Thesis

Surface enhanced Raman scattering based assays for DNA detection by

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Awarding institution
  • University of Strathclyde
Date of award
  • 2013
Thesis identifier
  • T13470
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Abstract
  • Surface enhanced Raman scattering (SERS) based molecular diagnostic assays for the detection of specific DNA sequences have been developed in recent years to compete with the more common fluorescence based approaches. Current SERS assays require either time consuming separation steps that increase assay cost and can also increase the risk of contamination, or are negative assays where the signal intensity decreases in the presence of target DNA. A new separation free SERS assay with an increase of signal intensity when target DNA is present using a specifically designed SERS primer has been developed in this thesis. The presence of specific bacterial DNA from Staphylococcus epidermidis was detected using Polymerase Chain Reaction (PCR) and SERS and indicates a new opportunity for exploration of SERS assays requiring minimal handling steps. SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption between single stranded and double stranded DNA onto nanoparticle surfaces. Seven parameters important for improved positive SERS assays for real applications were investigated using a model system for optimization experiments. This was followed by a PCR assay to detect pathogen DNA, and the introduction of a novel assay which utilizes the 5'·3' exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye labelled single stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However within the PCR assays it was found that uni- and bi-molecular SERS primers performed very similarly, the most reproducible results were obtained using the 5'·3' exonuclease digestion assay. The SERS based assays developed in this thesis offer new routes over conventional fluorescence based techniques.;SERS primers have been designed for multiplex pathogen detection. A selection of dyes for multiplex pathogen detection was made. Assay designs for the SERS primer digestion, and the SERS primer extension assay were made successfully, followed by cross reaction analysis of the oligonucleotides required for the multiplex detection. The results indicate that it is feasible to perform a multiplex detection using both types of assays. However it was decided to postpone the final multiplex detection part of the project and investigate assay systems that require less complex designs. Additionally, to simplify the SERS primer assay novel cationic silver nanoparticles were synthesised and tested for discrimination between double stranded and single stranded DNA. These silver nanoparticles with a positive surface charge produced poor discrimination between double and single stranded DNA. However these cationic silver nanoparticles provide a simplified SERS substrate for DNA detection.
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  • Strathclyde theses - ask staff. Thesis no. : T13470
DOI
Date Created
  • 2013
Former identifier
  • 991197

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