There is an urgent need for sensitive diagnostic tools to detect the early stages of cancer - the earlier the detection, the better the chance of successful treatment. Nanosensors based on nanoparticles have recently been shown to be effective in cancer mRNA detection in solution and in vitro. However, using these tools for ex vivo has yet to be studied. This thesis, therefore, describes the development of a novel gold nanorod-based fluorescent hairpin DNA probe (nanoprobe) for ex vivo cancer detection. The nanoprobe is composed of gold nanorods (GNRs) functionalized with a thiol-modified ssDNA oligonucleotide hairpin (hpDNA) that is linked to a fluorophore label at the 5' end. The hairpin sequence is designed to recognize specific target gene mRNAs. Different cancer cells have specific pathological profiles of mRNA expression. In the absence of cancer-associated mRNA, the probe hairpin remains in a closed conformation and fluorescence is quenched by the fluorophore's proximity to the gold surface via a fluorescence resonance energy transfer (FRET) mechanism. However, when the targeted mRNA is expressed at high copy number in a tumour cell, it encounters and hybridizes to the complementary DNA sequence within the hpDNA of the probe, opening up its structure and fluorescence becomes greatly enhanced as FRET no longer occurs. In this thesis, the optimal chemistry for GNR formation was identified, and GNRs were created in different aspect ratios (were assessed by transmission electron microscopy) by varying the silver added during synthesis. Specific GNR aspect ratios were selected for their optical properties before being functionalized with hpDNA. Nanoprobes were created in which the target recognition hpDNA loops were complementary to established cancer-associated mRNAs: c-myc avian myelocytomatosis viral oncogene homolog (MYC), Paired Box 8 (PAX8), and tumour protein TP63, transcript variant 2 (TP63). Testing nanoprobes on synthetic cDNA targets in solution revealed great specificity in nanoprobe quenched/fluorescent state that was assessed using fluorescence intensity and fluorescence lifetime measurements. qRT-PCR experiments were performed to investigate mRNA quantitation in ten different cell lines: flow cytometry experiments with the nanoprobes were also performed in parallel, and largely correlated, indicating the quantitative nature of the nanoprobe fluorescence output. Flow cytometry identified high- and low-expressing cell lines, for each of the MYC, TP63 and PAX8 nanoprobes and this allowed us to mix high- and low-expressing cell lines in different ratios to mimic the rarity of circulating tumour cells in patients. The nanoprobes showed high detection sensitivity in these studies; detecting even 1 positive cell among 105 negative cells in the mixed population. Side scatter measurements confirmed the uptake of both unmodified GNRs and nanoprobes by cells and their accumulation in the cytoplasm - confirmed also by fluorescence confocal imaging. Finally, the nanoprobes were successfully used for the detection of ex vivo mRNA level differences in blood samples obtained from cancer patients compared to healthy donors. Hence, a simple, stable, sensitive and adaptable fluorescent tool has been developed to study biomarker gene expression levels that may one day has clinical application in disease diagnostics.
|Date of Award||2 Feb 2021|
- University Of Strathclyde
|Supervisor||Ben Pickard (Supervisor) & Yu Chen (Supervisor)|