Abstract

Towards Integrated PCR for Biosensing by Electrochemical Control of DNA Hybridisation

Till Bachmann, Professor/Reader, University of Edinburgh

Our research aims to solve a fundamental challenge related to the need for target amplification to achieve low limits of detection with nucleic acid biosensors and the requirements for integration into contained enabling sample-in-answer-out or point-of-care-testing systems. The principle aim is to develop an alternative method for driving DNA amplification that facilitates integration of nucleic acid amplification and biosensing on one platform using electrochemistry. The presentation outlines the fundamental work on controlling denaturation and hybridization of double-stranded DNA using electrochemistry and describes the studies on amplification of DNA by means of electrochemical PCR (ePCR) using disposable screen-printed electrodes. Control of denaturation and hybridization of double-stranded DNA is fundamental to hybridization-based bioanalytical methods ranging from Southern blotting over DNA biosensors to next generation sequencing. We recently demonstrated reversible electrochemical control of DNA hybridization using an electroactive DNA binding compound. Using a model system of short synthetic DNA oligonucleotides, we showed that, at a constant temperature, redox-state switching of the compound alters its DNA binding properties thereby enabling external control of DNA hybridization. Cycles of electrochemically-controlled denaturation and hybridization were demonstrated with in situ UV-Vis and circular dichroism spectroelectrochemistry. As no extreme conditions were implicated, and the precise temperature-regulation of reversible denaturation and hybridization of DNA was circumvented, we further demonstrated the applicability of this novel method by developing an isothermal ePCR. Amplification of DNA by means of ePCR was exemplified with disposable screen-printed electrodes via exhaustive electrolysis using synthetic DNA oligonucleotides. The amplification was analysed with gel electrophoresis while standard PCR, carried out in thermal cyclers, were employed