Development and applications of a bienzymatic amperometric glycerol biosensor based on a poly(neutral red) modified carbon film electrode

A bienzymatic biosensor for glycerol has been designed, based on the co-immobilization of two enzymes, glycerol kinase and glycerol-3-phosphate oxidase, the glycerol kinase phosphorylating glycerol to glycerol-3-phosphate. The enzymes were immobilized by cross-linking with glutaraldehyde on carbon film electrodes with poly(neutral red) as mediator of the enzymatic reaction. The polymer film was made on the carbon film substrate by potential cycling from a solution containing 1 mM neutral red. Glycerol, as well as glycerol-3-phosphate, was determined in amperometric mode at -350 mV vs. saturated calomel electrode. Several buffers and pH values were tested for the immobilized enzymes' response and the optimum experimental conditions were found to be in phosphate buffer at pH 8.0. The linear response range to glycerol was directly dependent on the concentration of adenosine-5'-triphosphate (ATP). With 3 mM ATP, glycerol was determined in the range 5-147 mu M. A monoenzymatic glycerol-3-phosphate biosensor was also evaluated and found to have a working range from 20-700 mu M. Application of the bienzymatic biosensor to natural samples was tested by glycerol determination in dry white and red wines and the method was validated by spectrophotometric enzyme assay.