Synthesis, Characterisation and Application of Functionalised Thiophene-based Conducting Polymers as DNA Sensors

Abstract

Specific DNA detection is important for many applications in areas such as biology, forensics and medical diagnostics which all share the need for fast and inexpensive detection technology. Conducting polymers are now widely investigated as alternative substrates for rapid and label-free DNA sensors. Previous work in this laboratory investigated DNA sensors based on conducting polymer films made from pyrrole but the chemical versatility of thiophene motivated the study of thiophene based conducting polymer substrates in this thesis. Four thiophene conducting polymers were synthesized wherein two of these (TAA and TPDA) shared structural features with pyrrole homologues for comparison of their sensing properties. The following thiophene-containing monomers and the conducting polymers were synthesised and characterised: TAA (5’’:2’’’- terthiophene)-3’’-yl) acrylic acid), TPDA (5-(2’:2’’, 5’’:2’’’-terthiophene)-3’’–yl)] (2E,4E)penta-2,4-dienoic acid ), HTAA (3-[3’,3’’’-bis(hydroxymethyl)-2’:2’’,5’’:2’’’- terthiophene)-3’’–yl (E)acrylic acid ) and AAE ((E)-3-(2,3-dihydrothieno[3,4- b][1,4]dioxin-2-yl)acrylic acid). All polymers were studied by electrochemical impedance spectroscopy (EIS) which was used as the sensor readout to allow rapid and label-free detection. Initial results using PTAA and PTPDA film as DNA sensors in aqueous solution showed poor hybridisation responses that were difficult to consistently reproduce. This prompted a comprehensive EIS study investigating the change of polymers properties when immersed (20 hours) in aqueous electrolytes before attachment of DNA. EIS revealed that the microstructure of these films collapses in aqueous solution for about three hours accompanied by a dramatically decreased capacitance and hindered redox activity and conductivity. Subsequently, a gradual increase in film capacitance resulted from the oxidizing potential applied during the EIS experiment. Discovery of this microstructure collapse in aqueous solution explained the difficulty in obtaining reproducible hybridisation responses. This finding runs contrary to the usual assumption in the literature that conducting polymers are electrochemically stable and may be used straight after preparation for DNA sensing [1-7]. The collapse was largely ameliorated in PHTAA films and the increased electrochemical stability of this polymer was attributed to its greater hydrophilicity endowed by its hydrophilic group (CH2OH). The slight gradual capacitance increase due to film charging induced by the oxidizing potential applied during EIS was also observed in PHTAA film. Poor DNA selectivity of PTAA and PTPDA films originated partly from the collapse and the consequent disruption of the π-bonded “highway” of the polymer backbone. Another contribution towards poor selectivity was the charge storage region that built up underneath the film surface during the time (20 hours) the films were allowed to adjust their microstructure in aqueous solution. EIS was optimized for DNA sensing by examining the effect of the Fe(CN)6 3-/4- redox couple on the DNA sensing of PTAA film which was found to enhance sensing. Additionally, the effect of the oxidation state of PTAA film on its DNA-sensing ability was examined where the oxidized state best detected DNA compared to the reduced state. An alternative modelling scheme that measures the hybridisation response from the change in phase angle on the phase angle plot (rather than the change in real impedance) was also demonstrated. This scheme can be adapted for other electrochemical DNA sensors where the Nyquist plot does not sufficiently characterize the hybridisation response. Unlike the Nyquist plot, the phase angle plot sensitively conveyed useful information about film microstructure changes and oligonucleotide attachment on the film surface for the entire frequency range studied although frequencies in the 103-105 Hz region provided the best indicator of hybridisation.