Using Surface Analysis to Investigate how Adsorbed Protein Structure Changes with Controlled Changes in Surface Chemistry: A Case Study Involving Bare & Sodium Styrene Sulfonate-Grafted Gold Surfaces

Abstract

Protein adsorption on synthetic surfaces is an important and widely studied phenomenon in biomaterials research. Despite its importance, it is still poorly understood. Therefore, the ultimate goal of this thesis is to contribute toward a better understanding of the protein-biomaterial interface, with application toward the next generation of biomaterial implants. Narrowing this broad objective down to something more specific, this thesis represents a body of work focused on the interaction of blood plasma proteins with sodium styrene sulfonate (NaSS) grafted surfaces. The approach to achieving this objective is straightforward. First, proof-of-concept must be established that NaSS can be successfully grafted to a given surface. A thorough characterization of NaSS films grafted from titanium and silicon oxide surfaces using atom transfer radical polymerization (ATRP) is presented herein. Next, the grafting procedure must be optimized and scaled in order to reliably produce sufficient quantities of NaSS-grafted samples to meet the demands of protein adsorption studies. Using a 24 factorial experimental design and “activators are continuously regenerated by electron transfer” (ARGET) ATRP chemistry, an optimized NaSS grafting procedure was developed. The findings here may be generally applied as a starting point for grafting other polymers to a variety of surfaces. The last step is application—to use the NaSS films for their intended purpose. Surface analysis techniques were applied to characterize changes in adsorbed bovine serum albumin (BSA), bovine fibrinogen (Fgn), bovine immunoglobulin G (IgG), and bovine plasma films between bare and NaSS-grafted gold surfaces. All three proteins and plasma adsorb more readily to, and have a higher affinity for gold than NaSS surfaces. However, at higher concentrations NaSS adsorbs similar amounts (for plasma) or more (for BSA and Fgn) total protein than gold. The only protein that NaSS surfaces adsorb less of than gold is IgG, because IgG adopts a highly denatured conformation on NaSS. Each adsorbed IgG molecule takes up more space on NaSS compared to gold surfaces, resulting in less total protein adsorbed at all concentrations. Still, with the exception of BSA and plasma on gold surfaces, neither surface appeared to have saturated at the highest protein solution concentration studied. Using principal component analysis (PCA) of just the amino acid ToF-SIMS mass fragments, it was determined that all three proteins and plasma adsorb differently on NaSS and gold surfaces, and that the structure of the xii adsorbed protein films change with surface concentration. One difference between adsorbed films on both surfaces, determined using peak ratios for buried/surface amino acids for each protein, is that adsorbed proteins denature more on NaSS than gold. Also, using peak ratios of non-uniformly distributed amino acids, small differences in average orientations were found between the two surfaces for BSA and IgG films. Finally, principal component (PC) modeling, was used to track changes in adsorbed plasma films with time. On NaSS surfaces the plasma films appear to be more BSA-like at short adsorption times, and more Fgn-like at longer adsorption times. Similarly, on gold surfaces the plasma films on appear to start out more IgG-like and become more Fgn-like with increasing adsorption time. However, the PC model included only the three proteins studied here, where plasma is a complex mixture of hundreds of proteins. Therefore, while both gold and NaSS appear to adsorb more Fgn with time, further study is required to confirm that this is truly representative of the final state of the adsorbed plasma films.