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Title: Low-dimensional chemistry : photochemical routes to micrometre- and nanometre-scale assemblies of proteins and lipids
Author: Johnson, Alexander
ISNI:       0000 0004 5948 1240
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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The goal of the work described in this thesis was to develop methods for the fabrication of micrometre- and nanometre-scale assemblies of proteins and lipids using photosensitive monolayers as templates. Micrometre- and nanometre-scale polymer brush structures have been fabricated by patterning monolayers of (chloromethyl)phenyltrichlorosilane (CMPTS) by mask-based photolithography and interferometric lithography (IL) respectively. Atomic force microscopy (AFM) and secondary ion mass spectrometry (SIMS) imaging have been used to characterise the resulting structures. Proteins have been immobilised onto these patterns with a high degree of specificity as evidenced by confocal microscopy. Rupturing lipid vesicles onto these structures, well-defined and mobile patterned supported lipid bilayers (SLB) could be formed. The quality of these structures were confirmed by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Lipid ratchet patterns were fabricated from polymer brushes and used for electrophoresis experiments. These experiments showed that the lipids could be effectively moved and concentrated in the polymer brush trap structures by the application of an electric field. Single- and two-component polymer brush structures were fabricated from 2-nitrophenylpropyloxycarbonyl-protected aminosiloxane (NPPOC-APTES) films and used for the organisation of both proteins, and membrane proteins incorporated into supported lipid bilayers. NPPOC-APTES films were formed and characterised by surface spectroscopy and the photodeprotection kinetics were determined following exposure to 244 nm UV light. Single-component brush patterns were fabricated by photodeprotecting NPPOC-APTES films through a photo-mask and coupling Br to the deprotected regions prior to atom-transfer radical polymerisation (ATRP). These structures were used to immobilise proteins with a high degree of specificity as evident by confocal microscopy. Two-component structures were fabricated from poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) and poly(cysteine methacrylate) (PCysMA) polymer brushes. SIMS imaging of these samples confirmed the selective growth of the polymer brushes into separate regions of the patterns. Proteoliposomes containing proteorhodopsin were ruptured onto PCysMA and shown to be mobile, as evident by FRAP. DC traps were fabricated from POEGMA and PCysMA brushes and proteoliposomes were selectively ruptured onto the PCysMA regions of the pattern. Electrophoreses of these trap structures demonstrated that the proteins were mobile and could be manipulated by the application of an electric field. Micrometre and nanometre scale titania patterns were fabricated by mask-based photolithography and interferometric lithography (IL) respectively. These patterns were passivated by the adsorption of a protein resistant OEG-silane film, which was selectively removed from the titania by photocatalysis. Proteins were adsorbed onto the titania regions exposed after photocatalytic degradation with a high degree of specificity, as evidenced by confocal microscopy. Photocatalytic cleaning and subsequent refunctionalisation of the titania nanolines was also demonstrated and quantified using confocal microscopy measurements. POEGMA/titania micrometre-scale patterns were fabricated and imaged by AFM, which showed selective growth of the polymer brush on the silica regions of the pattern. Nanoscale patterns of POEGMA were fabricated and successfully used to immobilise GFP.
Supervisor: Leggett, Graham Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available