DNA diagnostic assays using Surface Enhanced Raman Scattering (SERS)

• Main Author: Harper, Mhairi
• Published: University of Strathclyde 2013
• Subjects: 540
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.588971

DNA is the prerequisite for all biological life and its discovery has revolutionised the understanding of biomolecular interactions and disease expression. This has enabled significant improvements in patient diagnosis and medical treatment to be carried out. The advancements in technology and instrumentation have continually progressed this knowledge and continue to push the boundaries of diagnostic and clinical advancements. One effective way to achieve this is through application of dye labelled DNA sequences and metallic nanoparticle suspensions. This research details an understanding of the interaction between dye labelled oligonucleotides and silver nanoparticle surfaces, which generate strong surface enhanced Raman scattering (SERS) responses through specific hybridisation events which correlate to the presence of targeted sequences. During this study, the attraction of oligonucleotides onto metal nanoparticles was shown to be driven through the DNA nucleobases. Therefore, the increased exposure of the base groups within single stranded DNA sequences generated a higher affinity for metal surfaces which in turn produced stronger SERS responses when compared to double stranded DNA. This principle was utilised within a DNA detection assay to successfully demonstrate the presence of target DNA sequences. Two novel DNA detection assays were also investigated which utilised SERS to determine the presence of sequences relating to the methicillin resistant Staphylococcus aureus (MRSA) strain. A solution based detection method was developed through coupling a TaqMan assay with SERS. This combination enabled highly specific detection of clinically relevant sequences of MRSA to be obtained with 7 fM limits of detection achievable. The multiple detection of different genomic S. aureus strains was achieved through the molecularly specific and narrow emission spectral profiles obtained. A contrasting DNA detection strategy which relies upon the hybridisation of comple mentary sequences on a solid substrate surface was shown. Silver nanoparticles were functionalised with specific DNA sequences and a variety of SERS active molecules, enabling the selective detection of target sequences from nitrocellulose membranes. This thesis has exploited SERS to enable the specific identification of DNA sequences to be achieved via utilisation of silver nanoparticles. Through SERS, an insight into the interactions of DNA and silver nanoparticles surfaces has been gained as well as enhancing the sensitivity and specificity achievable within SERS detection assays.