Biosensors and Bioelectronics 22 (2007) 17331738

Laccase immobilization in redox active layered double hydroxides: A reagentless amperometric biosensor
Christine Mousty , Laetitia Vieille, Serge Cosnier
Laboratoire dElectrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universit e Joseph Fourier, Grenoble, France Received 3 May 2006; received in revised form 27 July 2006; accepted 9 August 2006 Available online 4 October 2006

Abstract This paper describes a new system for amperometric determination of dissolved oxygen and its application for the detection of anionic toxic substances, which are known as enzyme inhibitors. This biosensor is based on the co-immobilization of laccase from Trametes versicolor and a redox active layered double hydroxide [ZnCrABTS] on a glassy carbon electrode. The electrochemical transduction step corresponds to the electrocatalytic reduction of O2 at 0.2 V by laccase as catalyst and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator. Such device provides a fast and a sensitive response for dissolved oxygen determination between 6 108 and 4 106 M and very low detection limits for azide (5.5 nM), uoride (6.9 nM) and cyanide (6.2 nM). 2006 Elsevier B.V. All rights reserved.
Keywords: Laccase; Layered double hydroxides; Anionic clay; Biosensor; O2 reduction; Inhibition

1. Introduction Laccases (EC 1.10.3.2) are multicopper oxidases widely distributed in plant and fungal species. They received particular attention because they present rather low substrate specicity and are able to oxidise phenols, anilines, benzenethiols, phenothiazines with the concomitant reduction of molecular oxygen (O2 ) to water (Xu, 1996). Laccases are mainly used in paper and textiles industries, for wastewater treatment, delignication and dye bleaching. They have also found applications in biofuel cell development as a cathode on which O2 is electroreduced to water (Barton et al., 2001; Farneth and DAmore, 2005; Gupta et al., 2004; Palmore and Kim, 1999; Rowinski et al., 2004; Tarasevich et al., 2003). Laccase-based biosensors, in the absence or in the presence of mediators, have been applied for the determination of a broad range of phenolic species (Ferry and Leech, 2005; Freire et al., 2001, 2002; Haghighi et al., 2003; JaroszWilkolazka et al., 2005). Recently, Leech et al. have developed the concept of biosensor devices for the reagentless detection of laccase inhibitors (Leech and Daigle, 1998; Leech and Feerick, 2000; Trudeau et al., 1997).

Corresponding author. Fax: +33 476 514 267. E-mail address: Christine.Mousty@ujf-grenoble.fr (C. Mousty).

Most applications require enzyme immobilization. Dur an et al. have published an overview of the different methods used for the immobilization of laccase (Dur an et al., 2002). In particular, the immobilization of laccase from Trametes versicolor was extensively studied for several years. It is reported that a high percentage of laccase activity is maintained after its immobilization on clays (kaolinite, montmorillonite) (Dodor et al., 2004; Gianfreda and Bollag, 1994; Ruggiero et al., 1989). On the other hand, for several years we have developed in our laboratory amperometric biosensors based on the immobilization of enzymes within clay matrices (Mousty, 2004). For this purpose, we used either cationic clays such as laponite or anionic clays. Anionic clays are hydrotalcite type like materials known as synthetic layered double hydroxides (LDH) (de Roy et al., 1992). Recently, we have successfully immobilized horseradish peroxidase (HRP) into redox active layered double hydroxide [ZnCrABTS] (Shan et al., 2003a). The organic electroactive dianions, 2,2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) intercalated within the LDH interlayer domain, remain redox active and play the role of electron shuttle between the redox centre of HRP and the electrode. The electrochemical transduction step corresponds to the reduction at 0.0 V of ABTS+ enzymatically formed in the presence of H2 O2 . This biosensor was also applied for the determination of cyanide (Shan et al., 2004).

0956-5663/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.08.020

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Scheme 1. Schematic reaction occurring at the laccase/[ZnCrABTS] modied electrode.

