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
