Members of the recently discovered family of short non-coding RNAs, termed microRNAs (miRNAs), regulate the expression of most genes encoded by the human genome by repressing translation of messenger RNAs to proteins. MiRNAs are stably expressed throughout the body and can be detected robustly and reproducibly by RT-qPCR in body fluids such as blood and urine. Alterations in circulating miRNA profiles have been associated with cancers of the brain, breast and liver, and miRNAs hold great promise as biomarkers of numerous other diseases. However, current methods for miRNA biomarker detection rely on laborious, expensive and expert techniques, and involve invasive biopsy acquisition. The research contained within this thesis focusses on the development of a non-invasive, inexpensive and rapid electrochemical analytical test to quantify miRNA in human urine samples. Therefore we describe how glassy carbon and disposable screen printed carbon electrodes (SPCEs), were modified through electropolymerisation of a naphthalene sulfonic acid derivative. DNA complementary to a target miRNA was attached and the sensor analysed via electrochemical methods using a ferri/ferrocyanide electrolyte. After hybridising with a miRNA target, this analysis was repeated and compared to the original DNA-only analysis to give a corresponding change. This was performed using buffered solutions and shown to be sensitive to 20 fM and selective against sequences with a single mismatch; urine analysis was also performed. The method was then adapted for use with screen printed electrodes, using a new chlorination solvent system, to a lowest detected concentration of 10 fM. The ink materials used for the production of the SPCEs were optimised and a new design developed to allow for multiple analyses on one sensor. A small number of diabetic kidney nephropathy (DKN) patient and healthy control urine samples were then analysed for biomarkers we have recently identified, comparing their relative expression levels.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:732259 |
| Date | January 2017 |
| Creators | Smith, Daniel |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/107718/ |
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