Amplification-free detection and live-cell imaging of nucleic acids
Brennan, Eoin (2015) Amplification-free detection and live-cell imaging of nucleic acids. PhD thesis, Dublin City University.
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A number of approaches which aim to achieve amplification free nucleic acids detection are reported. The first approach uses dual function platinum nanoparticles to detect DNA. These particles physically separate DNA capture and electrocatalytic detection, which allows for significant enhancement for the sensitivity of DNA detection. Platinum nanoparticles were grown in the defect sites of an alkane thiol monolayer. These particles could then be functionalised with a capture DNA strand, and subsequently released from the surface by sonication. Using this method attomolar limits of detection were achieved. A molecular beacon for the detection and quantification of miR-132 is also reported. The stem-loop structure comprises a sequence complementary to miR-132, modified with a 6-FAM dye and dabcyl quencher on either end. In the absence of the target, self-binding occurs bringing the luminophore and quencher into close proximity significantly decreasing the emission intensity. In the presence of miR-132, the signal is greatly enhanced, with a linear increase in intensity from a molar ratio of 0.25 to 2.00 of target. The structure could also efficiently differentiates between target and mismatched nucleic acid sequences. In the presence of a single base mismatch, the intensity is approximately a factor of 2 lower than a fully complementary target reflecting its lower association constant. The molecular beacon was then introduced into neuroblastoma cells by electroporation, allowing the miR-132 to be imaged within live cells. miR-132 appears to be localised within the nucleus of the cells where its concentration is of the order of 1μM. Transfection of the cells with an anti-miR-132, resulted in a decrease in the average emission intensity of 18%, however due to high variability in miR-132 between individual cells, confirmation of a statically significant difference was not possible. A displacement assay for the detection of miR-132, a biomarker of neuroblastoma, was also investigated. In this work, a capture strand of DNA complimentary to miR-132 was bound to a surface. A probe strand of DNA partially complimentary to this capture was attached to a large microsphere functionalised with Cy5 dye for visualisation of the particle. These microspheres where then bound to the surface by hybridization. This caused quenching of the dye excited state by the gold. When the fully complimentary miRNA target was added, it displaced the probe strand, and attached microsphere. This resulted in a “switching on” of dye emission from the particles. This has the potential for single molecule miRNA detection, with a single binding event can generate a large detectable signal.
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