Amperometric biosensors of food analysis relevance: Emphasis on the neurotoxin beta-ODAP in grass pea

Abstract: Enzyme-based amperometric biosensors have been developed and characterized in flow systems for a variety of analytes such as [beta]-N-oxalyl-[alfa],[beta]-diamionpropionic acid ([beta]-ODAP), L-glutamate, glucose, lactate, and sucrose, that have food analysis relevance. The principle of enzyme "wiring" using polymeric mediators poly(1-vinylimidazole) {(Os-4, 4' dimethylbipyridyl)2Cl3+/2+} and poly(4-vinlypyridine {Os(N,N'-dimethyl-2,2'-biimidazole)3}3+/2+ has been employed in most of the sensors developed, which resulted in the fabrication of sensitive, stable and reproducible biosensors with carbonaceous electrodes (spectrographic graphite and glassy carbon). Biosensor development, application and method validation for the analysis of the neurotoxin contained in Lathyrus sativus (grass pea), [beta]-ODAP, a causative agent for the disease called neurolathyrism has been emphasized in this thesis. The hydrogel based (the above polymers) bienzyme biosensor (glutamate oxidase and horseradish peroxidase) has been applied to real samples i.e. raw grass pea seed samples collected from lathyrism prone area of Ethiopia, and processed (solid-state fermentation) grass pea. Moreover, it has been successfully applied in the estimation of [beta]-ODAP degrading abilities of microbes isolated from cow rumen, which are adapted to grow on media containing [beta]-ODAP as the soul carbon source. The validation studies showed that the biosensors developed for this analyte are practical ones. In all these analyses, the biosensors were used as end column detection in a liquid chromatographic analysis set up. Additionally an attempt was made to replace the enzyme HRP, often used in conjunction with oxidases for the development of biosensors working in low potential regime for substrates of the oxidase, with the inorganic crystalline material Prussian Blue (PB). PB can be electrochemically deposited (from a mixture of ferric chloride and ferric hexacyanoferrate) on electrode surfaces and has catalytic activity for selective electrochemical reduction of hydrogen peroxide. Different glassy carbon materials and procedure for electrochemical deposition of PB have been investigated for its optimum performance. The stability problem of PB, especially at neutral and basic pH has been improved by adding tetrabutylammonium toluene-4-sulfonate (TTS) either in the electrolytic solution (in one of the electrodepostion steps) or in the carrier buffer during flow injection analysis. A fairly sensitive and stable biosensors could be developed for sucrose, Lactate, and [beta]-ODAP based on PB modified glassy carbon electrodes further modified by the respective oxidases