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Biosensors and Bioelectronics 22 (2007) 1733–1738

Laccase immobilization in redox active layered double hydroxides: A reagentless amperometric biosensor
Christine Mousty ∗ , Laetitia Vieille, Serge Cosnier
Laboratoire d’Electrochimie 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 [Zn–Cr–ABTS] 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 × 10−8 and 4 × 10−6 M and very low detection limits for azide (5.5 nM), fluoride (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 specificity 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, delignification 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 D’Amore, 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 [Zn–Cr–ABTS] (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).

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. An Ag/AgCl electrode saturated with KCl solution was used as reference electrode. The layered double hydroxide Zn2 Cr(OH)6 ABTS was synthesized by the coprecipitation method (Therias et al. / Biosensors and Bioelectronics 22 (2007) 1733–1738 Scheme 1. versicolor in the [Zn–Cr–ABTS] LDH matrix. The flow injection system consisted of an isocratic pump (Perkin-Elmer200LC) and a Rheodine 9725 injection valve with 20 ␮l loop. one can expect to have an efficient turn over of the O2 /laccase/[Zn–Cr–ABTS] system upon applied potential (Scheme 1). 2.4. The UV test was performed in 3 ml acetate buffer pH 5. versicolor has a very high activity with ABTS (Vianello et al.2.1. 2. each solution had a concentration of 4 mg ml−1 . phenol and aniline from Prolabo. Flow injection experiments were carried out using a BAS thin layer cell (radial flow). we report a new biosensor configuration based on the immobilization of laccase from T. Laccase and BSA were dissolved in water..1734 C.2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). and a Pt wire was placed in a separate compartment containing the supporting electrolyte.05 ml/min.5 ␮l) of aqueous mixtures containing a defined amount of clay (40 ␮g). catechol and dopamine were received from Sigma. In the present paper. Before use. 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). 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). Spectrophotometric measurements were carried out with a Varian Cary 1 UV-visible spectrophotometer. This O2 standard solution was obtained by bubbling for 1 h. Assay of laccase activity The activity of free laccase was determined by using ABTS as substrate. Mousty et al. Materials and solutions Laccase from T. The coating was . The electrolyte flow was fixed at 0.1 mM. versicolor (Tv) and glutaraldehyde (25%) were purchased from Fluka. as a counter electrode. Vianello and coworkers have reported that among different commercial laccase sources. Enzyme immobilization The clay colloidal suspension (2 mg ml−1 [Zn–Cr–ABTS]) was dispersed overnight under stirring conditions in deionised and decarbonated water. laccase from T. these electrodes were polished with 1 ␮m diamond paste and rinsed with water and acetone.0 containing 5 mM ABTS (ε420 = 36000 M−1 cm−1 ). 2004).3. The activity of laccase determined by this method was 12 U/mg. Cyclic voltamme- tries were recorded with Autolab 100 potentiostat. This biosensor configuration is applied for monitoring oxygen levels and for the determination of laccase inhibitors such as sodium azide. 2. One unit of laccase activity is defined as the amount oxidizing 1 ␮mol substrate per min. 2. Therefore. Bovin serum albumin (BSA). Oxygen concentration in the saturated supporting electrolyte was 1. All other chemical reagents were of analytical grade. laccase (5 ␮g) and BSA (5 ␮g) was spread on the surface of the glassy carbon electrode. 1996). Schematic reaction occurring at the laccase/[Zn–Cr–ABTS] modified electrode. 2. Experimental 2. A drop (22. For the oxygen detection. Water was doubly distilled in quartz apparatus. sodium fluoride and potassium cyanide. All electrochemical experiments were carried out in a conventional thermostated three-electrode cell (10 or 40 ml) at 30 ◦ C. the experiments were made in a glove box under argon atmosphere by successive additions of saturated O2 supporting electrolyte solution.

