| Characterization of the amount, identity, conformation, and orientation of adsorbed proteins is critical for understanding biological reactions at surfaces. These reactions impact a wide range of industries (biomedical devices, tissue engineering, food processing, and marine engineering, etc.). Static Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is uniquely qualified for this problem due to its chemical specificity, high mass resolution, analytical sensitivity, and extreme surface sensitivity. This dissertation presents the first detailed examination of adsorbed proteins using static ToF-SIMS. This analytical problem can be considered in two parts: (1) What are the capabilities and limitations of ToF-SIMS for characterizing adsorbed proteins? and (2) How can the relevant information be extracted from the ToF-SIMS data? This study has demonstrated that the ToF-SIMS spectra of different adsorbed proteins are unique and can be used to identify the adsorbed proteins. Furthermore, adsorbed serum albumins with very similar amino acid compositions (∼90% similarity) and sequence homology (∼75%) were differentiated using ToF-SIMS. Multivariate data analysis techniques were used to simplify the ToF-SIMS data for interpretation. The detection limit for low amounts of adsorbed protein ranged from 0.1–10 ng/cm2, depending on the substrate chemistry and morphology. ToF-SIMS measurements of the composition of several binary protein films agreed with independent radiolabeled protein adsorption measurements. However, the minor component of the film had to constitute 10 mass % of the adsorbed protein for detection by ToF-SIMS. For one binary system, discrepancies between the ToF-SIMS and radiolabeled protein adsorption measurements were attributed to a partial covering of one component by the other. These results were coupled with Electron Spectroscopy for Chemical Analysis (ESCA) measurements to develop models of the structure of the adsorbed proteins. ESCA, ToF-SIMS, and radiolabeled protein adsorption measurements were used to obtain composition information on protein films adsorbed from blood serum and plasma. This study has outlined the capabilities and limitations of ToF-SIMS for investigating adsorbed protein films and has established this technique as a powerful tool for the characterization of adsorbed protein films. |
