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Title: Protein nanoparticle conjugates for use in bioanalytical applications
Author: Gallagher, Jane
ISNI:       0000 0004 2743 4589
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2011
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Proteins are the commanders of the cell. These structures control and form the basis for cellular activity. It is hoped through the understanding of these components and their interactions the mutations and faults that cause disease can be identified and prevented. The aim of this research was to investigate the use of employing SERS in detection of protein interactions. It is hypothesised that this technique can be utilised in a way fluorescence spectroscopy cannot. Multiplexing provides the ability to visualise and observe proteins in the real time and the path they take through the cell. One of the most commonly used markers to examine the cellular environment is fluorescent proteins (FPs). Fluorescent Proteins are an important biomarker and are used extensively in the field of cellular biology for a better understanding of proteins and the impact of their actions within the cell. As one of the most widely used markers in cellular biology, it was thought that SERS could be employed in this instance. Ag-FP conjugates were prepared using two different methods: electrostatic attachment; and thioctic acid capped nanoparticles. This allowed for the label-free, solution based SERS detection of FPs. The detection and optimisation of this process are described within this document, which led to a competitive LOD. Furthermore the successful detection of these proteins has opened up the possibility of multiplexing of FPs using SERS. As shown in this research, vibrational peaks individual to each protein were present, indicating that very slight structural conformation changes can be detected using SERS. To transpose this Raman based technique into the cellular environment, there first must be a route into the cell. Cellular membranes are difficult to cross - even with successful entry; the cargo may not reach its eventual target within the cell. TAT peptide is a know cell penetrating peptide that has been shown to send 'cargo' across the cellular membrane and aid with cell localisation. The route to the conjugation of fluorescein labelled TAT was investigated. It was found thioctic acid capped nanoparticles provided a conjugation method to silver nanoparticles. When incubated with HeLa cells, subsequent SERS detection of these conjugates was obtained. This provided the platform for the use of other proteins to be conjugated to TAT to allow for in vivo detection. However the cellular environment is very different from 'bench' experiments, there are many processes occurring at any one time. To visualise these events, multiplexing must be implemented. For efficient multiplexing there has to be the development of novel dyes which will provide unique vibrational peaks that can be identified in this situation. Squaraine dyes provide a peak in a region where no other commercially available dye resonates. This type of dye has been synthesised, however solubility issues plagued the formation of a linker. SERS was obtained from this previously unpublished dye. A linker was successfully synthesised and conjugated to the A/G protein. This conjugate was used in a micro-assay from which SERS was obtained. This confirmed successful interaction between the protein and an antibody, confirming protein activity remained when conjugated to the linker and nanoparticle. This application based work was carried out to prove the benefits of these compounds in protein detection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available