Flow injection analysis by use of a biosensor based on a co-enzyme regeneration system

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Co-enzyme NAD and dehydrogenases were immobilized on insoluble Sepharose,and the regeneration system of the co-enzyme was established. The application of the regeneration system to flow injection analysis (FIA) was performed.1. A direct immobilization method of NAD onto Sepharose was developed by use of the bifunctional reagent, glutaraldehyde (GA). The immobilization mechanism depended on the pH of the reaction mixture of NAD and the GA-activated Sepharose. It was suggested that the structure of methylol type was formed between <alpha> (beta)-unsaturated aldehyde on teh GA-activated Sepharose and N^6-amino group on the adenine moiety of NAD in the acidic region (pH 3). In the neutral or weak alkaline region (pH 8), it was supposed that the immobilization was achieved by the force of ionic interaction between the phosphate moiety of NAD and the positive charge on the support. The immobilized NAD, which was prepared at pH 8, maintained a high co-factor activity and was applicable to co immobilization with alcohol dehydrogenase (ADH).2. The modified gel (R_3-Seph) was prepared by the reaction of CNBr-activated Sepharose with hexamethylenediamine and GA, followed by the same reaction for three times. The produced modified gel had a relatively long size of spacer, and it was applied for co-immobilization of ADH and NAD. The re-usability of the co-immobilized gel mainly depended on the reaction time of GA with Sepharose. The co-immobilized gel, which was prepared by the optimum condition (GA 5%, 2 hr), proposed the linear responses for 0 -0.8 mM ethanol by the batch system.3. The co-immobilized gel of ADH, NAD and diaphorase was prepared by the nearly same method described in section 2. The optimum condition for the R_3-Seph preparation was as follows: GA concentration 20%, reaction time 1 hr. The co-immobilized gel was applied to FIA method to determine ethanol. The FIA method was applicable to ethanol determination (0.1 - 0.5 M).