Novel enzymatic methods of DNA detection using enhanced Raman scattering

Over the years there have been a number of significant advances in DNA detection. In particular the advent of the Polymerase Chain Reaction (PCR) has enabled extremely low levels of DNA to be amplified and quantified, and along with fluorescence based detection measurements, has provided a reliable method of detecting genomic concentrations of DNA. However, throughout the years it has become apparent that although successful, these techniques have inherent complications that cannot be ignored in a research field as progressive as medical diagnostics. As a result, the future of biomolecule detection relies on the development of novel techniques that are able to eliminate the problems associated with conventional detection methods. Detection methods amenable to this goal include oligonucleotide nanoparticle conjugates for colorimetric DNA detection, and surface enhanced resonance Raman scattering (SERRS), which provides improved sensitivity in comparison to fluorescence along with the potential for multiplexed analysis. Additionally, the use of enzymes in DNA detection to provide a method of target cycling and signal amplification provides a path toward amplification free analysis. The overall aim of this research was to develop novel methods for the detection of DNA based on enzymatic activity. This primarily involved implementing the enzyme lambda exonuclease in a target cycling strategy, alongside DNA labelled with a donor and acceptor FRET pair for use in fluorescence detection. This enzyme was also utilised within a nanoparticle assembly system, whereby oligonucleotides were conjugated to gold and silver nanoparticles for use in SERRS analysis. Finally, alternative enzymatic processes were investigated for the detection of DNA using resonance Raman and surface enhanced resonance Raman scattering. The catalytic activity of a DNAzyme on certain peroxidase substrates was investigated and used within a catalytic beacon assay protocol for the detection of DNA. Also, the enzymatic activity of gold and silver nanoparticles was investigated and their potential as enzyme mimetics for analyte detection was examined.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2013

Author dc:creator

• McKeating, Kristy Stephanie

DOI

10.48730/zr51-zh37

Identifier

T13562

OAI identifier oai:identifier

oai:strathclyde:1z40ks82j