Detection of DNA by electrochemical impedance spectroscopy (EIS) has been reported by many authors and assays have been developed using lab setups. However, as for most detection assay methods there are issues to address to enable the development for the sensor market: Long time-to-result & high complexity for labelled assays and a lack of sensitivity and reproducibility for label-free assays. This work considers two different approaches to address the issues of time-to-result and assay complexity. The first part presents work on achieving rapid sequence-specific electrochemical detection of DNA hybridisation to complementary DNA on an electrode surface. To accomplish assay sensitivity to low DNA target concentrations, a signal amplification strategy is often necessary. One approach is to couple an enzyme to the hybridised target molecules and to deposit insoluble dyes in the subsequent enzymatic reaction, which enhances sensitivity through an increase in the impedance signal in presence of a redox mediator. The time typically taken for this process (20 – 40 min) precludes the use outside lab setups. Therefore, a protocol for sensitive detection in the presence of redox mediator is demonstrated on a practical timescale required for use in sensor applications. Based on these results a model for the fundamental understanding of the amplification reaction is presented which explains the retention of sensitivity at these enhanced timescales. This also enabled further optimisation of the assay for application in single base pair mismatch detection in biologically relevant sequences. Moreover, direct detection of the precipitate formation is demonstrated which enables real-time measurement of the enzymatic reaction without redox agent addition and with enhanced mismatch discrimination. The second part investigates the possibility to detect DNA non-sequence-specifically by non-Faradaic means. This approach aims at reducing assay complexity by establishing whether it is possible to sense the presence of polymeric DNA in solution by measuring changes in the properties of the electrochemical double layer without DNA surface hybridisation. In a sensor setup this approach could be linked to a polymerase chain reaction (PCR) to discriminate polymer from nucleotide monomer and thereby enable PCR progress to be monitored. In this work the response in the electrochemical double layer at the interface of blocked metal electrodes and solutions containing DNA are studied by means of EIS. Blocking layers were applied to the electrode surface to prevent unspecific adsorption of molecules and ions to the metal surface whilst preserving the sensitivity to detection of changes in the double layer. The characteristics of surface blocking layers on disposable electrodes are studied as they are key to understand the double layer properties at a blocked surface. A number of self-assembled monolayers are compared with respect to their temperature stability and their blocking characteristics at different potentials and ion concentrations. This established the basis to study the effect of the presence of, initially, a model polyelectrolyte and, ultimately, DNA on the double layer. Polyelectrolyte detection is successfully shown for the model polyelectrolyte, polyacrylic acid. DNA detection was more challenging and possible causes for deviation from the polyacrylic acid response are discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700046 |
| Date | January 2015 |
| Creators | Kaatz, Miriam |
| Contributors | Mount, Andrew ; Bachmann, Till |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17890 |
Page generated in 0.0017 seconds