Electrochemical biosensors have long been used for measuring pH changes in complex biological samples. This research work involves investigating iridium sensors as a potential tool for detecting viral RNA by means of pH sensing during transcription mediated amplification (TMA). The iridium oxide film was formed on gold wire by electrochemical deposition. When complementary DNA/RNA bases are hybridized, they release a pyrophosphate molecule which is eventually hydrolyzed, contributing to the pH change during the TMA reaction which in turn is measured in voltage (mV) by the iridium sensor. Firstly a standard method for bacteriophage MS2 RNA amplification method was established. A Real Time PCR assay for MS2 RNA using TaqMan probe chemistry was used to quantify the MS2 RNA and used as a gold standard for later project. We then used transcription mediated amplification (TMA) technique using reverse transcriptase and RNA polymerase enzymes for producing MS2 RNA amplicons and resulting protons generated during amplification were measured with iridium sensors. The iridium oxide sensor showed linear pH potential response and showed a near ideal Nernstian behaviour in the pH range 2 – 12 with a slope of -60mV/pH. Since TMA is an isothermal amplification technique the iridium sensors produced reproducible and reliable measurements. To increase the cation selective permeability and inhibit possible interferences the iridium oxide sensor was coated with 5% Nafion. The sensor showed the same linearity in voltage response against RNA concentrations in protein enriched samples (20 mg/ml albumin) compare to RNA in water. The iridium oxide sensor was a rapid method of viral RNA detection as the detection time for MS2 RNA was 10 minutes. The lower limit of detection of the iridium sensor was 5ng of MS2 RNA which was comparable to the established real time PCR sensitivity. To further enhance the sensor’s the sensitivity the iridium oxide sensor was prepared on a 3 mm × 3 mm glass slide which increased the slope for 5 ng of MS2 RNA from 3.25mV/min to 4.07mV/min resulting in an improved limit of detection.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:537202 |
| Date | January 2011 |
| Creators | Aziz, Shahid |
| Contributors | Cass, Tony |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/6954 |
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