文摘
The electrochemical oxidation of the adenine moiety inNAD+ and other adenine nucleotides at carbon pasteelectrodes gives rise to redox-active products whichstrongly adsorb on the electrode surface. Carbon pasteelectrodes modified with the oxidation products of NAD+show excellent electrocatalytic activity toward NADHoxidation, reducing its overpotential by about 400 mV.The rate constant for the catalytic oxidation of NADH,determined by rotating disk electrode measurements andextrapolation to zero concentration of NADH, was foundto be 2.5 × 105 M-1 s-1. The catalytic oxidation currentallows the amperometric detection of NADH at an appliedpotential of +50 mV (Ag/AgCl) with a detection limit of4.0 × 10-7 M and linear response up to 1.0 × 10-5 MNADH. These modified electrodes can be used as amperometric transducers in the design of biosensors basedon coupled dehydrogenase enzymes and, in fact, we havedesigned an amperometric biosensor for glycerol basedon the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilizedin a carbon paste electrode using an electropolymerizedlayer of nonconducting poly(o-phenylenediamine) (PPD).After partial oxidation of the immobilized NAD+, themodified electrode allows the amperometric detection ofthe NADH enzymatically obtained at applied potentialabove 0 V (Ag/AgCl). The resulting biosensor shows a fastand linear response to glycerol within the concentrationrange of 1.0 × 10-6-1.0 × 10-4 M with a detection limitof 4.3 × 10-7 M. The amperometric response remainsstable for at least 3 days. The biosensor was applied tothe determination of glycerol in a plant-extract syrup, withresults in good agreement with those for the standardspectrophotometric method.
