Intra-operative Cancer Diagnostic Method Assisted by Laser Technologies

Abstract

This thesis describes the development of a novel approach for early-stage cancer diagnostics by detecting the cancerous mutation genes based on the fluorescent lifetime and PCR technology. By designing the fluorescent PCR probe combination, multiple DNA sequences can therefore be detected within the same PCR reaction, which significantly improves the detection speed and maintains the level of sensitivity. These PCR probes report the presence of the mutation genes by emitting the corresponding fluorescence. And for detecting the fluorescence signals, a frequency-domain fluorescence lifetime measurement system was designed and built. Together with the experiment system, a fast-Fourier-transform-based analysis code was also produced for identifying each of the emitting component present in a fluorescence mixture.
The developed experiment system with the post-processing method was tested and optimised by running two sub-projects: firstly, a lifetime measurement of TOTO-1 dyed Lambda DNA sample stretched over different substrate surfaces (glass, rough gold and gold-cavity) was performed using the developed system. Shortened lifetimes with increased fluorescence intensity were measured over the gold-nanovoids, proving that the designed gold-cavity structure can provide suitable localised plasmon-polariton modes for resonant coupling with the TOTO-1 dyed DNA, which then leads to an increased excitation rate and reduced of non-radiative relaxation pathways. Secondly, lifetime measurement was performed for a series of oligonucleotide sequences alongside with quantum yield measurement, excitation/emission spectrum study and melting analysis for studying how the fluorescence emission properties vary with different sequences under both methylated and unmethylated conditions.
The suitable fluorophore combination was selected by building a mathematical model in the Matlab software. A case-by-case study was performed for a mixture of six fluorophores based on the simulation results following by tests of the experimental system for establishing the detection limit. These selected fluorophores with biomarkers from existing diagnostic protocol are used for producing a series of TaqMan probe, which then were used for the demonstration of detection of multiple biomarkers within a single reaction. iv There were also attempts to transform the lab-based experiment setup into a portable diagnostic system. The first system with a convective structure for running PCR was initially designed and built by Lawrence Coles, a third-year student in the research group. And the second system for running multiple PCR experiment in parallel was initially designed and built by the 2020-2021 MEng group-design project team (Gabriel Farhat Dominguez, Samuel McCarroll, Matthew Price and Dean Tracey). Both systems were then further improved and functionalised to a level that can perform PCR experiments. Successful PCR experiments have been achieved using these two systems. The PCR product was not only measured as the level of the fluorescence emission intensity changes but also analysed in a gel electrophoresis experiment.

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