Electrocatalytic sensing of NADH on a glassy carbon electrode modified with electrografted o-aminophenol film Hossam M. Nassef a , Abd-Elgawad Radi a, * , Ciara K. O’Sullivan a,b, * a Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain b Institucio ´ Catalana de Recerca i Estudis Avanc ¸ ats, Passeig Lluı ´s Companys 23, 08010 Barcelona, Spain Received 3 July 2006; received in revised form 27 July 2006; accepted 31 July 2006 Available online 6 September 2006 Abstract A simple and sensitive method for the electrocatalytic detection of NADH on a glassy carbon electrode modified with electrografted o-aminophenol film (o-AP) is presented. The modification of a glassy carbon electrode surface with an o-AP film was achieved by elec- trochemical reduction of the corresponding, in situ generated nitrophenyl diazonium cation. The functionalized electrode shows an effi- cient electrocatalytic activity towards the oxidation of NADH with activation overpotential, which is ca. 350 mV lower than that at the bare electrode. The stability and the electrocatalytic activity of the modified electrode have been critically addressed. The formation of an intermediate charge transfer complex is proposed for the charge transfer reaction between NADH and adsorbed o-AP. The second-order rate constant for electrocatalytic oxidation of NADH, k obs , and the apparent Michaelis–Menten constant K M , at pH 7.0 were evaluated with rotating disk electrode (RDE) experiments, using the Koutecky–Levich approach. Using the o-AP-GC electrode, at an applied potential of +150 mV (vs. Ag–AgCl) with amperometric detection of NADH, a calibration range from 7.5 · 10 7 to 2.5 · 10 6 with a detection limit of 1.5 · 10 7 M was obtained, and excellent reproducibility was demonstrated with an RSD% = 2.1, n = 10 using a con- centration of 1.0 · 10 6 M NADH. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Electrocatalytic oxidation; NADH; Diazonium salt; Surface modification; NADH sensor; Nanostructured selectively catalytic surface 1. Introduction The electrochemical detection of NADH is of great interest because the pyridine nucleotides NAD and NADP are ubiquitous in all living systems and are required for the reactions of more than 450 oxidoreduc- tases [1]. Although the formal potential of NADH/ NAD + couple in neutral pH at 25 °C is estimated to be 0.56 vs. SCE [2–4], significant overpotential is often required for the direct oxidation of NADH at bare elec- trodes [5,6]. In addition, the direct oxidation of NADH is often accompanied by electrode fouling due to the poly- merization oxidation products on the electrode surface [7]. Different attempts have been made to decrease the over- potential for the oxidation of NADH, by using mediators in homogeneous solution and by modifying the electrode surface [8–59]. Among the mediators used so far are quinones [8], diimines [9], ferrocene [10], thionine [11] oxometalates [12], polymetallophthalocyanines [13], ruthe- nium complexes [14], pyrroloquinoline quinone [15–17], fluorenones [18], and quinonoid redox dyes such as ind- amines, phenazines, phenoxazines and phenothiazines [19–23]. To design an NADH sensor, the mediator has to be immobilized on the electrode surface or within the electrode material. Mediators can be immobilized by chemisorption [24], by covalent attachment directly to 1388-2481/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2006.07.045 * Corresponding authors. Address: Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain. Tel.: +34 977 558740/8722; fax: +34 977 559621/8205. E-mail addresses: ciara.osullivan@urv.net, ckosulli@etse.urv.es (C.K. O’Sullivan). www.elsevier.com/locate/elecom Electrochemistry Communications 8 (2006) 1719–1725