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Title: Fluorescent gold nanorod probes for the detection of cancer mRNA biomarkers
Author: Craig, Gillian D.
ISNI:       0000 0004 8509 5159
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2020
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Cancer is a leading cause of death worldwide and high mortality rates are due to diagnosis at advanced disease stages, where the success of treatment is reduced. Current cancer diagnostic techniques rely on anatomical imaging or invasive tumour biopsies that lack the sensitivity to detect early disease stages. Circulating cancer biomarkers, including circulating tumour cells and cell free DNAs and RNAs offer the opportunity to non-invasively detect cancer signatures at early disease states. Current investigations into liquid biopsies for the detection of circulating tumour cells and cell free RNAs lack sensitivity, specificity and involve complex sample processing. Therefore, there is a need for an early cancer detection method to improve treatment success and patient outcomes. As cancer cells display different genetics from healthy cells, genetic biomarkers have become of great interest for cancer detection. RNA biomarkers are of particular interest, as they can be detected in the serum of cancer patients even at early disease stages. Here, the synthesis of fluorescent gold nanorod probes for the detection of cancer mRNA biomarkers in liquid biopsies will be reported. The nanoprobe consists of gold nanorods functionalised with fluorophore labelled hairpin DNA that is targeted against a cancer mRNA biomarker sequence. Due to the optical properties of gold nanorods, the fluorophore is quenched by energy transfer in the closed hairpin DNA conformation and fluorescence is only generated upon target mRNA hybridisation. This provides a high signal to background ratio due to the surface plasmon enhanced energy transfer in the hairpin DNA conformation and surface plasmon enhanced fluorescence following target hybridisation. Steady state and time resolved fluorescence spectroscopy of the nanoprobe incubated with non-target and target DNA has demonstrated the nanoprobe binding specificity. Flow cytometry of cell lines incubated with nanoprobes has demonstrated the nanoprobe is capable of specifically detecting target mRNA within individual target mRNA expressing cells and the fluorescence of the nanoprobe is proportional to target mRNA expression levels. Nanoprobe uptake into cells and target binding specificity in cancer cells has been demonstrated through fluorescence confocal microscopy and fluorescence lifetime imaging microscopy, with both the fluorescence emission and fluorescence lifetime of the nanoprobe increasing after incubation with target mRNA expressing cells. Liquid biopsies from cancer patients have also been investigated which demonstrated the presence of a fluorescence population in cancer positive samples following nanoprobe incubation, demonstrating the ability of nanoprobes to detect circulating tumour cells. Furthermore, the ability of nanoprobes to detect target mRNA in exosomes extracted from cancer cell lines was demonstrated, providing the potential for nanoprobes to detect cell free circulating mRNAs within liquid biopsies. These investigations have demonstrates the capability of nanoprobes for detecting cancer mRNA biomarkers in liquid biopsies and the potential clinical applications of nanoprobes in cancer detection. While nanoprobe optimisation was initially focused on one cancer biomarker, CMYC,nanoprobes have been designed against several other mRNA cancer biomarkers of interest, including HER1, HER2, SOX2, as well as wild type and mutational P53. Successful synthesis of these nanoprobes show the potential for multiplexed biomarker detection. Molecular sensing and imaging of mRNAbiomarkers demonstrated in this work provides the potential for a sensitive, noninvasiveand early cancer detection method from liquid biopsies.
Supervisor: Chen, Yu ; Birch, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral