laccase immobilitation in redox

8/13/2019 Laccase Immobilitation in Redox

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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, Universite 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 versicolorand 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 6108 and 4106 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; O2reduction; Inhibition

1. Introduction

Laccases (EC 1.10.3.2) are multicopper oxidases widely dis-tributed in plant and fungal species. They received particular

attention because they present rather low substrate specificity

and are able to oxidise phenols, anilines, benzenethiols, phe-

nothiazines with the concomitant reduction of molecular oxygen

(O2) towater (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 O2is electroreducedto 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 determi-

nation of a broad range of phenolic species (Ferry and Leech,

2005; Freire et al., 2001, 2002; Haghighi et al., 2003; Jarosz-

Wilkolazkaet 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:[email protected](C. Mousty).

Most applications require enzyme immobilization. Duran et

al. have published an overview of the different methods used for

the immobilization of laccase (Duran et al., 2002).In particu-lar, the immobilization of laccase fromTrametes versicolorwas

extensively studied for several years. It is reported that a high

percentage of laccase activity is maintained after its immobiliza-

tion 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 immobi-

lization 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 materi-

als known as synthetic layered double hydroxides (LDH) (de

Roy et al., 1992).Recently, we have successfully immobilized

horseradish peroxidase (HRP) into redox active layered dou-

ble hydroxide [ZnCrABTS] (Shan et al., 2003a).The organic

electroactive dianions, 2,2-azino-bis(3-ethylbenzothiazoline-6-

sulfonic acid) (ABTS) intercalated within the LDH interlayer

domain, remain redox active and play the role of electron shut-

tle between the redox centre of HRP and the electrode. The

electrochemical transduction step corresponds to the reduction

at 0.0V of ABTS+ enzymatically formed in the presence of

H2O2. 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
mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.bios.2006.08.020http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.bios.2006.08.020mailto:[email protected]


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1734 C. Mousty et al. / Biosensors and Bioelectronics 22 (2007) 17331738

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

In the present paper, we report a new biosensor configuration

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, lac-

case from T. versicolor has a very high activity with ABTS

(Vianello et al., 2004). Therefore, one can expect to have an effi-

cient turn over of the O2/laccase/[ZnCrABTS] system upon

applied potential (Scheme 1). This biosensor configuration is

applied for monitoring oxygen levels and for the determination

of laccase inhibitors such as sodium azide, sodium fluoride 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, phe-

nol and aniline from Prolabo. The layered double hydrox-

ide Zn2Cr(OH)6ABTS was synthesized by the coprecipitation

method (Therias et al., 1996).All other chemical reagents were

of analytical grade. Water was doubly distilled in quartz appa-ratus. 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 bubblingfor 1 h. Oxygenconcentration

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 e-

corder 401 system (CBO instrumentation). Cyclic voltamme-

tries were recorded with Autolab 100 potentiostat. All electro-

chemical experiments were carried out in a conventional ther-

mostated three-electrodecell (10or 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

containingthe supporting electrolyte, as a counter electrode.The

working electrodes were glassy carbon electrodes with a diam-

eter of 3 mm for cyclic voltammetry and FIA experiments and

5 mm for batch chronoamperometric experiments under rotation

conditions (500 rpm).Before use, these electrodeswere polished

with 1m diamond paste and rinsed with water and acetone.

Flow injection experiments were carried out using a BAS thin

layer cell (radial flow). The flow injection system consisted of

an isocratic pump (Perkin-Elmer200LC) and a Rheodine 9725

injection valve with 20l loop. The electrolyte flow was fixed

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 defined as the amount

oxidizing 1mol substrate per min. The UV test was per-

formed in 3 ml acetate buffer pH 5.0 containing 5 mM ABTS(420= 36000 M

1 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

(22.5l) of aqueous mixtures containing a defined amount of

clay (40g), laccase (5g) and BSA (5g) was spread on

the surface of the glassy carbon electrode. The coating was


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C. Mousty et al. / Biosensors and Bioelectronics 22 (2007) 17331738 1735

dried under vacuum at room temperature. The resulting elec-

trode 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 O2atmosphere.

