Development of Highly Sensitive and Selective DNA Sensors Based on Conducting Polymers

Abstract

The research reported in this thesis demonstrates novel methods for increasing the sensitivity and selectivity of the label free electrochemical DNA sensors. Electrochemically conducting polymers (ECPs) are used as the sensor substrate. Novel fabrication methods for these devices are demonstrated. A robust and versatile micro fabrication technique – Direct Writing of Conducting Polymers - was developed for the addressable patterning of CPs. Utilizing a modified Scanning Ion Conductance Microscope (SICM), localized electrodeposition of 2- and 3D poly(pyrrole) (PPy) micro structures was realised on various substrates. This method is very promising to fabricate CP arrays that can be utilised in biosensor arrays. Oligonucleotide hybridization to a complementary sequence that is covalently attached to an ECP coating of the working electrode of an electrochemical cell causes an increase in reaction impedance for the ferro-ferricyanide redox couple. The use of this effect to measure, in real time, the progress of DNA polymerase chain reaction (PCR) amplification of a minor component of a DNA extract was demonstrated. The forward primer was attached to the ECP. The solution contained other PCR components and the redox couple. Each cycle of amplification gave an easily measurable impedance increase. Target concentration could be estimated by cycle count to reach threshold impedance. As proof of principle, an electrochemical real-time quantitative PCR (e-PCR) measurement in the total DNA extracted from chicken blood of an 844 base pair region of the mitochondrial Cytochrome c oxidase gene, present at ∼1 ppm of total DNA, was demonstrated. Detection and semiquantitation of as few as 2 copies/μL of target could be achieved within less than 10 PCR cycles. A critical factor for manufacture of devices is the speed and ease of attachment of the recognition sequence to the electrode. A facile method is demonstrated here for the direct immobilization of recognition probes. New monomers are described that are carboxylate functionalized and which have very low oxidation potentials. Amine (-NH2) functionalized oligonucleotides (ONs) could be attached to the carboxylic acid (-COOH) groups of the newly synthesized monomers. Functionally-active ECP films were prepared by direct polymerization of ON-functionalised monomers. The deposition was under mild conditions and was completed in less than 1 second. Devices prepared in this way showed remarkable sensitivity, selectivity and detection limits down to aM levels when used in the label-free detection method. Although not realised in this thesis the direct writing methodology could be used for an industrial-scale fabrication of such sensors.