1 ␮g.. 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).The maximum current density measured in saturated O2 atmosphere at 10 different electrodes was 6. 1.. the amount of retained laccase was 83% (4. the laccase/BSA/clay biomembrane was rehydrated for 20 min into 0. Influence of pH on amperometric response of laccase + BSA/ [Zn–Cr–ABTS] bioelectrode under saturated O2 atmosphere in acetate buffer solution (Eapp = 0. Moreover. Results and discussion 3. the efficient immobilization of laccase from T. 2000).9 × 10−8 .. on spectrographic graphite electrodes (Haghighi et al. (b) 9.2 V for further experiments.1 M acetate buffer solution (pH 5). Finally. When different amounts of saturated oxygen solution were added into the batch cell containing deoxygenated electrolyte.2 ± 0.08 × 10−6 and (d) 1. characteristic of ABTS immobilized into the clay matrix [Zn–Cr–ABTS] (Fig. versicolor immobilized on carbon fiber electrodes (Freire et al. 2001). 2003a. 4). amounts of both proteins coated on the electrode surface. Calibration curves had been recorded at two different applied potentials: 0. we decided to apply 0. The reproducibility of the biosensor responses was investigated by repeated (n = 6) injections of 10 ␮l of saturated oxygen solution.0 and 0. This resulted in an electrocatalytic reduction wave at the modified electrode.0) (Farneth et al. Before use. an increase in the catalytic reduction current was observed (Fig..2 V for 1 h under O2 atmosphere. 1 (curve a).05 U).C. 1993). 0. but the biosensor response is more stable at 0. v = 10 mV s−1 . The analytical performance of laccase + BSA/[Zn–Cr– ABTS] electrodes for oxygen detection was investigated in a glove box under argon atmosphere. a chemical cross-linking is generally required (Freire et al. Successive injections of saturated oxygen solution (20 ␮l loop) into a constant flow of deoxygenated acetate buffer solution gave a cathodic current of 2.2 V and the linear range is wider at this potential (6 × 10−8 to 4 × 10−6 M) than that at 0. Cyclic voltammograms of GCE modified by laccase + BSA/ [Zn–Cr–ABTS] (5:5:20 ␮g) (a) under saturated O2 atmosphere and with successive additions of sodium azide. 2003b) in LDH matrix. The sensitivities are similar. the relative standard deviation was 8%. Fig. Different biosensor configurations have been tested by varying the relative Fig. an enzyme inhibitor (Fig. The maximum response was obtained at pH 5. Consequently.64 nA (RDS 8%. 2005. 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. Indeed. n = 8).2 V (Fig. With laccase.. the current returned to its initial value. With a biofilm containing 40 ␮g LDH.2 V).2 ␮g) were retained on the electrode surface. Since ABTS oxidation is known to be reversible and pH independent (Scott et al.0 which is the same pH value as that reported previously for laccase from T. the biosensor was stabilized at 0. curves b and c) caused a decrease in the electrocatalytic reduction wave to finally obtain a reversible signal. Removal of oxygen from the solution by outgassing with argon (not shown) or the additions of azide.08 × 10−5 M in acetate buffer solution (pH 5. 3B). The reproducibility of O2 detection was also determined under flow injection analysis (FIA) (Fig. 2004) and polyphenol oxidase (PPO) (Shan et al. Under the optimal conditions. showing good reversibility of the detection device. versicolor appears to be problematic. 5 ␮g laccase and 5 ␮g BSA. Jolivalt et al. 2.. 3A). The resulting electrode was placed in saturated glutaraldehyde vapour for 1 h for cross-linking of the membrane. After each injection. This value corresponds to a specific activity of immobilized laccase Tv 1 (Imax /molEnz ) of 1 × 105 ␮A cm−2 mol− Enz which is in good . 83% of laccase (4. 1. 2004) but it is slightly higher than that reported for the free enzyme (pH 3. The later value was determined by UV spectroscopy. (c) 1. This immobilization procedure differed slightly from that previously adopted for the immobilization of HRP (Shan et al. Oxygen detection Typical voltammogram of laccase/[Zn–Cr–ABTS] modified electrode in the presence of O2 is shown in Fig. the variation of the cathodic current with pH can be related to changes of laccase activity. 3. The influence 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.0 V (4 × 10−7 to 2 × 10−6 M). 2001).1. 2). the reticulation time must be increased to 1 h. namely 443 and 474 mA M−1 cm−2 . / Biosensors and Bioelectronics 22 (2007) 1733–1738 1735 dried under vacuum at room temperature. 2003) and on kaolinite (Dodor et al. whereas with 10 ␮g of laccase without BSA only 37% (3. 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 efficient mediated reduction of O2 . curve d).7 ␮A cm−2 . Mousty et al.0).7 ␮g) remained... 1. a coreticulation with BSA appeared to be necessary..