The amount of enzyme immobilized on the electrode surface

was calculated by the difference between amount of protein ini-

tially 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%

(4.1g, 0.05 U).

3. Results and discussion

3.1. Oxygen detection

Typical voltammogram of laccase/[ZnCrABTS] modified

electrode in the presence of O2is shown inFig. 1(curve a). The

redox mediator ABTS is oxidized by laccase and the regener-ation of the enzyme is achieved by the reduction of molecular

oxygen to water (Scheme 1).This resulted in an electrocatalytic

reduction wave at the modified 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 finally

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 efficient mediated

reduction of O2. This immobilization procedure differed slightlyfrom 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 efficient immobilization of

laccase from T. versicolorappears to be problematic, a chemical

cross-linking is generally required (Freire et al., 2001). Different

biosensor configurations have been tested by varying the relative

Fig. 1. Cyclic voltammograms of GCE modified by laccase + BSA/

[ZnCrABTS] (5:5:20g) (a) under saturated O2 atmosphere and with suc-

cessive additions of sodium azide, (b) 9.9 108, (c) 1.08106 and (d)

1.08

105

M in acetate buffer solution (pH 5.0), v =10mVs1

.

Fig. 2. Influence of pH on amperometric response of laccase + BSA/

[ZnCrABTS] bioelectrode under saturated O2 atmosphere in acetate buffer

solution (Eapp= 0.2 V).

amounts of both proteins coated on the electrode surface. With

a biofilm containing 40g LDH, 5g laccase and 5g BSA,

83% of laccase (4.2g) were retained on the electrode surface,

whereas with 10g of laccase without BSA only 37% (3.7 g)

remained.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. 2).Since ABTS oxidation is known to be

reversible and pH independent (Scott et al., 1993), the varia-

tion 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 fiber elec-

trodes(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 con-

taining 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

0.2V (Fig. 4). The sensitivities are similar, namely 443 and

474mAM1 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)thanthatat0.0V(4 107 to 2 106 M). Conse-

quently, we decided to apply 0.2 V for further experiments. The

reproducibility of the biosensor responses was investigated byrepeated (n = 6) injections of 10l of saturated oxygen solution,

the relative standard deviation was 8%. The reproducibility of

O2detection was also determined under flow injection analysis

(FIA) (Fig. 3B). Successive injections of saturated oxygen solu-

tion (20l loop) into a constant flow 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 O2atmosphere

at 10 different electrodes was 6.2 0.7A cm2. This value

corresponds to a specific activity of immobilized laccase Tv

(Imax/molEnz) o f 1

105A cm

2

mol1

Enz which is in good


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Fig. 3. (A) Amperometric responseat laccase + BSA/[ZnCrABTS] bioelectrode at 0.2 V in 40 ml deoxygenated acetate buffer solutionpH 5.0 to repeated injections

(arrows) of 5l saturated O2solution. (B) Amperometric response at laccase + BSA/[ZnCrABTS] bioelectrode at 0.2 V in a radial flow injection cell to repeated

injections of 20l saturated O2 solution.

agreement with the value of 1.5

10

5

A cm

2

mol

1

Enzreportedby (Farneth and DAmore, 2005)for purified laccase Tv and

ABTS immobilized in a silica-coated carbon paper macroelec-

trode.

The KappM calculated from a LineweaverBurk plot of the

calibration curve was 30M. This value can be compared to the

values reported for the biocatalysts system of laccase Tv/ABTS

in solution (KABTSM = 192M,KO2M = 262M) (Farneth et al.,

2005)or for laccase Tv immobilized on kaolinite with ABTS

in solution (KABTSM = 162M) (Dodor et al., 2004).The lower

KappM reflects the efficient 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 specific interactions between redox mediator and theactive 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 modified

electrodes has been reported previously, for instance with myo-

globin (Zhang et al., 2004a) and HRP (Zhang et al., 2004b).

