Investigation of fluorophore motifs for protease detection

Bywaters, Luke James (2018) Investigation of fluorophore motifs for protease detection. (PhD thesis), Kingston University, .

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There is an increasing and serious problem of antimicrobial resistance emerging where previously useful antibiotics are no longer effective. This causes a significant monetary and human cost in the healthcare system, and is a direct result of the misuse and over prescription of antibiotics. The development and implementation of rapid point of care diagnostics to conclusively identify problem pathogens such as 'Staphylococcus aureus' could slow the emergence of antibiotic resistance by correctly targeted antibiotic therapy. The rapid and sensitive protease detecting LGX probe is an example of such a diagnostic test. The synthesis of this rhodamine based fluorogenic probe and the investigation of other fluorescence motifs for protease detection has been the focus of this project. Scale up and optimisation of the synthetic route to the previously produced LGX probe was achieved, allowing further testing in a healthcare environment. Previous work has failed to address the complexities associated with the use of conventional fluorescence motifs for probe generation, as well as the ways in which their fluorescence may be modulated. Herein we address these problems, and those encountered with the scale up of the ‘LGX’ probe. The lessons learned from the LGX synthesis provided the basis for development of a number of novel analogues of the orphan fluorophore class Singapore Green. A library of nine simplified and highly active fluorescent Singapore Greens were generated. These were exhaustively investigated and it was determined how structural variations effect both their spectral and fluorescence properties including quantum yield and extinction coefficient. The spectral characteristics of Singapore Green fluorescent motifs were found to be highly favourable allowing them to provide the central core of future probes. This work has also provided the synthetic methodology required to produce ‘LGX’ in the quantities required for eventual utilisation in healthcare environments. Furthermore it has provided the several novel platforms for the development of future probes. This is of potentially considerable value as it may not only contribute to the reduction of the problem pathogen 'S. aureus' as well as slow the emergence of resistance, but also facilitate generation of other important protease detection systems.

Item Type: Thesis (PhD)
Physical Location: This item is held in stock at Kingston University library.
Uncontrolled Keywords: LGX; detection; protease detection; protease; rapid detection; bacterial detection; culture free detection; fluorescence detection; Staphylococcus aureus; S. aureus; Singapore Green; Tokyo Green; fluorophore; Rhodamine; fluorescein; probe; pro fluorophore; Photoinduced Electron Transfer; PeT; quantum yield
Research Area: Biological sciences
Faculty, School or Research Centre: Faculty of Science, Engineering and Computing
Faculty of Science, Engineering and Computing > School of Life Sciences, Pharmacy and Chemistry
Depositing User: Philip Keates
Date Deposited: 07 Nov 2019 17:23
Last Modified: 07 Nov 2019 17:23

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