An Amperometric Fructose Biosensor Based on Fructose Dehydrogenase Immobilized in a Membrane Mimetic Layer on Gold

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Abstract
A prototype amperometric fructose biosensor based on membrane-bound fructose dehydrogenase (Gluconobacter sp.) and the coenzyme ubiquinone-6 immobilized in a membrane mimetic layer on a gold electrode has been constructed and tested. A bare gold electrode first was modified through chemisorption of a mixture of octadecyl mercaptan and two short-chain disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochloride. The membrane-bound enzyme, coenzyme, and additional phospholipid were codeposited through a detergent dialysis protocol. The short-chain modifiers may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate and phospholipids enable hydrophobic interaction with the largely lipophilic, membrane-bound enzyme. At oxidizing potentials, the enzyme electrode responded with catalytic current densities up to 45 μA/cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 μA/cm(2)·mM and a detection limit of less than 10 μM. Biosensor measurements of d-fructose in apple and orange juice agreed to within a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of interfering ascorbic acid to the electrode surface. Only a 4% positive error was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be particularly useful for quantitation of fructose in citrus juice.