point correlated to different roughness) in determining the optimal conditions for protein condensation into nanopores, which enhances protein coverage beyond the limits of classical Langmuir isotherm model. As a result of our work, we propose a preliminary simplified model of the protein/surface adsorption based on the interplay of subtle morphological parameters (local aspect ratio, pore volume) and electrostatic interactions. Beside improving our understanding of the basic mechanisms of biocompatibility, the results of this study suggest that suitable nanostructured platforms, where nanoscale chemistry and morphology can be independently controlled, could be employed for detecting and controlling specific biological interactions for the investigation of surface-induced phenomena, such as protein competitive adsorption, aggregation and crystallization, and hydrophobic interactions.