Available online at www.sciencedirect.com Electrochimica Acta 53 (2008) 2161–2169 Detection of NADH via electrocatalytic oxidation at single-walled carbon nanotubes modified with Variamine blue A. Radoi a,∗ , D. Compagnone b , M.A. Valcarcel c , P. Placidi d , S. Materazzi d , D. Moscone a , G. Palleschi a a Dipartimento di Scienze e Tecnologie Chimiche, Universit` a “Tor Vergata”, via della Ricerca Scientifica 1, 00133 Rome, Italy b Dipartimento di Scienze degli Alimenti, Universit` a di Teramo, via C. Lerici 1, Mosciano S. Angelo, Teramo, Italy c Department of Chemistry, Universitat Aut` onoma de Barcelona, Edifici Cs./08193, Bellaterra, Barcelona, Spain d Dipartimento di Chimica, Universit` a “La Sapienza”, Piazzale A. Moro 5, 00185 Rome, Italy Received 15 May 2007; received in revised form 24 August 2007; accepted 16 September 2007 Available online 25 September 2007 Abstract Screen-printed electrodes (SPEs) modified with Variamine blue (VB), covalently attached to the oxidized single-walled carbon nanotubes (SWCNTs-COOH), were developed and used as chemical sensors for the detection of the reduced nicotinamide adenine dinucleotide (NADH). The Variamine blue redox mediator was covalently linked to the SWCNTs-COOH by the N,N ′ -dicyclohexylcarbodiimide (DCC) and N- hydroxysuccinimide (NHS) chemistry. Infrared Fourier transform (FT-IR) spectroscopy revealed the presence of the amide bands situated at 1623 cm -1 (I band), 1577 cm -1 (II band) and 1437 cm -1 (III band) demonstrating the covalent linkage of Variamine blue to SWCNTs-COOH. The heterogeneous electron transfer rate, k obs. , was 13,850 M -1 s -1 , and the k s and α were 0.8 s -1 and 0.56, respectively. The pH dependence was also investigated. SPEs modified with Variamine blue by using the DCC/NHS conjugation method, showed a variation of -36 mV per pH unit. A successful application was the development of a lactate biosensor obtained by the immobilization of the l-lactate dehydrogenase on the NADH sensor. © 2007 Elsevier Ltd. All rights reserved. Keywords: Variamine blue; Single-walled carbon nanotubes; NADH; l-Lactate biosensor 1. Introduction Single-walled carbon nanotubes (SWCNTs) have attracted great attention because of their unique structural, electronic, mechanical and optical properties [1]. To facilitate their appli- cations, many approaches were employed to functionalise them with different chemical groups [2–8]. Among these, the car- boxyl group is very attractive, since it can be readily used for further reactions [5]. Usually, the carboxylation is achieved via oxidation of defect sites, by strong oxidants, such as hydrogen peroxide, nitric acid, sulphuric acid or a mixture of the last two. The resultant carboxyl groups are localized at the defect sites of SWCNTs and the degree of the carboxylation is relatively low [3,8]. ∗ Corresponding author. Tel.: +39 06 72594404; fax: +39 06 72594328. E-mail address: radoiantonio@yahoo.com (A. Radoi). Nicotinamide adenine dinucleotide, both in its reduced and oxidized forms (NADH/NAD + ), is the coenzyme for about 300 dehydrogenases, being involved in a wide range of enzymatic reactions [9,10]. The oxidation of NADH at bare electrodes occurs via radical cation intermediates with formation of strong adsorptive secondary reaction products. The fouling process reported at ordinary electrodes (for example, graphite and its derivatives, Pt, glassy carbon, etc.) [11], the generation of inac- tive NAD + dimers, and the relatively high overpotentials [12] required for NADH oxidation are some of the main draw- backs described in many papers dealing with the electrochemical detection of NADH coenzyme. Considerable effort has been devoted toward the goal of identifying new electrode materials or mediators to allow the stable low potential determination of NADH. Carbon nanotubes [13,14] are one such novel material utilized for the stable low potential detection of NADH [15,16]. Traditionally, the mediators were usually adsorbed or cova- lently bound onto the electrode surface [17–20]. Using a wide 0013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2007.09.031