A problem driven approach to the miniaturizsation and automation of enzyme-based assays and an investigation of the dissipation of cyanazine and bromide in wetland mesocosms

Dissertation (Ph.D.)--University of Kansas, Chemistry, 1997.Analytical chemistry, as a discipline, has something of an identity crisis. Its role in the development of instrumental methods frequently relegates it into the category of a technology, while the curiosity-driven fundamental studies of the technology drive it into the category of a science. Fortunately, it is possible to do both science and technology simultaneously. Fundamental studies and innovation in technology lead to the same result, better analyses. For this reason, regardless of the strategy of the study, analytical chemistry at its best is problem driven research. This work reflects the approach that it is of paramount importance to answer fundamental and technological questions in the context of real world problems. Biological recognition elements have enjoyed popularity recently in analytical chemistry. Receptors such as antibodies have been demonstrated to achieve low limits of detection in diverse complex matrices from serum to ground water. Enzymes are an integral part of such bioassays, providing increased sensitivity as a result of the time dependence of their product production. The power of enzyme based immunoassays has been demonstrated since their introduction in 1971. However, enzymes alone have been shown to be valuable bioanalytical tools as exemplified by the plethora of research in the area of implantable amperometric glucose oxidase (GOx) based biosensors for glucose. Biosensors or biologically based analytical methods must continue to evolve to prove useful in the changing landscape. Recent trends in new drug development have made different demands on the analytical chemist. No longer are simply good limits of detection, sensitivities, or high theoretical plates satisfactory for pharmaceutical companies. As thousands of combinatorial libraries queue up for analysis, speed and sample throughput have become increasingly important. Joining drug companies are hundreds of superfund sites, with millions of complex, toxic samples to be analyzed in a limited time frame. New demands brought about by the political climate in hospitals are forcing clinical labs to consider innovations to improve the speed and cost effectiveness of routine screening. Other considerations, such as minimizing contact with biological fluids brought about by concerns over hepatitis and HIV, renew interest in fundamental research in sample handling and preparation. This work reflects the approach that it is of paramount importance to answer fundamental and technological questions in the context of real world problems. In Part One two related problems are described. Chapter One demonstrates that the enzyme linked immunosorbent assay (ELISA) may be used to assess the immunogenicity of the immobilized GOx on an implantable glucose sensor. Chapter Two addresses the problem of how enzyme based analytical methods such as those used in Chapter One are automated and miniaturized. Part Two presents in Chapter Three the results of the environmental analysis of pond water drawn from wetland mesocosms treated with a herbicide and a volume tracer. The significance of this work is two-fold. First, inherent in a field study of this scope is the analytical challenges associated with sampling, sample handling and analysis specifically with respect to throughput. Explicitly, and more importantly, are the valuable discoveries related to the non-conservative behavior of both compounds applied