Mechanistic Studies of the Electrocatalytic Oxidation of NADH and Ascorbate at Glassy Carbon Electrodes Modified with Electrodeposited Films Derived from 3,4-Dihydroxybenzaldehyde F. Pariente, † F. Tobalina, † G. Moreno, † L. Herna ´ndez, † E. Lorenzo, † and H. D. Abrun ˜a* ,‡ Departamento de Quı ´ mica Analı ´ tica y Ana ´ lisis Instrumental, Universidad Auto ´ noma de Madrid, Canto Blanco 28049, Madrid Spain, and Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301 Studies of the electrocatalytic oxidation of -nicotinamide adenine dinucleotide (NADH) at glassy carbon rotated disk electrodes modified with electrodeposited films derived from 3 ,4 -dihydroxybenzaldehyde (3 ,4 -DHB) in- dicate that the mechanism of such electrooxidation pro- ceeds via the formation of an intermediate complex. The reaction also appears to be strongly influenced by the presence of Ca 2 + and Mg 2 + ions as well as by pH. Ascorbate can also be electrocatalytically oxidized at these modified electrodes, giving rise to an electrochemical response very similar to that obtained for NADH. Due to this similarity, the presence of ascorbate in NADH deter- minations presents a severe interference that cannot be mitigated on the basis of electrochemical responses alone. However, this interference effect can be virtually sup- pressed by the presence of ascorbate oxidase in solution or immobilized on a nylon mesh which, in turn, is in contact with the electrode modified with the film of 3 ,4 - DHB. Using this approach, we describe the construction of an alcohol biosensor based on alcohol dehydrogenase and which is, furthermore, free from interference effects due to ascorbate. The ubiquitous use, in living cells, of -nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD + ) as coenzymes by a great number of dehydrogenases has generated numerous studies directed at understanding the factors that may control the redox activity of these biological cofactors. Although the revers- ible potential for NADH oxidation is estimated to be -0.32 V vs NHE, 1 the direct oxidation of NADH at bare electrodes, in general, requires high overpotentials that can be as large as 1.0 V. 2,3 At carbon electrodes, this high overpotential can be decreased by pretreatment, although these surface-modified electrodes are rapidly deactivated. 4 Oxidation of NADH at lower potentials can be achieved through redox mediators present in the solution or confined at the electrode surface. A number of electrode modifications have been reported for this purpose using different functionalities, such as thionine, 5 phenazine, 6 and phenoxazine 7,8 derivatives adsorbed onto carbon electrodes or as electrodeposited films. 9 It is also generally accepted that o-quinones can be quite active in the electrocatalytic oxidation of NADH, and, as a result, numerous derivatives incorporating such a group have been employed. 10 In these types of systems, the NADH is oxidized by the surface- confined mediator (M ox ) in a chemical step defined by a rate constant k 1 (M -1 s -1 ). NAD + and the reduced form of the mediator (M red ) are the products of this reaction: The mediator is subsequently reoxidized at the electrode surface according to eq 2: where the number of protons involved in the process, x, depends on the quinone structure and pH. The decrease in the overpo- tential is given by the potential difference in the direct electro- chemical oxidation of NADH at the bare electrode and the formal potential of the mediator, E°′ M . At pH 7.0, typical values for E°′ M from around -0.2 to +0.1 V have been reported for electrodes modified with a number of quinone functionalities. 10 In addition, at pH 7.0, the direct oxidation of NADH at unmodified electrodes typically takes place at about +0.70 V, so that the use of electrodes modified with quinone functionalities for the electrooxidation of NADH typically gives rise to significant diminutions in the overpotential. We recently 11 reported that the electrooxidation of 3,4-dihy- droxybenzaldehyde (3,4-DHB) on glassy carbon electrodes gives † Universidad Auto ´noma de Madrid. ‡ Cornell University. (1) Clark, W. M. Oxidation Reduction Potentials of Organic Compounds; The Williams and Wilkins Co.: Baltimore, MD, 1960. (2) Moiroux, J.; Elving, P. J. J. Anal. Chem. 1978 , 50, 1056. (3) Jaegfeldt, H. J. Electroanal. Chem. 1980 , 110, 295. (4) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1976 , 48, 1240. (5) Hajizadeh, K.; Tang, H. T.; Halsall, H. B.; Heinemann, W. R. Anal. Lett. 1991 , 24, 1453. (6) Torstensson, A.; Gorton, L. J. Electroanal. Chem. 1981 , 130, 199. (7) Gorton, L.; Torstensson, A.; Jaegfeldt, H.; Johansson, G. J. Electroanal. Chem. 1984 , 161, 103. (8) Persson, B.; Gorton, L. J. Electroanal. Chem. 1990 , 292, 115. (9) (a) Persson, B.; Lan, H. L.; Gorton, L.; Okamoto, Y.; Hale, P. D.; Boguslavsky, L. I.; Skotheim, T. S. Biosens. Bioelectron. 1993 , 8, 81. (b) Persson, B.; Lee, H. S.; Gorton, L.; Skotheim, T.; Bartlett, P. Electroanalysis 1995 , 7, 935. (10) (a) Tse, D. C. S.; Kuwana, T. Anal. Chem. 1978 , 50, 1315. (b) Jaegefeldt, H.; Torstensson, A.; Gorton, L.; Johansson, G. Anal. Chem. 1981 , 53, 1979. (c) Ueda, C.; Tse, D. C. S.; Kuwana, T. Anal. Chem. 1982 , 54, 850. (d) Jaegefeldt, H.; Kuwana, T.; Johansson, G. J. Am. Chem. Soc. 1983 , 105, 1805. (11) Pariente, F.; Lorenzo, E.; Abrun ˜ a, H. D. Anal. Chem. 1994 , 66, 4337. NADH + M ox 9 8 k 1 NAD + + M red (1) M red y \ z k s M ox + 2e - + xH + (2) Anal. Chem. 1997, 69, 4065-4075 S0003-2700(97)00445-9 CCC: $14.00 © 1997 American Chemical Society Analytical Chemistry, Vol. 69, No. 19, October 1, 1997 4065