Optimisation methods of enzyme integration with transducers for analysis of irreversible inhibitors

Manufacturing and application of biosensors cause some problems, the main o f which are: (1) optimisation of enzyme immobilisation process to preserve bio-molecular enzyme structures and maximal value of enzyme activity and (2 ) development of approaches to accomplish a repeated analysis of substances which are irreversible inhibitors of the above mentioned molecules. In thi s paper both problems are analysed. For stabilisation of the structure of b eta-glucose oxidase, urease and cholinesterases during their immobilisation , the following approaches were examined: (1) application of one or a combi nation of the following chemical substances: protein, saccharose, glycerol, and specific substrates or their analogues; (2) optimisation of crosslinki ng methods including application of bi-functional reagents in aqueous and v aporous phases and (3) proper adjustment of technological operation times. Optimisation of these processes allows to preserve about 70-80% of initial enzyme activity. For the repeated analysis of organophosphorus pesticides a nd heavy metal ions, which are irreversible inhibitors of enzymes, the foll owing approaches were applied: (1) treatment of enzyme membrane by special reactivating reagents and (2) application of easily replaceable enzymatic m embrane. Methods of preservation of enzymatic sensors with use of sodium az ide, EDTA and DTT were tested. Optimal conditions of sensor's preservation and reactivation were chosen. (C) 1999 Elsevier Science S.A. All rights res erved.