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Title: Nucleic acid detection using oligonucleotide cross-linked polymer composites
Author: Ferrier, David Christopher
ISNI:       0000 0004 7224 8013
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2017
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There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.
Supervisor: Hands, Philip ; Smith, Stewart ; Shaver, Michael Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: microRNA ; biomarkers ; diagnostic tools ; carbon/- monomer mixture ; cross-linker density