This thesis outlines a study of the physical changes that hybridisation imposes on single-stranded DNA (ssDNA) immobilised by one end to a substrate, and of how such physical changes can be exploited to detect specific sequences of DNA in a target solution. The system studied was composed of a mixed monolayer of 20mer ssDNA with C6 alkanethiolate modifications on their 3??? ends and short-chain hydroxyterminated alkanethiolates, on a gold substrate. It was prepared using the self-assembly properties of alkanethiols on gold. Atomic force microscopy images showed that the end-immobilised ssDNA is flexible enough to lie over the diluent hydroxy-terminated self-assembled monolayer (SAM). Hybridisation was shown to cause the DNA to become more rigid and stand up off the substrate due to an increase in persistence length. Such physical changes of the DNA upon hybridisation were significant enough to be exploited in the development of a DNA recognition interface. The recognition interface was designed with the view of keeping it both simple to make and simple to use, and was coupled with electrochemical transduction. A label-free recognition interface was developed that relied on the oxidation of the sulfur head group of the alkanethiolate SAM to detect hybridisation (firstly air oxidation and then electrochemical oxidation). It produced a positive signal upon hybridisation with complementary target DNA. Improvements in the reliability and robustness of the recognition interface were made using a labelled approach. The labelled version employed electroactive molecules as labels on the 5??? ends of the probe DNA strands. Two labels were investigated ??? anthraquinone and ferrocene. The flexibility of the ssDNA ensured that the redox labels were able to directly access the underlying gold electrode. Hybridisation was expected to remove the labels from the electrode due to an increase in the DNA???s persistence length, and thus perturb the electrochemical signal. The use of ferrocene as a label provided a ???proof-of-concept??? for the system. The labelled recognition interface provides a foundation for the future development of a simple, reliable, and selective DNA hybridisation biosensor.
| Identifer | oai:union.ndltd.org:ADTP/188125 |
| Date | January 2006 |
| Creators | Mearns, Freya Justine, Chemistry, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. Chemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Freya Justine Mearns, http://unsworks.unsw.edu.au/copyright |
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