Protein engineering strategies, such as directed evolution, generate large libraries of enzyme variants, typically in the range of 106-108 variants. However, the availability of rapid, robust high-throughput screening methods has often limited the impact of directed evolution in discovering enzymes with enhanced catalyst performance. Raman spectroscopy is an established analytical technique, providing molecular specific information, permitting analysis in aqueous solutions and as such is an attractive, alternative screening method for biological systems. Although an inherently weak physical phenomenon, enhanced Raman scattering techniques, such as surface enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can be used to overcome the associated sensitivity issues. Herein, we successfully monitored xanthine oxidase (XO) catalysed conversions of xanthine to uric acid, before extending to hypoxanthine, using two contrasting Raman scattering enhanced approaches. Firstly, a SERS-based assay was developed utilising silver nanoparticles to measure analytes directly and quantitatively on micromolar scale, in the absence of chromogenic substrates or lengthy chromatography. Secondly, a UVRR approach was developed enabling monitoring of the XO-mediated reaction in real-time and without the need to quench the system. Significantly, both methods demonstrated over >30 fold reduction in acquisition times (when compared to conventional HPLC analysis), and offered excellent medium-term reproducibility and accuracy of results over significant time periods. Furthermore, investigations were made into developing this SERS-based assay into an enantiomeric screen using another vibrational spectroscopy approach, Raman optical activity (ROA), along with circular dichroism (CD). Successful chiral reduced nanoparticles were synthesised, with multiple characterisation techniques employed, affording enantiopure Au-cysteine and Ag-tyrosine colloids. However, it was not possible to generate consistent and reproducible SEROA responses, with these techniques ultimately being unsuccessful in analysing these chiral sensitive nanoprobes, and thus differentiating between the D- and L- forms. Finally, a novel SERS-based approach, in combination with the standard addition method (SAM), was developed for the routine analysis of uric acid (end product in XO catalysed reaction(s) and biomarker for various diseases), at clinically relevant levels in urine samples from patients. Results were highly comparable and in very good agreement with HPLC analyses, with an average < 9% difference in predictions between the two analytical approaches across all samples analysed, and a 60-fold reduction in acquisition time (when compared with HPLC). Together, the research presented in this thesis demonstrates the suitability of Raman enhanced techniques for quantitative analysis, measuring the analytes directly using a portable Raman instrument and, most importantly, offering significant reductions in acquisition times when compared to established analytical techniques.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:727985 |
| Date | January 2017 |
| Creators | Westley, Chloe |
| Contributors | Goodacre, Royston |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/raman-spectroscopy-and-its-enhancement-techniques-for-the-direct-monitoring-of-biotransformations(4ff7ebac-048b-4d81-b13c-7087a2028464).html |
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