Silica Surface Modifications for Protein Separation

Proteomics has emerged as the next phase of understanding diseases and ailments after the completion of the human genome project. There are countless examples of protein deficiencies leading to serious ailments. Therefore, proteins have been studied as biotherapeutics and have potential applications in the drug discovery process. The advancement of such biological applications demands simultaneous advancement in protein separations on both the analytical and preparative scales. Current challenges include reducing cost and increasing the speed and the yield of production while maintaining the structure of the proteins.;The current standard separation method used in the industry is liquid chromatography. It is one of the few scalable, nondestructive methods that can be used for both analytical and preparative separations. The stationary phases and proteins used in this research are based on industry and research practices. The most commonly-used stationary phase material is silica, due to its chemical and mechanical stability. Chemical functionalizations of silica surfaces, which are heavily documented, allow control of the strength and nature of interactions with the stationary phase. Thus, silica was used as a base material. Similarly, lysozyme is a commonly-used sample material due to its low cost and availability. Consequently, there is a wealth of scientific literature on the properties lysozyme, and it is used as the test protein for the majority of the research reported here.;Silica was functionalized with amino acids using a peptide synthesis method and the effect of amino acid functionalization on lysozyme adsorption was determined. The net charge difference between the surface and the adsorbed protein was the main driving force for the adsorption of lysozyme onto carboxyl- and amine-functionalized silica. Net charge difference did not affect lysozyme adsorption onto amino acid-functionalized silica. Rather, specific interactions between lysozyme and the amino acid functionalities determined adsorption behavior.;Multicomponent multilayer adsorption has been successfully modeled. The predicted mechanism of adsorption was mathematically validated. Multicomponent chromatographic models can now utilize type II adsorption models as well as type I and linear adsorption. This model can further be tested for other proteins.;The effect of surface curvature on protein adsorption was studied. A pore size effect is observed for lysozyme adsorption onto different functionalizations. The difference in pore size changed the shape of the adsorption isotherm. Entropic considerations such as the range of motion of the protein attachment to the surface were found to the impact the energy of adsorption.;Finally, selective surfaces for metalloproteinase adsorption from venom were synthesized and tested. The aspartic acid functionalized surface was eight times more selective for metalloproteinase over other proteins in solution. The strength of adsorption onto the aspartic acid surface is consistent with disintegrins modes of attachment. This shows that adsorption to the surfaces is specific and can mimic biological interactions.