. 2001) and laccase modified electrodes (Barton et al.0 to repeated injections (arrows) of 5 ␮l saturated O2 solution. The use of redox active clay as a host matrix for enzyme prevents the mediator leaching and enhances specific interactions between redox mediator and the active site of the enzyme. Mousty et al. 2003.. Tsujimura et al. in solution (KM M 2005) or for laccase Tv immobilized on kaolinite with ABTS ABTS = 162 ␮M) (Dodor et al. 2001)... 1998. Normalized inhibition curves were obtained by plotting (Ioxy − Iinh )/(Ioxy − Ideoxy ) × 100% versus inhibitor Fig. 2003).2. As shown in Fig.3 × 10−6 to 2. 2004). 2004b). show a sensitive linear response at lower concentration of oxygen (0.06–4 ␮M). especially in water effluents. 5A).2 × 10−5 M for myoglobin anchored on multi-walled carbon nanotubes (Zhang et al. addition of inhibitors resulted in a rapid decrease in the electrocatalytic reduction current (Fig. presented in this work.2 V in 40 ml deoxygenated acetate buffer solution pH 5.6 × 10−5 M for a poly(nile blue) modified electrode without enzyme (Ju and Shen. 4. 1997). 2005. 2000. NaF and KCN) at the laccase + BSA/[Zn–Cr–ABTS] modified electrode. The same effect has been previously reported for HRP encapsulated in the [Zn–Cr–ABTS] matrix (Shan et al.1736 C. 1. Leech and Feerick... which was measured by the mediated reduction of O2 . (A) Amperometric response at laccase + BSA/[Zn–Cr–ABTS] bioelectrode at 0.2 V in a radial flow injection cell to repeated injections of 20 ␮l saturated O2 solution. app The KM calculated from a Lineweaver–Burk plot of the calibration curve was 30 ␮M. 1 agreement with the value of 1. Tarasevich et al. was based on their modulation effect on the enzyme activity.. On the other hand. 2004. 2001. 2005) for purified laccase Tv and ABTS immobilized in a silica-coated carbon paper macroelectrode. The lower in solution (KM app KM reflects the efficient electrical wiring of laccase by ABTS intercalated in the LDH structure. (B) Amperometric response at laccase + BSA/[Zn–Cr–ABTS] bioelectrode at 0. This value can be compared to the values reported for the biocatalysts system of laccase Tv/ABTS ABTS = 192 ␮M. 1999.. Electrocatalytic reduction of oxygen at enzyme modified electrodes has been reported previously...2 V..5 × 105 ␮A cm−2 mol− Enz reported by (Farneth and D’Amore. The biosensor consumed only oxygen present in the solution. The detection principle of inhibitors. 3. In chronoamperometric measurements under saturated oxygen conditions at 0. / Biosensors and Bioelectronics 22 (2007) 1733–1738 Fig. 2004a) and HRP (Zhang et al. We have applied the same concept to detect inhibitors (NaN3 . 2004. 3. 3A). Gupta et al. . such as sodium azide. 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. Farneth and D’Amore. The catalytic current disappeared completely when the inhibitor concentration was high enough to stop totally the catalytic cycle of laccase. 2003a). the four-electron reduction of O2 to water was achieved with bilirubin oxidase (Mano et al.. O2 calibration curve (experimental conditions as in Fig. 2004a) and between 9 × 10−6 and 2 × 10−4 M for laccase immobilized in liquid crystals (Rowinski et al. K O2 = 262 ␮M) (Farneth et al. the increase of sodium azide concentrations modified the signals in cyclic voltammetry.. Rowinski et al. Palmore and Kim. Linear dependences of the electrode response versus O2 concentration were reported up to 2. Sensitive assays of these inorganic ions are of practical interest because they are environmental toxins. which allows this system to be considered for monitoring oxygen levels. These heme proteins catalyzed the reduction of O2 to hydrogen peroxide. Trudeau et al. Compared to these other electrodes the results. 2004) or 1.. for instance with myoglobin (Zhang et al.

