A solid state pH sensor for RNA detection
Author(s)
Aziz, Shahid
Type
Thesis
Abstract
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.
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.
Date Issued
2011
Date Awarded
2011-07
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Cass, Tony
Creator
Aziz, Shahid
Publisher Department
Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)