Surface Analysis of Adsorbed Proteins: A Multi-Technique Approach to Characterize Surface Structure

Adsorbed proteins on surfaces are important in many applications, including medical implants, sensors, marine materials, and in vitro substrates for cell culture and other uses. Understanding the protein structure on the surface would allow better control of the interaction of the material with the surrounding environment and a more reproducible system response. Protein adsorption is a complex process, and characterization requires the combination of multiple analysis techniques. In this thesis, adsorbed protein amounts were measured using x-ray photoelectron spectroscopy (XPS) and radiolabeled proteins. Protein conformation and orientation were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS), near edge x-ray absorption fine structure (NEXAFS), sum frequency generation (SFG), and enzyme-linked immunosorbent assays (ELISA). Surface type influenced the adsorbed surface concentration of albumin and fibronectin on surfaces of glass, polystyrene, titanium, and sulfonated polystyrene as measured by XPS and radiolabeled protein adsorption. More albumin adsorbed onto polystyrene than glass. More albumin and fibronectin adsorbed onto sulfonated polystyrene than titanium. ToF-SIMS also showed differences in structure of the proteins adsorbed onto the different surfaces. The A1 domain of von Willebrand Factor adsorbed in similar amounts onto glass, tissue culture polystyrene, and polystyrene surfaces, as measured by XPS. However, ToF-SIMS showed differences in solution exposure of A1 domain amino acids when adsorbed onto the three surfaces and NEXAFS showed the most ordered beta-sheet structure when A1 was adsorbed onto polystyrene. ELISA showed lowest binding of antibodies recognizing a nonlinear epitopes within A1 when A1 was adsorbed onto polystyrene. Functional studies using a parallel plate flow chamber measured platelet binding to A1 adsorbed onto the three surfaces. At high shear (20dyne/cm2), platelets showed most detachment from A1 adsorbed onto glass. At low shear (0.2dyne/cm 2), platelets showed most detachment from A1 adsorbed onto polystyrene. Surface analysis was also useful in characterizing collagen substrates created under different experimental conditions, including material source and pH. Collagen obtained from different sources exhibited altered adsorption behavior, both in amount adsorbed as measured by XPS and interaction with the A1 domain of von Willebrand Factor as measured by ToF-SIMS. SFG was used to identify differences in ordering of collagen adsorbed from solutions at different pH values. Collagen adsorbed at pH 8.0 showed higher SFG amide signal than collagen adsorbed at pH 6.5, suggesting greater ordering of the peptide backbone at pH 8.0. The differences in SFG signal were not due to the amount adsorbed protein, as XPS showed more collagen adsorbed onto tissue culture polystyrene at pH 6.5 than pH 8.0. These studies demonstrated that the surface type can have a large impact on adsorbed proteins, both in amount adsorbed and surface structure. These studies also showed that surface analysis is very useful in creating defined in vitro protein substrates. In all cases, it was crucial to use multiple analysis techniques to understand these systems.