The main drawback of O2 biosensors remains the lifetime. n = 10. After the regeneration. and Iinh is the current observed upon inhibition in oxygenated electrolyte. 500 ␮M KCN) (Trudeau et al. Mousty et al. Inset shows the biosensor responses for low inhibitor concentrations with the following linear fits NaN3 : Y = . 1998. bulirubin oxidase-ABTS (Tsujimura et al. / Biosensors and Bioelectronics 22 (2007) 1733–1738 1737 Fig. the LOD for fluoride was reported between 8 × 10−5 and 5 × 10−4 M for biosensors based on the immobilization of cholinesterase on nylon. This decrease in the cathodic current recorded at 0. = 0. additions of phenol derivatives caused an increase in the reduction current due to the reduction of the quinoid form of the substrates.1 V.. Fig. For instance. The same observation has been previously reported by Leech and Feerick (2000) with the laccase/redox osmium hydrogel system. Farneth et al. were detected at very low detection limits. is the standard deviation b b of the blank. potassium cyanide and sodium fluoride (acetate buffer solution pH 5. A loss in signal of 20 %/h was observed. 2001) but also with laccase-osmium complex (Leech and concentration. as suggested by Leech.996.06X (R = 0.9 ± 0. = 0. .43 ± 0.6 ␮M NaN3 . n = 13. n = 10. 500 ␮M Table 1 Parameters of calibration curves of inhibitors to be determined using the laccase + BSA/[Zn–Cr–ABTS] bioelectrode Inhibitors NaN3 KCN NaF C50 (␮M) 3 23 153 LODa (nM) 5.30).85E8 ± 0. For cyanide.20 + 2. 43 ␮M NaF) (Leech and Feerick. fluoride and cyanide. = 0. S.24).C.2 V can be explained by a competitive process between the phenol derivatives and ABTS at the laccase T1 site. 3. For instance. S. sodium fluoride and potassium cyanide. 1996). azide was detected at different biosensors based on laccase with a LOD = 1–2.2 V). the effect of instability on the inhibition curves can be minimized by normalization of the response (Leech and Daigle.18 + 1. caused a decrease in the reduction current corresponding respectively to 39%. the present detection limit is close to the value that we had previously detected at the HRP/[Zn–Cr–ABTS] electrode. The same feature was observed when laccase is immobilized in a redox hydrogel (5.23).996. At this applied potential. However. NaF:Y = 0.03E8 ± 0. namely 5 nM (Shan et al. This confirms that inhibition of laccase Tv by azide is reversible (Leech and Feerick. This interference effect seems to follow the sequence of the Km values reported for these substrates (Haghighi et al.5 nM and (b) six successive injections 15 nM NaF.. dopamine.5 ± 0. 5. NaF.06X (R = 0. where Ioxy and Ideoxy are the currents in the presence and absence of oxygen. 2004).3. (A) Amperometric response at laccase + BSA/[Zn–Cr–ABTS] bioelectrode under saturated O2 atmosphere (Eapp = 0.5 ␮M (Leech and Daigle.D. S. azide. Leech and Feerick. paper or cellulose nitrate (Evtugyn et al. compared to the detection limits reported in the literature.51 ± 0. 5B shows these curves for sodium azide. These inorganic ions.2 V) to: (a) four successive injections 7.. for example catechol. 2000).. where S. 1997).. Similarly.39 ± 0. KCN: Y = 3.4.996. mean value for two different biosensors. 1995).D. in the nanomolar range. These biosensor configurations require the entrapment of both enzyme and mediator into a matrix in which oxygen and water can freely diffuse. 2005). 2000). 1998). 