Conformational changes of proteins adsorbing on biomaterial surfaces affect biocompatibility. Titanium is among the most successful biomaterials; however, the mechanisms leading to its biocompatibility are not yet understood. The primary objective of this study was to investigate the conformation of human plasma fibrinogen (HPF) adsorbed on titanium oxide surfaces. A method for preparing ultraflat titanium substrates was developed. This allowed high-resolution investigation of both the titanium oxide surfaces and the adsorbed state of HPF. The titanium oxide surfaces were first imaged with an atomic force microscope in air and subsequently incubated in a solution of HPF in phosphate buffer and imaged in fluid with tapping mode AFM. The titanium oxide surfaces exhibited a root-mean-squared (RMS) roughness of (0.29 ± 0.03) nm over (1.00 × 1.00) μm2 areas. Different degrees of molecular order were found on the titanium oxide surface. In crystalline surface areas square lattices with parameters a 0 = b 0 ≈ 0.5 nm were resolved, which is consistent with the (001) planes of the titanium dioxide TiO2 rutile. Repetitive scanning of TiO2 surface areas resulted in the abrasion of the outermost TiO2 layers by the AFM tip to a depth varying between 0.3 and 1.0 nm. When imaging adsorbed HPF, individual molecules and aggregates, often joined through their D domains, were resolved, and the typical HPF multiglobular structure was observed. The mean length and height of single molecules were (46 ± 3) nm and (1.4 ± 0.2) nm, respectively. Additional features (chain segments) adjacent to the D and E domains were resolved and attributed to the α chains and their C-termini. The combination of AFM and the ultraflat titanium preparation method has proven successful for the high-resolution study of both the TiO2 surface and the adsorbed HPF.

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