In the present paper, we report a new biosensor conguration based on the immobilization of laccase from T. versicolor in the [ZnCrABTS] LDH matrix. Vianello and coworkers have reported that among different commercial laccase sources, laccase from T. versicolor has a very high activity with ABTS (Vianello et al., 2004). Therefore, one can expect to have an efcient turn over of the O2 /laccase/[ZnCrABTS] system upon applied potential (Scheme 1). This biosensor conguration is applied for monitoring oxygen levels and for the determination of laccase inhibitors such as sodium azide, sodium uoride and potassium cyanide. 2. Experimental 2.1. Materials and solutions Laccase from T. versicolor (Tv) and glutaraldehyde (25%) were purchased from Fluka. Bovin serum albumin (BSA), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), catechol and dopamine were received from Sigma, phenol and aniline from Prolabo. The layered double hydroxide Zn2 Cr(OH)6 ABTS was synthesized by the coprecipitation method (Therias et al., 1996). All other chemical reagents were of analytical grade. Water was doubly distilled in quartz apparatus. For the oxygen detection, the experiments were made in a glove box under argon atmosphere by successive additions of saturated O2 supporting electrolyte solution. This O2 standard solution was obtained by bubbling for 1 h. Oxygen concentration in the saturated supporting electrolyte was 1.1 mM. 2.2. Apparatus The amperometric measurements were performed with a Tacussed PRG-DL potentiostat in conjunction with a Kipp and Zonen BP 91 XY/t recorder (O2 determination) or with an ecorder 401 system (CBO instrumentation). Cyclic voltamme-

tries were recorded with Autolab 100 potentiostat. All electrochemical experiments were carried out in a conventional thermostated three-electrode cell (10 or 40 ml) at 30 C. An Ag/AgCl electrode saturated with KCl solution was used as reference electrode, and a Pt wire was placed in a separate compartment containing the supporting electrolyte, as a counter electrode. The working electrodes were glassy carbon electrodes with a diameter of 3 mm for cyclic voltammetry and FIA experiments and 5 mm for batch chronoamperometric experiments under rotation conditions (500 rpm). Before use, these electrodes were polished with 1 m diamond paste and rinsed with water and acetone. Flow injection experiments were carried out using a BAS thin layer cell (radial ow). The ow injection system consisted of an isocratic pump (Perkin-Elmer200LC) and a Rheodine 9725 injection valve with 20 l loop. The electrolyte ow was xed at 0.05 ml/min. Spectrophotometric measurements were carried out with a Varian Cary 1 UV-visible spectrophotometer. 2.3. Assay of laccase activity The activity of free laccase was determined by using ABTS as substrate. One unit of laccase activity is dened as the amount oxidizing 1 mol substrate per min. The UV test was performed in 3 ml acetate buffer pH 5.0 containing 5 mM ABTS (420 = 36000 M1 cm1 ). The activity of laccase determined by this method was 12 U/mg. 2.4. Enzyme immobilization The clay colloidal suspension (2 mg ml1 [ZnCrABTS]) was dispersed overnight under stirring conditions in deionised and decarbonated water. Laccase and BSA were dissolved in water, each solution had a concentration of 4 mg ml1 . A drop (22.5 l) of aqueous mixtures containing a dened amount of clay (40 g), laccase (5 g) and BSA (5 g) was spread on the surface of the glassy carbon electrode. The coating was

