Feature Article Fast-Scan Cyclic Voltammetry and Scanning Electrochemical Microscopy Studies of the pH-Dependent Dissolution of 2-Electron Mediators Immobilized on Zirconium Phosphate Containing Carbon Pastes Florentina D. Munteanu, a Marcus Mosbach, b Albert Schulte, b Wolfgang Schuhmann, b Lo Gorton* a a Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-22 100, Lund, Sweden; e-mail: Lo.Gorton@analkem.lu.se b Analytische Chemie ± Elektroanalytik & Sensorik, Ruhr-Universit‰t Bochum, D-44780 Bochum, Germany Received: October 18, 2001 Final version: December 21, 2001 Abstract A detailed study is presented on the leaking of two different 2-electron-transfer redox mediators from zirconium phosphate-modified carbon-paste electrodes. Two redox mediators commonly used in bioelectrochemistry, viz. methylene green and riboflavin, were adsorbed on zirconium phosphate and further integrated into carbon paste. Fast scan cyclic voltammetry and scanning electrochemical microscopy were used to characterize the leakage of these redox mediators at different pH conditions. It could be shown that an exposure to acidic pH-values significantly increased the rate of their leakage from the mediator-zirconium phosphate-modified carbon paste, whereas a tight adsorption was observed at neutral pH. Keywords: Methylene green, Riboflavin, Leakage, Zirconium phosphate, Modified carbon-paste electrodes, Fast-scan cyclic voltammetry, Scanning electrochemical microscopy (SECM) 1. Introduction Nicotinamide adenine dinucleotide (NADH) is involved as a soluble cofactor in several hundred enzyme reactions catalyzed by the NAD  /NADH dependent dehydrogenases [1,2],thelargestgroupofredoxenzymesknown.Oneofthe major problems to solve for successful technical applica- tions of these enzymes is the regeneration of the soluble redox cofactor. Due to the redox nature of the cofactor, studies of a possible electrochemical regeneration have beendoneformanyyearswithspecialfocusontheoxidation of NADH. However, coenzyme electrode reactions are possible only at relatively high overpotentials [3, 4] or in the presence of a suitable redox mediator [1, 2]. The electron transfer between the coenzyme and most other compounds is extremely slow under normal conditions. The high overpotential for the electrochemically induced oxidation ofNADHmayalsoresultinelectrodefoulingbecauseofthe adsorption of NAD  at the electrode surface or due to the formation of radical intermediates generated as the redox reaction [5±8]. Various types of redox mediators have been used with more or less success to catalyze the electrochemical oxidation of NADH [1, 2, 9]. From previous investigations it became clear that mainly mediators of the 2 electron- proton acceptor type have fairly high reaction rates with NADH and also yield enzymatically active NAD  as reaction product (in contrast to the 1 electron-no-proton acceptor type [9]). However, the E8'-value of these 2 electron-proton acceptor type of mediators is pH-depend- ent both for the mediator in solution and when immobilized onto conventional electrode material. Therefore, any change in the pH of the sample solution may affect the rate constant for the reaction between NADH and the mediator which in turn can lead to additional inaccuracies when used for analytical purposes. For this reason, it is of importance to discover strategies to circumvent the draw- backof2electron-protonacceptortypemediatorsandgreat interest has been paid to develop new sensors and biosensors with a higher sensitivity, selectivity, and stability [10 ± 15]. At the same time, great efforts have been made in finding materials to which enzymes and mediators can be strongly bound in order to avoid their leaking under the experimental conditions [16, 17]. Various inorganic materi- als (i.e., composites [18 ± 20], silica gel [21 ± 23], or zeolites [24, 25]) can be used in order to modify conventional electrodes.Inalmostallcasesthesensitivityofthesensorsis significantly increased, concomitantly with an increase in their stability. Zirconium phosphate (ZP) is known to be a good ion exchanger because it is a strongly acidic material [26, 27]. In addition, it is a good ion conductor due to its phosphate groups [28]. These properties make it a very attractive material to be employed as a matrix for the immobilization of mediators facilitating the electron transfer between the enzyme cofactor and the electrode. ZP presents good adsorption properties to bind most of the phenoxazines, phenothiazines, phenazines, and flavins and is known to 1479 Electroanalysis 2002, 14, No. 21 ¹ 2002 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 1040-0397/02/2111-1479 $ 17.50+.50/0