Full Paper Rutin Determination at an Amperometric Biosensor Christine Mousty, a * Serge Cosnier, a * Marta Sanchez-Paniagua Lopez, b Enrique Lopez-Cabarcos, b Beatriz Lopez-Ruiz c a Laboratoire d)Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, ICMG FR CNRS 2607, Universite ´ Joseph Fourier, Grenoble, France b Departamento de Química-Física II, Facultad de Farmacia, Universidad Complutense, 28040, Madrid, Spain c Seccio ´n Departamental de Química Analítica, Facultad de Farmacia, Universidad Complutense, 28040, Madrid, Spain *e-mails: Christine.Mousty@ujf-grenoble.fr; Serge.Cosnier@ujf-grenoble.fr Received: July 10, 2006 Accepted: September 18, 2006 Abstract The feasibility of amperometric detection of rutin at a biosensor using polyphenol oxidase (PPO) as bioelement was investigated. The biosensor was fabricated by entrapment of PPO within a laponite clay film coated on an electrode surface. The amperometric detection of rutin was carried out at  0.1 V via the direct electrochemical reduction of the product of the enzymatic reaction or its mediated reduction by a phenothiazine redox mediator (Azure B) intercalated within the clay matrix. The biosensor response was optimized with regard to pH, temperature, applied potential (E app ) , and clay film thickness. The biosensor performance was discussed depending on the detection principle. Keywords: Polyphenol oxidase, Tyrosinase, Amperometric biosensor, Rutin, Clay electrode DOI: 10.1002/elan.200603718 1. Introduction Flavonoids are a group of polyphenolic compounds present invegetables,teaandwine.Thesephenoliccompoundshave received a great scientific interest due their properties including antimicrobial [1] and antioxidant activity [2]. Quercetin is a flavonoid very abundant in vegetables and fruits.Thiscompoundisusuallypresentasglycoside,suchas quercetin-3-rutinoside (rutin, known also as vitamin P, Scheme 1) that is found in tomato and it has been suggested that is an activating factor of vitamin C [3]. Analytical methods, such as HPLC [4 – 6], capillary electrophoresis [7, 8], and chemiluminescence [9] have been applied to the determination of quercetin and rutin at subnanomolar concentrations in pharmaceutical samples. However, these highly sensitive standard methods are expensive, time consuming and require formal training that considerably limits their wide spread use. In comparison with centralized and sophisticated analyt- ical systems, the electrochemical transduction ensured a faster, easier and less expensive detection. Since all flavo- noids are electroactive, easily subjected to either oxidation or reduction, they can be determined by electrochemical methods. For instance, determination of rutin has been performed by voltammetry at different electrodes such as glassy carbon electrode [3, 10], carbon paste electrode [3, 11], singlewalled carbon nanotubes modified electrode [12] and b cyclodextrin incorporated carbon nanotube-modified electrode [13]. In all cases, the rutin detection principle was based on the measurements of the oxidation current at ~0.4 V achieving the lowest detection limit (1  10 8 M) with the singlewalled carbon nanotubes [12]. Very recently, a rutin-copper (II) complex preconcentrated in hanging mercury drop electrode was analyzed by cathodic stripping square-wave voltammetry with a detection limit of 0.5 nM [14]. However, the detection principle generally involves an electrochemical oxidation that may induce interfering effects due to the presence of easily oxidizable species in the sample solution [14]. An alternative approach consists to use amperometric biosensors based on copper oxidases, such as laccase or tyrosinase (polyphenol oxidase, PPO). In the presence of oxygen, these enzymes catalyze the oxidation of phenols and o-diphenols to o-quinones allowing an electrochemical detection of the enzyme substrate by reduction instead of Scheme 1. Structure of Rutin. 253 Electroanalysis 19, 2007, No.2-3, 253–258 # 2007 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim