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Durham University

Department of Biosciences

Profile

Publication details for Dr Martin Schröder

M. Schröder & P. Friedl (1997). Enzymatic determination of D-glucose in cell culture media using glucose oxidase, horseradish peroxidase and 3,3 ',5,5 '- tetramethylbenzidine. The Genetic Engineer and Biotechnologist 17(4): 157-163.

Author(s) from Durham

Abstract

An enzymatic assay for the determination of D-glucose using glucose oxidase, horseradishperoxidase (HRP) and 3,3',5,5'-tetramethylbenzidine (TMB) in cell culture media containing bovine serum is described. The major advantages of this assay compared to other enzymatic assays are as follows. (1) The substrate TMB is a safer chemical than other substrates for HRP. (2) No interference of serum proteins is observed in this assay, in contrast to that reported for enzymatic assays employing the reduction of nicotine adenine dinucleotide phosphate to its reduced form by glucose-6-phosphate dehydrogenase and hexokinase. (3) The procedure is straightforward-no preparation of the samples or the reagents is required. The problems of insolubility and instability of TMB in aqueous solutions which are often encountered have been overcome by dissolving TMB in dimethylsulfoxide prior to dilution in aqueous buffers. The sensitivity is similar to other assays, and D-glucose concentrations as low as 5 mM can be determined. The utilisation of the glucose oxidase/HRP assay for the determination of D-glucose consumption curves by cultured bovine aortic endothelial cells is shown. Similar results were obtained when the glucose consumption curve from this assay was compared to that determined with the hexokinase/glucose-6-phosphate dehydrogenase assay.