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dried under vacuum at room temperature. The resulting electrode was placed in saturated glutaraldehyde vapour for 1 h for cross-linking of the membrane. Finally, the laccase/BSA/clay biomembrane was rehydrated for 20 min into 0.1 M acetate buffer solution (pH 5). Before use, the biosensor was stabilized at 0.2 V for 1 h under O2 atmosphere. The amount of enzyme immobilized on the electrode surface was calculated by the difference between amount of protein initially adsorbed and that detected in the washing buffer solution. The later value was determined by UV spectroscopy. Under the optimal conditions, the amount of retained laccase was 83% (4.1 g, 0.05 U). 3. Results and discussion 3.1. Oxygen detection Typical voltammogram of laccase/[ZnCrABTS] modied electrode in the presence of O2 is shown in Fig. 1 (curve a). The redox mediator ABTS is oxidized by laccase and the regeneration of the enzyme is achieved by the reduction of molecular oxygen to water (Scheme 1). This resulted in an electrocatalytic reduction wave at the modied electrode. Removal of oxygen from the solution by outgassing with argon (not shown) or the additions of azide, an enzyme inhibitor (Fig. 1, curves b and c) caused a decrease in the electrocatalytic reduction wave to nally obtain a reversible signal, characteristic of ABTS immobilized into the clay matrix [ZnCrABTS] (Fig. 1, curve d). The optimization of the procedure for laccase immobilization was carried out in order to improve the enzyme retention on the electrode surface and to obtain the most efcient mediated reduction of O2 . This immobilization procedure differed slightly from that previously adopted for the immobilization of HRP (Shan et al., 2003a, 2004) and polyphenol oxidase (PPO) (Shan et al., 2003b) in LDH matrix. With laccase, a coreticulation with BSA appeared to be necessary. Moreover, the reticulation time must be increased to 1 h. Indeed, the efcient immobilization of laccase from T. versicolor appears to be problematic, a chemical cross-linking is generally required (Freire et al., 2001). Different biosensor congurations have been tested by varying the relative

Fig. 2. Inuence of pH on amperometric response of laccase + BSA/ [ZnCrABTS] bioelectrode under saturated O2 atmosphere in acetate buffer solution (Eapp = 0.2 V).

Fig. 1. Cyclic voltammograms of GCE modied by laccase + BSA/ [ZnCrABTS] (5:5:20 g) (a) under saturated O2 atmosphere and with successive additions of sodium azide, (b) 9.9 108 , (c) 1.08 106 and (d) 1.08 105 M in acetate buffer solution (pH 5.0), v = 10 mV s1 .

amounts of both proteins coated on the electrode surface. With a biolm containing 40 g LDH, 5 g laccase and 5 g BSA, 83% of laccase (4.2 g) were retained on the electrode surface, whereas with 10 g of laccase without BSA only 37% (3.7 g) remained. The inuence of the pH on the amperometric response of the biosensor was studied in the pH range between 4 and 7 in acetate buffer solution (Fig. 2). Since ABTS oxidation is known to be reversible and pH independent (Scott et al., 1993), the variation of the cathodic current with pH can be related to changes of laccase activity. The maximum response was obtained at pH 5.0 which is the same pH value as that reported previously for laccase from T. versicolor immobilized on carbon ber electrodes (Freire et al., 2001), on spectrographic graphite electrodes (Haghighi et al., 2003) and on kaolinite (Dodor et al., 2004) but it is slightly higher than that reported for the free enzyme (pH 3.0) (Farneth et al., 2005; Jolivalt et al., 2000). The analytical performance of laccase + BSA/[ZnCr ABTS] electrodes for oxygen detection was investigated in a glove box under argon atmosphere. When different amounts of saturated oxygen solution were added into the batch cell containing deoxygenated electrolyte, an increase in the catalytic reduction current was observed (Fig. 3A). Calibration curves had been recorded at two different applied potentials: 0.0 and 0.2 V (Fig. 4). The sensitivities are similar, namely 443 and 474 mA M1 cm2 , but the biosensor response is more stable at 0.2 V and the linear range is wider at this potential (6 108 to 4 106 M) than that at 0.0 V (4 107 to 2 106 M). Consequently, we decided to apply 0.2 V for further experiments. The reproducibility of the biosensor responses was investigated by repeated (n = 6) injections of 10 l of saturated oxygen solution, the relative standard deviation was 8%. The reproducibility of O2 detection was also determined under ow injection analysis (FIA) (Fig. 3B). Successive injections of saturated oxygen solution (20 l loop) into a constant ow of deoxygenated acetate buffer solution gave a cathodic current of 2.64 nA (RDS 8%. n = 8). After each injection, the current returned to its initial value, showing good reversibility of the detection device.The maximum current density measured in saturated O2 atmosphere at 10 different electrodes was 6.2 0.7 A cm2 . This value corresponds to a specic activity of immobilized laccase Tv 1 (Imax /molEnz ) of 1 105 A cm2 mol Enz which is in good

