The University of ManchesterDavid Paul CowcherDoctor of PhilosophyThe Development of Enhanced Raman Scattering for the Trace Analysis of Biomolecules27th September 2013Raman spectroscopy is an established analytical technique for determining molecular structure, whose major drawback is lack of sensitivity. Enhanced Raman scattering techniques, such as surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS), utilise nanoscale substrates to enhance the Raman signal through the interaction of surface charges with the incident electromagnetic radiation.Here, nanoparticle-based SERS was used to detect dipicolinic acid (DPA), a biomarker for bacterial spores. Whilst this has been demonstrated previously, the use of a different nanoparticle aggregation mechanism and the inclusion of an internal standard has enabled a SERS detection method to be developed that is quantitative to almost an order of magnitude lower than previously reported. Moreover, for the first time, a nanoparticle-based SERS method was applied to the detection of viable Bacillus spores.Investigations were made into the possibility of SERS enhancement using deep UV laser excitation at 244 nm using a novel boron nitride surface material. This semiconductor has a band gap of comparable magnitude to the laser excitation wavelength and therefore had the potential to impart a SERS enhancement via a chemical enhancement mechanism. Whilst initial results looked promising using Rhodamine 6G as a test analyte, it was not possible to demonstrate reproducibly and no enhancement was observed on other analytes that were tested.TERS was shown to be able to discriminate between glycosylated and non-glycosylated forms of protein molecules, based on the measurement of just a few molecules at a time. This was achieved even without control of the protein interaction with the TERS substrate. The vibrational peak positions in TERS experiments were shown to be highly dependent on the analyte's orientation relative to the TERS tip, giving variable and complex spectral data. As such, the data processing and analysis methods had to be carefully considered in order to eliminate bias.Lastly, a novel SERS detector for high-performance liquid chromatography (HPLC) was built and tested. It was shown to be able to quantify purine bases from mixtures in tandem with, and in lower amounts than the conventionally used UV absorbance detection, even when the analyte peaks were co-eluting. This quantitative analysis is conducted on-line and in real-time, making it applicable to high throughput applications.Together the four research projects presented in this thesis make a significant contribution to the field of enhanced Raman scattering and promote its sensitivity and reproducibility as a quantitative analytical technique for the trace analysis of biomolecules.
|Date of Award||1 Aug 2014|
- The University of Manchester
|Supervisor||Royston Goodacre (Supervisor)|
- Data analysis
- Protein glycosylation