Revue Roumaine de Chimie, 2006, 51(1), 25–30 FORMATE DEHYDROGENASE-MODIFIED CARBON PASTE ELECTRODES FOR AMPEROMETRIC DETECTION OF FORMATE Florentina-Daniela MUNTEANU, a Delia GLIGOR, b Ionel Cătălin POPESCU b and Lo GORTON ∗a a Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden b Department of Physical Chemistry, Faculty of Chemistry and Chemical Engineering, “Babeş-Bolyai” University, Arany Janos 11, 400028 Cluj-Napoca, Roumania Received July 5, 2005 A simple and inexpensive amperometric biosensor for formate detection was obtained by immobilization of formate dehydrogenase (FDH) on graphite powder, modified with a new phenothiazine derivative (DDDP; 16H, 18H-dibenzo[c,1]-7,9-dithia-16,18-diazapentacene). Two different enzyme environments were used: FDH was adsorbed on DDDP-modified graphite powder in the presence of polyethylenimine (PEI) as well as in its absence. In both cases, when 10 mM NAD + were added in the supporting electrolyte (phosphate buffer, pH 7.0), the biosensor developed a response to formate, obeying Michaelis-Menten kinetics with a linear domain up to 3 mM formate. The biosensor sensitivity, calculated as the ratio I max /K M app , was better in the second case (0.962 mA/M), but the best detection limit (3 µM) was observed in the first case. INTRODUCTION Redox enzymes have been widely used in amperometric biosensor research. 1-7 The largest group of redox enzymes known today is the nicotinamide dinucleotide (NAD + /NADH) dependent dehydrogenases. 8-11 Coupling the electrocatalytic oxidation of NADH by an appropriate mediator to the reaction catalyzed by the NAD + dependent dehydrogenase enzymes enables the construction of amperometric biosensors for a large variety of biologically important species in biotechnology and analysis. 12-14 NAD + -dependent formate dehydrogenase (FDH; formate:NAD + oxidoreductase, EC 1.2.1.2) from methyloprotic bacterium Pseudonomas sp. is one of the most extensively characterised NAD + -dependent dehydrogenases. 15,16 FDH is composed of two identical subunits each comprising two domains: a coenzyme binding domain and a substrate-binding domain. 17 FDH was purified from several sources including eukaryotes, archaea and bacteria. In general, FDHs from aerobic organisms reduce NAD + , have a high K M for formate and are insensitive to O 2 . 18 The optimum pH range for substrate binding is 5.5–10.5. 19 FDH catalyzes the oxidation of formate to carbon dioxide. Formate reacts with FDH in the presence of NAD + , which is reduced in the process to NADH. In turn NADH should be reoxidized at the electrode surface in a biosensor format. Formate as substrate for FDH is one of the cheapest hydrogen sources and the oxidation product CO 2 can be easily removed from the reaction mixture. 18,20 Formate is a common substance found in the atmosphere, museum cabinets, exhaust emissions, natural and seawaters, sediments 21,22 and is a product of degradation of metal-cyano compounds by fungi. 22 A limited number of devices for the real-time determination of formate, based on a biosensor using FDH and a chemically modified electrode, have been recently introduced. 14 These biosensors are mainly based on electrocatalytic oxidation of NADH with modified electrodes such as polypyrrole/ferrocyanide 23,24 and 3,4-dihydroxibenzaldehyde. 25 The major drawbacks of these biosensors are the low sensitivity, poor stability and slow response. A gaseous biosensor for formic acid has been developed using Meldola’s blue as mediator, the biosensor suffering from very short lifetime due to its constructional limitation. 21 ∗ Corresponding author: lo.gorton@analykem.lu.se