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Fig. 3. (A) Amperometric response at laccase + BSA/[ZnCrABTS] bioelectrode at 0.2 V in 40 ml deoxygenated acetate buffer solution pH 5.0 to repeated injections (arrows) of 5 l saturated O2 solution. (B) Amperometric response at laccase + BSA/[ZnCrABTS] bioelectrode at 0.2 V in a radial ow injection cell to repeated injections of 20 l saturated O2 solution.
1 agreement with the value of 1.5 105 A cm2 mol Enz reported by (Farneth and DAmore, 2005) for puried laccase Tv and ABTS immobilized in a silica-coated carbon paper macroelectrode. app The KM calculated from a LineweaverBurk plot of the calibration curve was 30 M. This value can be compared to the values reported for the biocatalysts system of laccase Tv/ABTS ABTS = 192 M, K O2 = 262 M) (Farneth et al., in solution (KM M 2005) or for laccase Tv immobilized on kaolinite with ABTS ABTS = 162 M) (Dodor et al., 2004). The lower in solution (KM app KM reects the efcient electrical wiring of laccase by ABTS intercalated in the LDH structure. The use of redox active clay as a host matrix for enzyme prevents the mediator leaching and enhances specic interactions between redox mediator and the active site of the enzyme. The same effect has been previously reported for HRP encapsulated in the [ZnCrABTS] matrix (Shan et al., 2003a). Electrocatalytic reduction of oxygen at enzyme modied electrodes has been reported previously, for instance with myoglobin (Zhang et al., 2004a) and HRP (Zhang et al., 2004b). These heme proteins catalyzed the reduction of O2 to hydrogen peroxide. On the other hand, the four-electron reduction of O2 to water was achieved with bilirubin oxidase (Mano et al., 2003; Tsujimura et al., 2001) and laccase modied electrodes (Barton

et al., 2001; Farneth and DAmore, 2005; Gupta et al., 2004; Palmore and Kim, 1999; Rowinski et al., 2004; Tarasevich et al., 2003). Linear dependences of the electrode response versus O2 concentration were reported up to 2.2 105 M for myoglobin anchored on multi-walled carbon nanotubes (Zhang et al., 2004a) and between 9 106 and 2 104 M for laccase immobilized in liquid crystals (Rowinski et al., 2004) or 1.3 106 to 2.6 105 M for a poly(nile blue) modied electrode without enzyme (Ju and Shen, 2001). Compared to these other electrodes the results, presented in this work, show a sensitive linear response at lower concentration of oxygen (0.064 M), which allows this system to be considered for monitoring oxygen levels. 3.2. Determination of inhibitors Leech and coworkers have developed a reagentless enzyme sensor of laccase activity based on the immobilization of the enzyme in a redox osmium hydrogel quoted on the electrode surface (Leech and Daigle, 1998; Leech and Feerick, 2000; Trudeau et al., 1997). The detection principle of inhibitors, such as sodium azide, was based on their modulation effect on the enzyme activity, which was measured by the mediated reduction of O2 . The biosensor consumed only oxygen present in the solution. We have applied the same concept to detect inhibitors (NaN3 , NaF and KCN) at the laccase + BSA/[ZnCrABTS] modied electrode. Sensitive assays of these inorganic ions are of practical interest because they are environmental toxins, especially in water efuents. As shown in Fig. 1, the increase of sodium azide concentrations modied the signals in cyclic voltammetry. The catalytic current disappeared completely when the inhibitor concentration was high enough to stop totally the catalytic cycle of laccase. In chronoamperometric measurements under saturated oxygen conditions at 0.2 V, addition of inhibitors resulted in a rapid decrease in the electrocatalytic reduction current (Fig. 5A). Normalized inhibition curves were obtained by plotting (Ioxy Iinh )/(Ioxy Ideoxy ) 100% versus inhibitor

Fig. 4. O2 calibration curve (experimental conditions as in Fig. 3A).

