A biosensor for the determination of amylase activity

Abstract

A new biosensing flow injection method for the determination of alpha-amylase activity has been introduced. The method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, co-crosslinked by gelatin-glutaraldehyde together with bovine serum albumine. alpha-Glucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-D-glucose by mutarotase. beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol l(-1) maltose, which means 2 nkat ml(-1) in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3:1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml(-1). A linear range of current response was 0.1-3 mmol l(-1) maltose, with a response time of 35s. The biosensor was stable at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 degrees C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months).