Development of some capillary electrophoretic separations and enzyme-based sensors for substances of pharmaceutical and biomedical interest

摘要

The development of Capillary Electrophoresis (CE) over the past decade has witnessed continued expansion, and it is now recognised as a powerful analytical technique. In Chapter 1 the concepts of CE are introduced and the various separation modes available to CE are discussed. Combinatorial Chemistry is a technique that is rapidly re-shaping the drug discovery process. This novel and cost-effective approach is capable of synthesising vast numbers of chemical variants all at one time, followed by the screening of these molecules for their bio-activity. This growing interest in combinatorial chemistry by the pharmaceutical industry has influenced the development of new analytical techniques for the analysis of such complex molecules. In Chapter 2 a CE method was developed for the separation of NSG-peptoid combinatorial libraries. In total, six libraries were examined. The separation power and versatility of CE makes it an ideal tool for the analysis of such libraries. A particular attraction is the ease with which the selectivity can be manipulated to bring about the efficient separation of these libraries. Chapter 3 discusses a CE method for the separation and analysis of products and intermediates of L-Dopa oxidation with Diode Array Detection (DAD). L-Dopa is one of the most important pre-cursors in the melanogenesis pathway. In this study both the tyrosinase enzyme and sodium periodate were used to catalyse several key steps in this pathway. Because of the instability of several of the intermediates formed, the detection and isolation of these compounds has remained problematic. Herein the advantages offered by CE are discussed and comparisons made with spectroelectrochemical techniques. Cyclic voltammetry and chrononcoulometric methods were used for the characterisation of the final polymeric product, melanin, and its utility as a sensor/biosensor assessed.ududRecent advances in biosensor development are discussed in Chapter 4, in particular, for the detection and monitoring of toxins. The suitability o f electroactive redox polymers as artificial mediators is examined and the concepts of charge propagation through such polymers. Recent trends have been to ‘wire’ a redox enzyme to an electrode via the use of these redox polymer chains. The development of a ‘reagentless’ immobilised enzyme inhibition sensor for respiratory poisons is described in Chapter 5. This system was based on the co-immobilisation of tyrosinase and a redox polymer, [Os(bpy)2 (PVT)ioCl]Cl (OsPVI), where bpy is the 2,2’-bipyridine ligand and the (PVI)io is poly-N-vinylimidazole indicating a ratio of co-ordinated redox sites to free pendant groups of 1:10. The hydrogel enzyme electrode allows ‘reagentless’ sampling of the enzyme activity by electrochemicaliy ‘switching on’ the enzymatic reduction of oxygen through electrochemical reduction of the immobilised osmium redox couple. This tyrosinase basedudsensor is capable of detecting any modulator of enzyme activity. Both homogenous and heterogeneous enzyme inhibition were investigated. The respiratory poison azide, which inhibits the tyrosinase enzyme, was selected as a model inhibitor to demonstrate the feasibility of the approach. It is envisaged that this system can be extended for the detection of other inhibitors of tyrosinase such as cyanide. Finally, Chapter 6 discusses the conclusions reached during the course of this research and looks at possible areas for future research.