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Fig. 5. (A) Amperometric response at laccase + BSA/[ZnCrABTS] bioelectrode under saturated O2 atmosphere (Eapp = 0.2 V) to: (a) four successive injections 7.5 nM and (b) six successive injections 15 nM NaF. (B) Normalized inhibition curves for laccase + BSA/[ZnCrABTS] bioelectrode for sodium azide, potassium cyanide and sodium uoride (acetate buffer solution pH 5.0 under saturated O2 conditions, Eapp = 0.2 V). Inset shows the biosensor responses for low inhibitor concentrations with the following linear ts NaN3 : Y = .4.51 0.20 + 2.36E8 0.06X (R = 0.996, n = 13, S.D. = 0.30); KCN: Y = 3.43 0.19 + 2.03E8 0.06X (R = 0.996, n = 10, S.D. = 0.24); NaF:Y = 0.3.39 0.18 + 1.85E8 0.06X (R = 0.996; n = 10; S.D. = 0.23).

NaF, 500 M KCN) (Trudeau et al., 1997). The same feature was observed when laccase is immobilized in a redox hydrogel (5.6 M NaN3 , 43 M NaF) (Leech and Feerick, 2000). These inorganic ions, azide, uoride and cyanide, were detected at very low detection limits, in the nanomolar range, compared to the detection limits reported in the literature. For instance, azide was detected at different biosensors based on laccase with a LOD = 12.5 M (Leech and Daigle, 1998; Leech and Feerick, 2000) or on catalase (LOD = 25 M) (Sezgin urk et al., 2005). Similarly, the LOD for uoride was reported between 8 105 and 5 104 M for biosensors based on the immobilization of cholinesterase on nylon, paper or cellulose nitrate (Evtugyn et al., 1999) and 8 107 M for a biosensor based on lever esterase (Marcos and Townslend, 1995). For cyanide, the present detection limit is close to the value that we had previously detected at the HRP/[ZnCrABTS] electrode, namely 5 nM (Shan et al., 2004). As reported previously for other LDH biosensors, these low detection limits can be related to the possible accumulation of anions into the anionic clay in the vicinity of enzyme. Additions of laccase substrates, for example catechol, dopamine, aniline and phenol at a concentration of 10 M, caused a decrease in the reduction current corresponding respectively to 39%, 9%, 8% and 2% of the inhibition response observed with NaN3 at the same concentration. This decrease in the cathodic current recorded at 0.2 V can be explained by a competitive process between the phenol derivatives and ABTS at the laccase T1 site. This interference effect seems to follow the sequence of the Km values reported for these substrates (Haghighi et al., 2003; Xu, 1996). It can be discriminated from real inhibition process by applying a cathodic potential of 0.1 V. At this applied potential, additions of phenol derivatives caused an increase in the reduction current due to the reduction of the quinoid form of the substrates. The same observation has been previously reported by Leech and Feerick (2000) with the laccase/redox osmium hydrogel system. 3.3. Lifetime The stability of the biosensor in oxygenated electrolyte was examined by recording the current response at 0.2 V for 4 h. A loss in signal of 20 %/h was observed. However, as suggested by Leech, the effect of instability on the inhibition curves can be minimized by normalization of the response (Leech and Daigle, 1998). For instance, the regeneration of the biosensor was investigated by placing the inhibited electrode in decarbonated water for 40 min. After the regeneration, the intensity of catalytic current of O2 reduction has decreased but the same sensitivity for further azide determination was observed. This conrms that inhibition of laccase Tv by azide is reversible (Leech and Feerick, 2000). The main drawback of O2 biosensors remains the lifetime. These biosensor congurations require the entrapment of both enzyme and mediator into a matrix in which oxygen and water can freely diffuse. The lack of stability was reported for bio-systems with laccase-ABTS (Farneth and DAmore, 2005; Farneth et al., 2005), bulirubin oxidase-ABTS (Tsujimura et al., 2001) but also with laccase-osmium complex (Leech and

