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    Controlled synthesis of silver nanostructures for use in surface-enhanced Raman spectroscopy : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nanoscience, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Otter, Sam
    Surface-enhanced Raman scattering (SERS) has been extensively researched in the past few years, with further research being done into the development of noble metal substrates that have been shown to drastically increase the enhancement provided by SERS. Noble metals are exceptionally good at enhancing Raman signals stemming from their innate optical properties. The innate enhancement factor of these noble metals has been essential for SERS research, as without them, the appearance of SERS peaks will be indistinguishable from the background noise. Research has shown that the Raman enhancement provided by noble metals substrates can be improved upon by using controlled, uniform nanostructures with sharp corners and edges instead of standard substrates or heterogeneous mixtures of random nanoparticles. Both silver and gold have been shown to adopt several unique nanostructure shapes ranging from nanocubes to complex nanoflowers. This thesis discusses the synthesis and characterization of three novel silver nanostructures along with the results of using the nanostructures in a variety of Raman spectroscopy experiments. The nanostructures produced were nanocubes, nanoplates and nanowires with all three being used as substrates in several solution based Raman spectroscopy experiments, which included conventional SERS, single molecule SERS, microfluidic SERS and Raman tweezers. The primary hypothesis of the project is that different regions of the substrate will provide different degrees of enhancements due to a difference in localized surface plasmon resonance (LSPR) intensity. In the case of the nanostructures used in this project, there should be a difference in LSPR between the face, edges and corners of the three structures, as each region will contain different degrees of LSPR interaction. To test this hypothesis the data collected from the SERS experiments was processed using several statistical analysis techniques, including Euclidean distance mapping, principal component analysis (PCA) and self-organizing mapping, to test the validity of the hypothesis. For this hypothesis to be correct the data should fit into a discrete number of groups that represent the different regions of the substrate. The experimental data did not appear to support the original hypothesis, indicating that a more nuanced explanation may be required to describe the generation of SERS signal from controlled substrates.
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    Characterisation of the filamentous bacteriophages end-caps : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2021) León Quezada, Rayén
    Ff (f1, fd, and M13) filamentous phage of Escherichia coli have collectively been the workhorse of phage display technology over the past few decades. Their use has expanded in the recent years into nanotechnology, where they serve as filament-like templates (≥ 880 nm x 6 nm) for assembly of nanostructures such as nanowires, nanorings, and more complex assemblies including nano-scale batteries, among others. The filament end-caps are the key to improving phage display applications and design of novel Ff-built nanostructures. Furthermore, proteins pIII and pVI, that form the distal end-cap, are of key importance for understanding the Ff biology. They mediate the Ff assembly-release and entry, two opposing processes that involve, respectively, excision and insertion of the virion out of and into the inner membrane of E. coli. The mechanisms of these two processes are a mystery, and the only path towards understanding is to determine the structures of the pIII-pVI complex. While the atomic resolution structure of the Ff filament shaft made of the major coat protein pVIII has been determined, the structure of the phage end-caps remains unresolved, as they constitute only 2% of the virion mass. To enable the end-cap structural analyses we developed methodology for high-efficiency production and purification of short Ff-derived nanorods, where the end-caps are highly enriched, accounting for up to 38% of the total virion mass. Furthermore, methods for Ff-derived nanorod production have been majorly improved in this thesis by engineering a novel system, resulting in at least 200-fold increase relative to the systems described previously. The nanorod purification was also improved by including an anion exchange chromatography step. Highly pure and concentrated 50 nm and 80 nm nanorods were analysed structurally and biochemically to characterise the pIII-pVI complex. Intact nanorods were structurally characterised by cryo-EM single-particle analysis (cryo-EM SPA) that resulted in 2D classes of the filament end-caps. Furthermore, a preliminary 3D model of the pIII-pVI cap was generated at a resolution of 5 Å. Further refinement of the 3D model is under way. Besides the intact particles, analysis was expanded to purified pIII-pVI complex obtained from the DOC-chloroform-disassembled nanorods by size exclusion chromatography. Under native conditions, protein pIII could be detected in a complex larger than 720 kDa, indicating that multiple copies of pVI and pIII form a multimer-dimer that includes a substantial amount of the shaft protein, pVIII. Applications of nanorods benefit from precise control of the nanorod lengths, which is very difficult to achieve when it comes to non-biological materials. In this thesis, Ff-derived nanorods of novel sizes were designed by eliminating specific DNA regions from the nanorod replication cassettes that controls the length of the nanorod ssDNA backbone. This work showed that the DNA segment between the packaging signal and (-) ori is not essential for replication and resulted in production of 40 nm nanorods, shortest ever constructed to date. Two novel lengths, 40 and 70 nm, were added to the of sub-100-nm nanorod collection produced using this system.