AN AMPEROMETRIC FRUCTOSE BIOSENSOR BASED ON FRUCTOSE DEHYDROGENASE IMMOBILIZED IN A MEMBRANE MIMETIC LAYER ON GOLD

A prototype amperometric fructose biosensor based on membrane-bound fr uctose 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 throu gh chemisorption of a mixture of octadecyl mercaptan and two short-cha in disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochlori de. The membrane-bound enzyme, coenzyme, and additional phospholipid w ere 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 ox idizing potentials, the enzyme electrode responded with catalytic curr ent densities up to 45 mu A/ cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 mu A/cm(2).mM and a detection limit of less than 10 mu M. Biosen sor measurements of D-fructose in apple and orange juice agreed to wit hin a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of inter fering ascorbic acid to the electrode surface, Only a 4% positive erro r was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be par ticularly useful for quantitation of fructose in citrus juice.