Identifier: TDX:3045
Authors: Magriñá Lobato, Ivan
Abstract:
This thesis is focused on the development of electrochemical DNA sensors, which are decentralized analytical devices for use at the point-of-need for the detection of DNA sequences. These sensors can find widespread application in a number of diverse areas, including clinical diagnostics, disease monitoring, food analysis, forensics, bioterrorism and environmental control. However, electrochemical DNA sensors still have a number of problems that need to be addressed, including a need for the amplification of the target sequence, the need for post-amplification generation of single stranded DNA, target hybridization with a complementary capture probe and finally further reporting steps to detect the hybridized target DNA sequence. In this thesis we focused on the elimination of some of these steps to bring electrochemical DNA sensors closer to the point-of-need. To eliminate the reporting step we used ferrocene labelled dATP (dAEFcTP) to obtain already labelled target amplicons. To eliminate the single stranded generation step we followed two different strategies, the use of tailed primers instead of regular primers, or, alternatively performing DNA amplification directly on the electrode surface. The incorporation of dAEFcTP has been demonstrated in primer extension and amplification experiments. dAEFcTP and tailed primers have been used to develop a singleplex electrochemical sensor to detect K. armiger in seawater samples, and a duplex electrochemical sensor to detect B. anthracis in real samples. The effect of the dAEFcTP:dATP ratio on DNA amplification yield, ferrocene peak intensity and limit of detection has been studied, as well as the effect of the electrolyte solution on the ferrocene peak intensity. Finally, the use of dAEFcTP in combination with isothermal recombinase polymerase amplification was explored.
Repositori URV