2000) or on catalase (LOD = 25 ␮M) (Sezgin¨ urk et al. these low detection limits can be related to the possible accumulation of anions into the anionic clay in the vicinity of enzyme. Xu.19 + 2.D.2 V for 4 h.D.1 6.1 a Detection limit calculated for 3S. The lack of stability was reported for bio-systems with laccase-ABTS (Farneth and D’Amore. Lifetime The stability of the biosensor in oxygenated electrolyte was examined by recording the current response at 0.06X (R = 0.D. 2003. aniline and phenol at a concentration of 10 ␮M. Additions of laccase substrates. 9%.36E8 ± 0. 2005... (B) Normalized inhibition curves for laccase + BSA/[Zn–Cr–ABTS] bioelectrode for sodium azide. 2005). As reported previously for other LDH biosensors. the intensity of catalytic current of O2 reduction has decreased but the same sensitivity for further azide determination was observed.0 under saturated O2 conditions.3. The apparent C50 values were lower when the enzyme was immobilized compared to the values reported for laccase Tv in solution (14 ␮M NaN3 .1 6. Eapp = 0. It can be discriminated from real inhibition process by applying a cathodic potential of −0. the regeneration of the biosensor was investigated by placing the inhibited electrode in decarbonated water for 40 min. C50 values (the concentration causing 50% activity reduction) and detection limits (LOD) estimated from these curves are presented in Table 1. 1999) and 8 × 10−7 M for a biosensor based on lever esterase (Marcos and Townslend.2 ± 0. 8% and 2% of the inhibition response observed with NaN3 at the same concentration.

C.J. H. Mano. S. Jaffrezic. H.. Chem. Soil Sci. J. Enzyme Microb. J. Kano. Evtugyn. Kapustin. 390–396. Stebe. Daigle. Ruggiero. we have described the development of a laccase biosensor based on the entrapment of the enzyme into redox active layered double hydroxides [Zn–Cr–ABTS]. 2004..E. the coimmobilization of laccase and redox active clay on the electrode surface must be improved. 113–119. Chem. F. Chem. Bioelectron. Sarkar. D. 361–370.. 2004b. S..J. Talanta 66. In the former case. P. Van Nostrand Reinhold.A. 2006). M. Mousty. Ikeda.. Shan. as the cathodic catalyst.. Rajendran... 1981–1988. Soil Sci.T. Xu. 190–196. J. S. X. Robson. J.. D.. Sci. 2005.. 15290–15291. Y.O.L... C.. Anal. Budnikov.E. Shimizu. Palmore.R. Chim. A. No pH change was observed in the soaking solution but we have observed by UV spectroscopy leaching of the biofilm. Cambria.. H. C. 86–87.. Biosens. 125. 69. S. A. Townslend. T.. 907–931. G.-W. L. N. Chem. Forano. Barhoumi. 103–113. Trudeau.. N. 2003a. F. Martelet. G. 4914– 4920.. P. Technol. M..L.. N. Anal. D.-M. 1998. 581.P. R. 6710–6714. J. 159–177. 69–75. Jarosz-Wilkolazka. H. Bard. D. 1672–1681. Anal.. Biosens. K. R. Electroanal. Cambria.C. M. 2004. C. Bollag. 1182– 1185.. Farneth.E. 210–217. 1995... L. 2001.A. 58. Commun. Cosnier. L. Freire. pp.A.. Biosens. Gierke. Phys. 76. Sezgin¨ urk. 2003a). 108–170.. He. Espenson..-M. Kubota. Mougin... Kim. P.. Marty.. Rowinski.. (Eds.. Kim.. J. W.1738 C.-M. W. Our main goal will be the improvement of the immobilization of laccase in the clay matrix coated on a larger surface area electrode.. Rosa. M.. R. 2000. D. Shen. Diner. Electrochem. J. E. Shin. Forano. K. L.). 2002. 1989. El Malki. C. M. 387–393. G. Electroanalysis 16. D. Gupta. Am. Hwang. N. Cosnier. 315–321. This biosensor offers a fast and a sensitive response in presence of dissolved oxygen and can be used to detect laccase inhibitors. J. / Biosensors and Bioelectronics 22 (2007) 1733–1738 de Roy. W. 684–688. 