The aim of this research was to progress the application of surface enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy for the detection of longchain DNA fragments and amplification products.The use of SERS for the detection of DNA has significant potential in terms of sensitivity and multiplex target detection from nanoparticles. Current methods are usually performed with short chain, synthetic DNA fragments or require additional separation steps for the detection of longer chain DNA fragments. An integrated method for the rapid and sensitive detection of long-chain DNA fragments (â¥ 100-base) with minimal preparation and separation steps was demonstrated. Key to this was the DNA sequence-specific assembly of silver nanoparticles labelled with a Raman tag to provide an enhanced signal from the tag and hence molecular recognition of the target DNA. Effective detection of long-chain DNA was achieved with head-to-head probes and by adding polyethylene glycol 10000 to the hybridisation buffer. This gave a 34-fold discriminatory enhancement factor when applied to a synthetic target. A structured approach toward maximising hybridisation procedures and SERS response has been described, followed by an initial demonstration of SERS detection of a single-stranded DNA target amplified by asymmetric PCR which was used without further separation. This has implications for future developments in using SERS for DNA detection due to the new-found ability to integrate SERS with asymmetric PCR.Furthermore, an alternative strategy for the detection of a recombinase polymerase amplification (RPA) product was demonstrated using tailed primers, an enzyme linked reporter conjugate and resonance Raman spectroscopy. The amplification ofDNA by RPA could be achieved within 20 minutes and a colorimetric change could be detected using resonance Raman spectroscopy with discriminatory value at the lowest concentration of 100 aM. This demonstrated the first application of resonance Raman spectroscopy for the detection of an RPA amplification product and indicates significant potential for the integration of resonance Raman spectroscopy with an emerging rapid isothermal nucleic acid amplification technique.The research described here has successfully demonstrated the sensitive and rapid detection of long-chain DNA fragments and amplification products using both SERS and resonance Raman spectroscopy. Furthermore, the methods presented require minimal sample preparation steps for the detection of the long-chain DNA fragments and amplification products. This indicates significant progression from current literature which commonly describes the detection of short, synthetic DNA fragments or utilises methods requiring complex physical separation or purification steps for the detection of longer DNA fragments. The findings therefore advance the potential for the application of Raman spectroscopy for the detection of biologically relevant nucleic acid target sequences, and it hoped that this will encourage further development of Raman spectroscopy for the application of DNA detection in future.
|Date of Award||17 Feb 2020|
- University Of Strathclyde
|Sponsors||EPSRC (Engineering and Physical Sciences Research Council)|
|Supervisor||Duncan Graham (Supervisor) & Karen Faulds (Supervisor)|