concentration, where Ioxy and Ideoxy are the currents in the presence and absence of oxygen, and Iinh is the current observed upon inhibition in oxygenated electrolyte. Fig. 5B shows these curves for sodium azide, sodium uoride and potassium cyanide. C50 values (the concentration causing 50% activity reduction) and detection limits (LOD) estimated from these curves are presented in Table 1. The apparent C50 values were lower when the enzyme was immobilized compared to the values reported for laccase Tv in solution (14 M NaN3 , 500 M
Table 1 Parameters of calibration curves of inhibitors to be determined using the laccase + BSA/[ZnCrABTS] bioelectrode Inhibitors NaN3 KCN NaF C50 (M) 3 23 153 LODa (nM) 5.5 0.1 6.2 0.1 6.9 0.1

a Detection limit calculated for 3S.D. where S.D. is the standard deviation b b of the blank, mean value for two different biosensors.

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C. Mousty et al. / Biosensors and Bioelectronics 22 (2007) 17331738 de Roy, A., Forano, C., El Malki, M., Besse, J.-P., 1992. In: Occelli, M.L., Robson, H.E. (Eds.), Synthesis of Microporous Materials, Expanded Clays and Other Microporous Solids. Van Nostrand Reinhold, New York, pp. 108170. Dodor, D.E., Hwang, H.-M., Erkunwe, S.I.N., 2004. Enzyme Microb. Technol. 35, 210217. Dur an, N., Rosa, M.A., DAnniblae, A., Gianfreda, L., 2002. Enzyme Microb. Technol. 21, 907931. Evtugyn, G.A., Ivanov, A.N., Gogol, E.V., Marty, J.-L., Budnikov, H.C., 1999. Anal. Chim. Acta 385, 1321. Farneth, W.E., DAmore, M.B., 2005. J. Electroanal. Chem. 581, 197205. Farneth, W.E., Diner, B.A., Gierke, T.D., DAmore, M.B., 2005. J. Electroanal. Chem. 581, 190196. Ferry, Y., Leech, D., 2005. Electroanalysis 17, 113119. Freire, R.S., Dur an, N., Kubota, L.T., 2001. Talanta 54, 681686. Freire, R.S., Dur an, N., Kubota, L.T., 2002. Anal. Chim. Acta 463, 229238. Gianfreda, L., Bollag, J.-M., 1994. Soil Sci. Soc. Am. J. 58, 16721681. Gupta, G., Rajendran, V., Atanassov, P., 2004. Electroanalysis 16, 1182 1185. Haghighi, B., Gorton, L., Ruzgas, T., Jonsson, L.J., 2003. Anal. Chim. Acta 487, 314. Jarosz-Wilkolazka, A., Ruzgas, T., Gorton, L., 2005. Talanta 66, 12191224. Jolivalt, C., Brenon, S., Caminade, E., Mougin, C., Ponti e, M., 2000. J. Membr. Sci. 180, 103113. Ju, H., Shen, C., 2001. Electroanalysis 13, 789793. Leech, D., Daigle, F., 1998. Analyst 123, 19711974. Leech, D., Feerick, K.O., 2000. Electroanalysis 12, 13391342. Mano, N., Fernandez, J.L., Kim, Y., Shin, W., Bard, A.J., Heller, A., 2003. J. Am. Chem. Soc. 125, 1529015291. Marcos, J., Townslend, A., 1995. Anal. Chim. Acta 310, 173180. Mousty, C., 2004. Appl. Clay Sci. 27, 159177. Palmore, G.T.R., Kim, H.H., 1999. J. Electroanal. Chem. 464, 110117. Rowinski, P., Bilewicz, R., Stebe, M.-J., Rogalska, E., 2004. Anal. Chem. 76, 283291. Ruggiero, P., Sarkar, J.M., Bollag, J.-M., 1989. Soil Sci. 147, 361370. Scott, S.L., Chen, W.-J., Bakac, A., Espenson, J.H., 1993. J. Phys. Chem. 97, 67106714. Sezgin urk, M.K., G oktug, T., Dinc kaya, E., 2005. Biosens. Bioelectron. 21, 684688. Shan, D., Cosnier, S., Mousty, C., 2003a. Anal. Lett. 36, 909922. Shan, D., Cosnier, S., Mousty, C., 2003b. Anal. Chem. 75, 38723879. Shan, D., Cosnier, S., Mousty, C., 2004. Biosens. Bioelectron. 20, 390396. Tarasevich, M.R., Bogdanovskaya, V.A., Kapustin, A.V., 2003. Electrochem. Commun. 5, 491496. Therias, S., Mousty, C., Forano, C., Besse, J.P., 1996. Langmuir 12, 4914 4920. Trudeau, F., Daigle, F., Leech, D., 1997. Anal. Chem. 69, 882886. Tsujimura, S., Tatsumi, H., Ogawa, J., Shimizu, S., Kano, K., Ikeda, T., 2001. J. Electroanal. Chem. 496, 6975. Vial, S., Forano, C., Shan, D., Mousty, C., Barhoumi, H., Martelet, C., Jaffrezic, N., 2006. Mater. Sci. Eng., C, 387393. Vianello, F., Cambria, A., Ragusa, S., Cambria, M.T., Zennaro, L., Rigo, A., 2004. Biosens. Bioelectron. 20, 315321. Xu, F., 1996. Biochemistry 35, 76087614. Zhang, L., Zhao, G.-C., Wei, X.-W., Yang, Z.-S., 2004a. Chem. Lett. 33, 8687. Zhang, Y., He, P., Hu, N., 2004b. Electrochim. Acta 49, 19811988.