2000. M. M. Heller.. Acta 49... Mater. 1998).N. 2001. Bollag. C.. Zhang. Gorton.. Caminade.. Haghighi. 20.D... 491–496. S....H. F. 27. J. 75. this system can be applied.. Acta 310. 97. 1999..-C.5 nM) for azide and (6. to fabrication of biofuel cell. D.I. Gianfreda. M.. Daigle. Shan. Y. Dinc ¸ kaya.. D. C. 2005.. 1996. this bioelectrode provides the lowest detection limits (5. S. D’Anniblae. Brenon.. Feerick.T. W. Electroanalysis 12. Chem. New York. H. S. 7608–7614. Gogol. Chen.... J. M. Mousty.. Rogalska. Ruzgas.B. 2004. Electroanalysis 13. plays the role of redox mediator performing the electrical wiring of laccase.. Eng. 2005. H. S. Chem.R. A. S. Bogdanovskaya. Chem... 229–238. Gianfreda.-L. Chem. Synthesis of Microporous Materials. 2004. The slow dissolution of laccase can dislodge a part of the clay film from the electrode surface. In: Occelli.. 173–180... E.C. 1992. J. A. V.-H. 2003b. Electroanal. Dur´ an. Membr.T. Erkunwe. Moreover based on its good electrocatalysis for oxygen reduction. Scott. 3872–3879. J. Chem. Soc. Cosnier.. Tarasevich.-J.. Farneth. However. 2004a. Ferry. The use of this electroactive nanohybrid material as a host matrix for enzyme prevents the mediator leaching and enhances specific interactions between the redox mediator and the active site of the enzyme. A. E. Acknowledgment This work is supported by the ACI Program Nanohybrides Enzymes-HDL 2003-NR0005 from the Research Ministry of France. Analyst 123.S. Dur´ an.H. Y. Appl... L. Electrochim. 2001.. 882–886. 4.. H... 2005. Ju. 180. F. Acta 487.. Talanta 54.. Chem.-P. Enzyme Microb.. Leech. Forano. 1997. E.. 2003. 909–922. Kubota. 20. D’Amore. Zhao. J. J.. J. B. 2005.. G.. Besse.. Daigle. Ponti´ e.. Wei. Lett. J.. 283–291. Shan. 13–21. Mousty et al. Mousty. Tatsumi.2 nM) for fluoride reported with electroenzymatic sensors. 1339–1342.V. Ivanov. Expanded Clays and Other Microporous Solids. Bioelectron. H. Ogawa... Kim.. Leech.. C. Therias. 2003.. 2003. Bioelectron. 464. C. 36. 35.. 2004. Electroanal. J. 110–117..T... S. Hu. Consequently. Yang. V... A. S.. Anal. Electroanal. Zhang. 197–205. B. 3–14. A. T.L. 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. 581.. 681–686.. Conclusion In this work... C. Clay Sci. 1971–1974. Fernandez. Electroanalysis 17. Tsujimura. Ragusa... Vianello.. 21.. Freire. Bakac. This increase of pH value can damage the enzyme. 1219–1224. Sci... 2004. Vial.A.. D.V. Marcos. 147. intercalated within LDH layers.. we have previously reported very good operational stability for HRP biosensor using the same [Zn–Cr–ABTS] matrix (Shan et al. Acta 463.. 2002.. Anal.E.. 1999. A. 2001. S. Ruzgas. J. In particular. Mousty.. J. Leech. Soc. T. Jonsson. A. N.K. C. G. This can probably be envisaged with the coprecipitation method that we have already realized with urease (Vial et al. Gorton.. T. C. the authors suggest that the chemical stability of ABTS limits the system lifetime..B. Anal. 5. Chim. Jolivalt.. C. Zhang.. L... Chem...-S. Rigo. B 105. Dur´ an.N. N. Atanassov. F. Biochemistry 35. G¨ oktug. Langmuir 12.. 1994.. Mousty.... Phys. 1993. Binyamin. Am. 11917–11921. 33. 21. L. Besse. Heller. 789–793. 496. Z. Acta 385. L. 2006. Mousty. Anal.M.S. References Barton. P. Lett.... T. . A.. Bilewicz.. Dodor. Technol. Zennaro. D’Amore. ABTS.. A..-J.. Y. A.. L. Anal.. Chim. M. Shan. 1996. Leech. Chim. C.