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Title: Fabrication of protein nanostructures
Author: Xia, Sijing
ISNI:       0000 0004 5371 4103
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Plants can convert light energy to chemical energy with high efficiency through photosynthesis. Our group are trying to adapt the photosynthetic mechanisms of plants to anthropogenic purposes in order to develop biologically-inspired systems for solar energy collection and may provide a solution for energy shortage. We aim to reconstruct a synthetic low-dimensional system of the complete photosynthetic pathway of the bacterium Rhodobacter sphaeroides on a chip. The main focus of this thesis is to develop the methodologies to fabricate multiple protein patterns on silica surfaces and finally those biochips with multiple protein patterns will be used in the study on photosynthesis in this low dimensional chemistry (LDC) programme. Different photochemical techniques, including mask-based UV lithography, interferometric lithography (IL) and scanning near-field photolithography (SNP) have been used as tools for the fabrication of micrometre and nanometre scale structures on the self-assembled monolayers (SAMs) of silanes. SAMs of 3-aminopropyltriethoxysilane (APTES) were prepared on silica surfaces. The amount of water in reaction solvent was investigated using Karl Fisher Titration method. Water content ranged from ca. 67 ppm to 93 ppm was found out in the solvent of toluene. The wettability, roughness and thickness of APTES films were measured using, respectively, contact angle goniometer, atomic force microscope (AFM) and ellipsometry. Their variations depending on the reaction time and adsorbate's concentration were found out. The contact angle could go up to ca. 55o and the roughness was lower than 0.5 nm. Surface composition of APTES films was obtained by X-ray photoelectron spectrometer (XPS), the results of which combined with the thickness data suggested the formation of multiple layers. SAMs of aminopropyltriethoxysilane protected by oligo (ethylene glycol) modified 2-nitrophenylethoxycarbonyl (OEG-NPEOC-APTES) were prepared on silica surfaces. The effect of UV exposure on OEG-NPEOC-APTES films was studied by XPS and their deprotection rates under 244 nm and 325 nm laser were found out. The methodologies to fabricate micrometer and nanometer scale patterns by mask-based UV lithography, IL and SNP were developed. Single and multiple proteins were immobilized on Micro- and nano-patterned OEG-NPEOC-APTES SAMs to form very nice protein patterns. Attachment of aminobutylnitrilotriacetic acid (ABNTA) to UV laser selectively deprotected OEG-NPEOC-APTES SAMs had been realized to enable the spatially selective fabrication of specific protein-binding surface sites. The efficiency of patterning and the binding affinity of Histidine (His) - tagged proteins was investigated by ellipsometry and by fluorescence measurements using confocal laser scanning microscopy. The results showed that an exposure dose of ca. 5 Jcm–2 is sufficient to ensure the formation of a monolayer of site-specifically oriented protein. The methodology to prepare micrometer and nanometer scale patterns of His-tagged GFP and CPCA onto NTA/Ni2+ functionalized structures had been developed. SAMs of 4-azido-3-(triethoxypropylsilane) benzamide (aryl azide) had been prepared on silica surfaces. The effect of UV exposure on aryl azide films was obtained by XPS and contact angle measurements. Their deprotection rate under 244 nm and 325 nm laser were found out. An exposure dose of ca. 0.8 Jcm-2 under 244 nm laser and that of ca. 33 Jcm-2 under 325 nm laser were sufficient to convert eighty percentage of azide group to amide. The photoreactions of aryl azide SAMs with different amines were investigated by XPS and contact angle measurements. The results confirmed the reaction between amine and azide groups under irradiation. The methodology to pattern aryl azide SAMs at the nanometer scale was achieved by using IL followed by passivation with octadecylamine. Membrane proteins were photochemically coupled to the unmodified regions through flood exposure successfully.
Supervisor: Leggett, Graham Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available