These heme proteins catalyzed the reduction of O2to hydrogen

peroxide. On the other hand, the four-electron reduction of O2to water was achieved with bilirubin oxidase (Mano et al., 2003;

Tsujimura et al., 2001)and laccase modified electrodes (Barton

Fig. 4. O2calibration curve (experimental conditions as inFig. 3A).

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 myo-

globin anchored on multi-walled carbon nanotubes (Zhang et al.,

2004a) andbetween9 106 and2 104 M forlaccase immo-

bilized in liquid crystals (Rowinski et al., 2004)or 1.3106

to 2.6 105 M for a poly(nile blue) modified electrode with-

out 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 (0.064M),

which allows this systemto be considered formonitoring 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 reduc-

tion 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]modified electrode. Sensitive assays of these inorganic ions

are of practical interest because they are environmental tox-

ins, especially in water effluents. As shown in Fig. 1, the

increase of sodium azide concentrations modified the signals

in cyclic voltammetry. The catalytic current disappeared com-

pletely when the inhibitor concentration washigh 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


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C. Mousty et al. / Biosensors and Bioelectronics 22 (2007) 17331738 1737

Fig. 5. (A) Amperometric response at laccase + BSA/[ZnCrABTS] bioelec-

trode 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 fluoride (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 fits

NaN3: Y= .4.510.20 + 2.36E80.06X (R = 0.996, n = 13, S.D.= 0.30);

KCN: Y= 3.430.19 + 2.03E80.06X (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).

concentration, where Ioxy and Ideoxy are the currents in the

presence and absence of oxygen, andIinhis the current observed

upon inhibition in oxygenated electrolyte. Fig. 5B shows

these curves for sodium azide, sodium fluoride and potassium

cyanide. C50 values (the concentration causing 50% activity

reduction) and detection limits (LOD) estimated from these

curves are presented inTable 1.The apparentC50 values werelower when the enzyme was immobilized compared to the val-

ues reported for laccase Tv in solution (14M NaN3, 500M

Table 1

Parameters of calibration curves of inhibitors to be determined using the lac-

case + BSA/[ZnCrABTS] bioelectrode

Inhibitors C50(M) LODa (nM)

NaN3 3 5.50.1

KCN 23 6.20.1

NaF 153 6.90.1

a Detection limit calculated for 3S.D.b where S.D.b is the standard deviation

of the blank, mean value for two different biosensors.

NaF, 500M KCN) (Trudeau et al., 1997). The same feature

was observed when laccase is immobilized in a redox hydrogel

(5.6M NaN3, 43M NaF) (Leech and Feerick, 2000).These

inorganic ions, azide, fluoride 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 = 12.5M (Leech and Daigle, 1998; Leech and Feerick,

2000)or on catalase (LOD = 25M) (Sezginurk et al., 2005).

Similarly, the LOD for fluoride 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) and8 107 M for a biosensorbased on lever esterase

(Marcos and Townslend, 1995).For cyanide, the present detec-

tion 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 10M,

caused a decrease in the reduction current corresponding respec-

tively 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 sub-

strates (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 reductionof the quinoid form of the substrates. The same observation has

been previously reported byLeech 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 inves-

tigated by placing the inhibited electrode in decarbonated waterfor 40 min. After the regeneration, the intensity of catalytic cur-

rent of O2 reduction has decreased but the same sensitivity for

further azide determination was observed. This confirms that

inhibitionof laccase Tv by azide is reversible(LeechandFeerick,

2000).

The main drawback of O2 biosensors remains the life-

time. These biosensor configurations 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


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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+ + 4e2H2O. This increase of pH value candamage

the enzyme. No pH change was observed in the soaking solu-

tion but we have observed by UV spectroscopy leaching of the

biofilm. The slow dissolution of laccase can dislodge a part of

the clay film from the electrode surface. Consequently, the co-

immobilization 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 lac-

case biosensor based on the entrapment of the enzyme intoredox active layered double hydroxides [ZnCrABTS]. ABTS,

intercalated within LDH layers, plays the role of redox media-

tor 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 specific interac-

tions 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 fluoride

reported with electroenzymatic sensors. Moreover based on its

good electrocatalysis for oxygen reduction, this system can beapplied, 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.

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