Daigle, 1998). In the former case, the authors suggest that the chemical stability of ABTS limits the system lifetime. However, we have previously reported very good operational stability for HRP biosensor using the same [ZnCrABTS] matrix (Shan et al., 2003a). Another possibility can consist in a shift of local pH within the matrix as a result of the oxygen reduction O2 + 4H+ + 4e 2H2 O. This increase of pH value can damage the enzyme. No pH change was observed in the soaking solution but we have observed by UV spectroscopy leaching of the biolm. The slow dissolution of laccase can dislodge a part of the clay lm from the electrode surface. Consequently, the coimmobilization of laccase and redox active clay on the electrode surface must be improved. This can probably be envisaged with the coprecipitation method that we have already realized with urease (Vial et al., 2006). 4. Conclusion In this work, we have described the development of a laccase biosensor based on the entrapment of the enzyme into redox active layered double hydroxides [ZnCrABTS]. ABTS, intercalated within LDH layers, plays the role of redox mediator performing the electrical wiring of laccase. The use of this electroactive nanohybrid material as a host matrix for enzyme prevents the mediator leaching and enhances specic interactions between the redox mediator and the active site of the enzyme. This biosensor offers a fast and a sensitive response in presence of dissolved oxygen and can be used to detect laccase inhibitors. In particular, this bioelectrode provides the lowest detection limits (5.5 nM) for azide and (6.2 nM) for uoride reported with electroenzymatic sensors. Moreover based on its good electrocatalysis for oxygen reduction, this system can be applied, as the cathodic catalyst, to fabrication of biofuel cell. Our main goal will be the improvement of the immobilization of laccase in the clay matrix coated on a larger surface area electrode. Acknowledgment This work is supported by the ACI Program Nanohybrides Enzymes-HDL 2003-NR0005 from the Research Ministry of France. References
Barton, S.C., Kim, H.-H., Binyamin, G., Zhang, Y., Heller, A., 2001. J. Phys. Chem. B 105, 1191711921.