A Fast and Direct Amperometric Determination of Hg 2 + by a Bienzyme Electrode Based on the Competitive Activities of Glucose Oxidase and Laccase Serge Cosnier,* a Christine Mousty , b Amandine Guelorget, a Marta Sanchez-Paniagua Lopez, a, c Dan Shan. a, d a DØpartement de Chimie MolØculaire UMR-5250, ICMG FR-2607, CNRS UniversitØ Joseph Fourier, BP-53, 38041 Grenoble, France fax: (33)476514267, tel: (33)476514998 b Laboratoire des MatØriaux Inorganiques, UniversitØ Blaise Pascal CNRS UMR-6002, 24, Avenue des Landais. 63171 Aubire cedex, France c Present address: Secc. Dptal. Quimica Analitica, Facultad de Farmacia, Universidad Complutense, 28040, Madrid, Spain d Present address: School of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, China *e-mail: Serge.Cosnier@ujf-grenoble.fr Received: April 7, 2011; & Accepted: May 11, 2011 Abstract A new concept of enzyme inhibition-based biosensor involving the appearance of an amperometric signal for an in- hibition by mercury was developed. The bienzyme sensor was composed of two layers of clay materials. The inner layer was constituted of layered double hydroxides entrapping laccase wired by ABTS. The outer laponite layer contained glucose oxidase (GOD). GOD catalyzed the glucose oxidation with the reduction of O 2 into H 2 O 2 . This induced a drastic decrease of the biosensor response to O 2 by the electrically wired laccase. HgCl 2 inhibited the O 2 consumption by GOD leading to a signal increase of the electroenzymatic reduction of O 2 . Keywords: Biosensor, Inhibitor determination, Laccase, HgCl 2 , Clay DOI: 10.1002/elan.201100200 Biosensors based on the principle of an enzyme inhibition process were applied to a wide range of significant pollu- tants such as organophosphorous pesticides, organochlor- ine pesticides, derivatives of insecticides, heavy metals and glycoalkaloids [1, 2]. In general, the inhibitive mea- surement of these toxic analytes was performed via an in- cubation procedure [3–7]. As a consequence, the biosen- sor functioning is time-consuming, requires formal train- ing and is not really suitable for on-site monitoring. Since the rapid detection and monitoring in environmental field of these environmental pollutants, in particular for the sustainable use of water, constitutes a societal challenge, it is of great importance to develop biosensors based on new determination strategies. With this aim in view, we have conceived an innovative bienzyme sensing system based on the antagonist activi- ties of two enzymes, separately immobilized. The outer enzyme layer consumes the substrate of the enzyme locat- ed in the inner layer decreasing or suppressing its electro- chemical signal. The injection of a pollutant that inhibits selectively the outer enzyme layer leads thus to the ap- pearance of a biosensor signal. In order to illustrate this concept, a biosensor based on the immobilization of glu- cose oxidase (GOD) and laccase, was designed for the de- termination of HgCl 2 . Indeed, inorganic mercury (Hg 2 + ) was always used for gold mining in Africa, South Ameri- ca and Asia and the combustion of fossil fuels increases its released amount into the environment [8]. The biosensor configuration results from the immobili- zation of GOD in the outer layer and the immobilization and electrical wiring of laccase in the inner layer. GOD and laccase were separately entrapped into cationic clay, laponite, and anionic clay, redox active layered double hydroxides (LDHs, Zn 2 Cr-ABTS), respectively. The or- ganic electroactive dianions, 2,2’-azino-bis 3-ethylbenzo- thiazoline-6-sulfonic acid (ABTS 2À ) intercalated within the LDHs interlayer domain, remain active and play the role of electron shuttle between the redox centre of lac- case and the electrode surface [9–11]. In presence of glu- cose, GOD catalyzes the oxidation of this sugar into glu- conolactone and the concomitant reduction of O 2 into H 2 O 2 . GOD immobilized in the outer clay layer should thus induce a depletion of O 2 in the inner layer contain- ing laccase. Taking into account that the electrically wired laccase catalyzes the reduction of O 2 into H 2 O generating thus a cathodic current proportional to the O 2 concentra- tion, the GOD activity should lead to a drastic decrease of the biosensor response. In order to investigate the val- idity of our concept, HgCl 2 was selected as an inhibitor model of the GOD activity [12]. Owing to the cationic nature of this water pollutant, GOD was entrapped into a laponite matrix that presents cationic exchange properties 1776  2011 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Electroanalysis 2011, 23, No. 8, 1776 – 1779 Short Communication