Thesis

Fluorescence photophysics, characterisation and application of labelled glucose/galactose binding protein

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Awarding institution
  • University of Strathclyde
Date of award
  • 2013
Thesis identifier
  • T13508
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Department, School or Faculty
Abstract
  • There is currently a pressing need for reliable, minimally-invasive, continuous glucose monitoring for the treatment of diabetes mellitus. One promising glucose sensor is bacterially-derived glucose binding protein (GBP). In this project, the modified triple mutant H152C/A213R/L238S-GBP (mGBP) was investigated for viability as a functioning glucose sensor. Labelling the protein with the environmentally-sensitive dye BADAN produced repeatable, nearly linear, changes in both fluorescence intensity and fluorescent lifetime measurements. The robustness of the GBP-BADAN sensor response was tested and the results showed no interference from the sugars fructose and lactose- while it was also shown that alterations in temperature and pH level had deleterious effects on the sensor function. The dyes Texas Red and iodoacetamide nile red (IANR) were attached to the GBP binding site to investigate the possibility of producing an alternative, longer-wavelength sensor system than when using BADAN. The ostensibly environment-insensitive Texas Red dye produced a protein-dye complex with no discernible response to glucose, but a significant response was observed for the GBP labelled with IANR: a 2-exponential lifetime decay relative amplitude change of 18% was recorded. These data provided further evidence for the proposed mechanisms of labelled GBP, while showing proof-of-concept for a long-wavelength fluorescence lifetime based glucose sensor. Finally, methods of protein encapsulation were examined. Silica sol gels derived from both tetramethyl orthosilicate (TMOS) and tetraethyl orthosilicate (TEOS) precursors were found to be ineffective environments for the GBP-BADAN sensor, as the fluorescence response was significantly impaired. Encapsulation in a hydrogel produced using 9-fluorenylmethoxycarbonyl (FMOC-YL) was also examined, and this arrangement was found to provide an environment where both mGBP-BADAN and mGBP-IANR could function. mGBP-IANR in the FMOC-YL hydrogel was found to have a binding constant of 41.99 (±3.78)mM. It was concluded that mGBP-IANR in an FMOC-YL hydrogel could potentially provide a route to a functioning glucose biosensor.
Resource Type
DOI
Date Created
  • 2013
Former identifier
  • 996233

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