An AFM investigation of the interfacial properties of biocompatible nanostructured titanium oxide films

Cluster-assembled nanostructured Titanium Oxide (ns-TiOx) deposited by Supersonic Cluster Beam Deposition (SCBD) [1] recently proved to be a very promising biomaterial, allowing the adhesion and proliferation of cancer and primary cells, with no need of additional coating with extra-cellular matrix proteins [2], and the adhesion of proteins, such as streptavidin, with no need of additional coatings of polycations [3]. The intrinsic nanostructure of this material, with fine granularity, high porosity and specific area, coupled to the chemical reactivity of the surface is likely a key element in determining the biological affinity of the material with nanometer-sized biomolecules, such as proteins.[4] Little is known however of the specific role played by each of these surface properties in the interaction of proteins with nanostructured biocompatible materials. Here we present an atomic force microscopy (AFM) study of the morphological and adhesive properties of ns-TiOx biocompatible surfaces. AFM provides nanometer spatial resolution in both imaging and force spectroscopy, and it is therefore the technique of choice for the investigation of biologically relevant surface properties of materials. AFM in force-volume mode (FV-mode) has been used to characterize the local adhesive properties of ns-TiOx, in an effort to establish a correlation between the macroscopic wetting behavior of ns-TiOx films and those local morphological and chemical properties, which can be relevant in protein adhesion processes. We have developed a patterning strategy based on the combined use of SCBD and nanosphere lithography, for the production of sub-micrometer patterns of ns-TiOx on glass or other reference substrates (Fig.1). The main objective of developing such patterns is to have reference and target materials simultaneously in the same experiment. In FV-mode, we have studied the interaction of silicon nitride (Si3N4) tips with ns-TiOx and glass, as reference. The results obtained in aqueous medium suggest that ns-TiOx interacts with Si3N4 via chelation mechanisms, in addition to hydrogen bonding: these mechanisms are responsible for stronger adhesion of the AFM tip with ns-TiOx surface as compared to the reference glass substrate. The presence of reference during the investigation of the target material helped us resolving ambiguities that show up when reference and target material are investigated independently. Local adhesion measurements in aqueous media allowed us to get insights on basic interaction mechanisms between surface groups that are expected to play a major role in the interactions between proteins and biocompatible surfaces.