Fructose determination using immobilized enzymes in a flow system with special emphasis on the effect of isomerism

A flow injection method for the determination of fructose has been developed using immobilized hexokinase, glucose phosphate isomerase and glucose-6-phosphate dehydrogenase. The produced NADH was monitored amperometrically with a graphite electrode modified by an adsorbed Nile-blue derivative. The response was linear from the detection limit, 1μM to 3 mMD-fructose for a sample volume of 25μl. Glucose, the only interfering substrate, was eliminated with a reactor containing immobilized glucose oxidase, mutarotase and catalase. The experiments indicate that hexokinase is selective for theβ-fructo-furanose isomer. The other isomers will spontaneously isomerize toβ-fructo-furanose with time, if the latter is consumed. The fructose response factor of an enzyme reactor or biosensor will therefore depend on the isomer distribution in the original sample solution. The effect of pH and temperature can be controlled, but complexing ligands like borate interfere in a complex way and should be absent.