NADH Oxidation at the Honey-Comb Like Structure of Active Carbon: Coupled to Formaldehyde and Sorbitol Dehydrogenases Carmen E. Campbell and Judith Rishpon* Department of Biotechnology, Tel-Aviv University, Ramat-Aviv, 69978, Israel; e-mail: rishpon@post.tau.ac.il Received: February 11, 2000 Final version: April 12, 2000 Abstract We have demonstrated a simple electrode configuration for the oxidation of NADH on active carbon cloth poised at 100 mV (vs. SCE). This design is unique in that it does not require a soluble mediator for NADH oxidation. The response time is on the order of several seconds; the sensor response is linear, selective, and sensitive. The configuration allows for the implementation of virtually any of the dehydrogenase enzymes dependent on NAD  cofactor. Here we demonstrate the implementation of two such enzymes: formaldehyde dehydrogenase and sorbitol dehydrogenase for the detection of formaldehyde and sorbitol, respectively. The linear range of the formaldehyde and sorbitol sensors is up to 250 mM and the detection limit 2 mM. Keywords: Dehydrogenase, NADH, Active carbon 1. Introduction In their recent review article, Lobo and colleagues have summarized various amperometric biosensors based on NAD(P)- dependent dehydrogenase enzymes. They outline the advantages and disadvantages of using dehydrogenase enzymes in biosensor design. One advantage is that oxygen is not involved in the enzymatic reaction and therefore it does not interfere in the detection step. Another advantage is that the dehydrogenase enzymes make up the majority of the oxidoreductases and include more than 250 different NAD  dependent enzymes. They concluded that the main drawback, however, in using dehydrogenase based systems is that the enzymatically generated reduced cofactor NAD(P)H must be efficiently reoxidized [1]. It has long been established that the electrocatalytic oxidation of NADH can be acheived electrochemically [2]. This electro- catalytic oxidation requires a high overpotential where unwanted side reactions can occur and therefore was not suitable for use in biosensor design. However, the requirement of the NAD  cofactor for dehydrogenase based biosensors led researchers to investigate the mediated electron transfer of NADH oxidation. Several studies demonstrate that NADH electrooxidation is possible on different conductive materials with high applied potentials which can be decreased by the use of soluble mediators [1, 3–7]. Furthermore, the electrocatalytic oxidation of NADH at chemically modified electrodes has been investigated [4–6]. In 1958, Hallum and Drushel presented a model which suggests that active carbon has a honey-comb like structure with surface groups which resemble mediators of NADH oxidation like ortho- and para-quinone [8]. They claim that 18 % of the oxygen is tied up as 1,4-quinone. The carbon with the high surface area and immobilized quinone-like groups was further investigated and found to be a stable mediator for direct oxidation of NADH [1–3, 6, 7, 9–11]. In this work we present a novel approach exploiting the honey-comb like structure of active carbon for sensitive and selective oxidation of NADH at applied potentials between 0 and 100 mV. The 1,4-hydroxyquinone mediator is a component of active carbon and thus is the driving force for NADH oxidation to occur at 100 mV (vs. SCE) where unwanted side reactions do not occur. The high surface area of active carbon cloth and localized mediator are two advantages of using active carbon cloth as electrodes for direct NADH oxida- tion at low applied potentials. The use of carbon cloth for elec- trodes has also been investigated for oxidase based biosensor design because of the high surface area and thus high signal to noise ratio [12]. Dehydrogenase based sensors have been investigated for a number of analytes including; glutamate, lactate, ethanol, and LDH [1, 3, 7, 11, 13–15]. Additionally, formaldehyde dehy- drogenase has been integrated into sensors which have been proposed for the detection of formaldehyde [16–18]. One configuration depends upon the generation of H  and thus could be affected by nonspecific proton generating reactions [16]. Another device requires the use of a soluble toxic mediator, 1,2- naphthoquinone-4-sulfonic acid and demonstrates a response of ca. 15 nA=mM NADH [17]. A novel device integrates the enzyme into a flow system which depends on the use of cyclohexanedione, ammonium acetate, and concentrated hydrochloric acid [18]. The current study demonstrates a simple system which incor- porates the dehydrogenase enzyme onto an active nylon membrane positioned next to an active carbon cloth electrode and its appli- cation in the detection of formaldehyde and sorbitol. The sensor is designed so that NADH, the enzymatically reduced cofactor of NAD  is concentrated at an active carbon cloth electrode surface for direct oxidation at an electrode potential of 100 mV (vs. SCE). The resulting current is measured using chronoamperometry. This sensing device requires low concentrations of enzyme as compared to spectrophotometric methods, is sensitive to mmol=L changes in substrate concentration with a lower detection limit of 2 mmol=L, is specific, and stable over several days. 2. Experimental 2.1. Materials NAD  , NADH, and formaldehyde dehydrogenase [EC 1.2.1.46] (FDH) from P. putida with a specific activity of 17 Electroanalysis 2001, 13, No. 1 # WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 1040-0397/01/0101–0017 $17.50.50=0