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Nanoscale surface patterning as a means of controlling protein immobilisation

Williams, Sophie Elizabeth 2008. Nanoscale surface patterning as a means of controlling protein immobilisation. PhD Thesis, Cardiff University.

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It would be desirable to synthesise a molecularly imprinted polymer with specific high-affinity protein recognition sites as a durable, cost-effective replacement for antibodies in biotechnology. A novel protein imprinting approach was proposed as an outline for these investigations. The focus of this project was consideration of fundamental aspects of surface nanometer-scale patterning and protein-surface interactions with the aim of preparing an ordered array of surface protein. This was in part achieved during the course of the work. An equilibrium dialysis method was validated for the measurement of ligand-protein binding parameters. Human serum albumin (HSA) and ethacrynic acid (ETH) were chosen as the ligand-protein pair to be surface-immobilised. Molecular modelling suggested a good fit for ethacrynic acid in the covalent HSA binding 'cleft', however, the covalent HSA-ETH complex was not successfully isolated. A derivative of the ligand, ETH-glycine, was synthesised to a very high purity but a low yield. A gas-phase silanisation method was developed to deposit functional aminopropyletriethoxysilane (APS) groups onto silicon wafer surfaces. The dispersion of APS could not be sufficiently controlled, by changing the evaporative distance or the APS evaporation concentration, and hence it was not possible to bring about gold nanoparticle (AuNP) patterning at the nanometer scale using this approach. However AuNP patterning could be achieved by incubating APS monolayer surfaces with different dilutions of a commercially available AuNP solution. Subsequent development of a protein imprinting strategy would require that non-specifically adsorbed HSA can be removed from PNA silicon surfaces. This was found to be difficult to achieve using mild conditions. Controlled gas-phase deposition of APS could not be used to directly facilitate dispersed ligand attachment. AuNP patterning can potentially be used as an indirect method for controlling surface dispersion of immobilised ligand. Controlled surface orientation and patterning of HSA, using the specific interaction with ETH, remains a significant challenge.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Pharmacy
Subjects: R Medicine > RM Therapeutics. Pharmacology
ISBN: 9781303183782
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:33

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