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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
A new enzyme electrode based on ascorbate oxidase immobilized in gelatin for specific determination of l-ascorbic
1Department of Biochemistry, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey.
Abstract:
A biosensor for the specific determination of l-ascorbic acid in fruit juices and vitamin C tablets was developed using ascorbate oxidase (EC 1.10.3.3) from cucumber (Cucumis sativus L.) in combination with a dissolved oxygen probe. Ascorbate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The phosphate buffer (50 mM, pH 7.5) and 35 degrees C were established as providing the optimum conditions. The biosensor response depends linearly on l-ascorbic acid concentration between 5.0x10(-5) and 1.2x10(-3) M with a response time 45 s. The biosensor is stable for more than 2 months, while more than 200 assays were performed. The results obtained for fruit juices and tablets were compared with DCIP (2,6 dichlorophenolindophenol) method.
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