Improving the Surface Activity of Attached Biomolecules

Abstract

This thesis studies the effects of changing the mode of binding on the activity of surfaceattached biomolecules such as antibodies and antimicrobial proteins. The principal analysis techniques used were X-ray and neutron reflectometry, which were used to characterise reactive polymer coatings for biomolecule deposition (PMMA and APTES) and to structurally analyse attached antibody, surface linker and protein layers. PMMA films were uniform and reproducible when measured in air, but formed hydrated layers at the Si/PMMA interface when exposed to aqueous buffer. These layers were consistent and did not expand significantly over extended periods of time. APTES films were less consistent and required tight control of water in the deposition solution for films to be reproducible but also appeared stable in aqueous solution. Antibodies were physisorbed to PMMA films, forming layers with a ‘flat-on’ orientation at various concentrations. BSA was added to all layers and was observed filling the spaces in between antibody layers on PMMA films. Comparison of the layer thickness with the molecular dimensions of BSA suggests that tertiary structure is disrupted at the surface. The antigen capture efficiency of antibodies covalently attached to PMMA and three different linker surfaces, chosen to produce improved flexibility and higher surface hydration, was assessed. Antibodies covalently bound to all surfaces were more efficient and captured a higher amount of antigen than physisorbed antibodies. The structure of PAMAM dendrimers attached to APTES films was assessed in aqueous solution, and it was found that the G4.5 dendrimer formed a thicker layer with higher hydration than on the PMMA film. The killing mechanism of the antimicrobial protein hydramacin-1 (HM-1) was studied and it’s fusogenic nature confirmed. HM-1 and lysozyme were attached to APTES and PMMA films with and without a PEG-based linker molecule and the protein layers characterised using NR. Antimicrobial activity was observed for both proteins bound to APTES-PEG films however no activity was observed for proteins bound to any of the other surfaces prepared.