Evaluation of some fluorescent dyes and molecular tethers for a fibre optic DNA biosensor

This thesis reports studies of two fluorescent probes that could be used to detect DNA hybridization by a fibre optic nucleic acid biosensor that is being developed by our research group. The two dyes that were examined were propidium iodide and pyrylium iodide. When compared in solution to ethidium bromide (EB), pyrylium iodide showed the most promising attributes because of the longer wavelength of excitation (570 nm compared to 515 nm for EB) and a larger fluorescence increase (approximately 4000% higher than EB) upon intercalation into dsDNA. Different loading ratios of these dyes in dsDNA were investigated and compared to similar experiments which used EB. The optimum loading ratios of pyrylium iodide, propidium iodide, and EB in an AT 20mer was 1:1, 2:1, and 2:1, respectively.;In addition, a new molecule, 10-[10-(4,4'-dimethoxytrityloxy)-decyloxy]-1-decanol (LCAT), was synthesized for use as a linker to covalently attach DNA to a surface, and as a tether to attach a fluorescent probe to DNA. Thin films of this linker were prepared using a reverse phosphoramidite method. 20mers composed of thymidylic acid icosanucleotides were successfully grown by automated DNA synthesis on this linker using standard phosphoramidite chemistry. LCAT-dT20 was characterized by anion exchange HPLC, negative ion electrospray mass spectrometry, ellipsometry, XPS, and fluorescence microscopy. This molecule was also successfully used a tether.