Ethanol Biosensors and Electrochemical Oxidation of NADH P. C. Pandey, S. Upadhyay, B. C. Upadhyay, and H. C. Pathak Chemistry Department, Analytical Chemistry Division, Banaras Hindu University, Varanasi 221005, India Received November 20, 1997 Comparative studies of the electrochemical oxida- tion of reduced nicotinamide coenzyme (NADH) at the surfaces of chemically modified graphite paste elec- trodes (CMEs) are reported. Three different electroac- tive materials, tetracyanoquinodimethane (TCNQ), tetrathiafulvalene (TTF), and dimethyl ferrocene (dmFc), were used to construct three different chemi- cally modified paste electrodes. The oxidation of NADH was examined on the basis of cyclic voltammet- ric measurements. The results show that all three me- diators (TCNQ, TTF, and dmFc) behave as efficient mediators of the oxidation of NADH. The typical re- sponse curves of NADH at the CMEs surfaces are re- ported. Incorporating alcohol dehydrogenase and electroactive materials (TCNQ, TTF, and dmFc) within the graphite paste electrodes has led to the develop- ment of ethanol biosensors. Typical response curves for the ethanol analysis are reported. Comparative studies on the mediated electrochemical responses of the biosensors to ethanol are discussed. © 1998 Academic Press A number of reports suggest that direct electrochem- ical oxidation of NADH introduces a large overvoltage at unmodified (naked) metal electrodes. Additionally, direct electrochemical oxidation of NADH at a naked metal surface does not allow regeneration of enzymat- ically active NAD + which is especially useful as a con- tinuously supplied cofactor in enzyme-catalyzed or- ganic synthesis (1, 2) or in electrochemical detection of dehydrogenase-based catalyzed reactions. Electro- chemical oxidation of NADH leading to enzymatically active NAD + at mild potentials can be facilitated at mediator-modified electrode surfaces (3, 4). A number of mediators have been tested for the electrochemical oxidation of NADH incorporated into carbon paste elec- trodes (5–10) or physically adsorbed on carbon elec- trode surfaces (11, 12). In some cases the redox groups were covalently attached to the polymeric matrix phys- ically adsorbed on an electrode surface (7, 12). The components of an organic metal, tetrathiaful- valene (TTF) 1 or tetracyanoquinodimethane (TCNQ), have been used as efficient mediators for the regener- ation of several oxidoreductase-catalyzed reactions (13–15), dehydrogenase-based catalyzed reactions (9, 10), and other reduced analytes, i.e., photoreduced an- thraquinone (16). Similarly, a derivative of ferrocene, dimethyl ferrocene (dmFc), has been used as an effi- cient mediator for several enzymatic reaction-based systems, some of which have been commercialized (17). We have recently developed electrochemical biosensors for glucose and peroxide (18). However, the following studies are still needed to understand the electrochem- ical oxidation of NADH, which is a key step in design- ing a dehydrogenase-based electrochemical biosensor; (i) study of the regeneration of a biologically active NADH during electrochemical probing of dehydroge- nase-based catalyzed reactions, (ii) study of the mech- anistic behavior of the interaction between NADH and mediator (TCNQ/TTF/dmFc), and (iii) study of the com- parative responses of a dehydrogenase and the media- tor (TCNQ, TTF, dmFc)-modified biosensor. We have recently made a comparative study of the regeneration of a glucose oxidase and a peroxidase using these three mediators based on the mediated bioelectrochemistry within graphite paste electrodes (18). We found that TCNQ incorporated into the paste acts as a good me- diator for probing a glucose oxidase-catalyzed reaction; TTF, on the other hand, was a better mediator for probing a peroxidase-catalyzed reaction. Although dmFc acts as an efficient mediator for probing both 1 Abbreviations used: TTF, tetrathiafulvalene; TCNQ, tetracyano- quinodimethane; dmFc, dimethyl ferrocene; ADH, alcohol dehydro- genase. 0003-2697/98 $25.00 195 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY 260, 195–203 (1998) ARTICLE NO. AB982679