ANALmcA

CHIMICA

ACTA
Analytica Chimica Acta 311(1995) 255-263

A biosensor as warning device for the detection of cyanide,

chlorophenols, atrazine and carbamate pesticides
Jean-Luc Besombes f;Serge Cosnier b, Pierre Labbe bY
*, Gilbert Reverdy a
Laboratoire de Chimie Molkulaire
b Laboratoire dElectrochimie

et Environnement, ESIGEC, Universiti de Savoie, Campus Scientifique Savoie Technolac,

73376 Le Bourget du Lac, France
Organique et de Photochimie R&ox, URA CNRS 1210, Vniversitt? Joseph Fourier Grenoble I, BP 53,
38041 Grenoble Ceder 9, France

Received 22 September 1994; revised 5 December 1994; accepted 12 December 1994

Abstract
The determination of cyanide, chlorophenols, atrazine, dithiocarbamate and carbamate pesticides is described, utilizing an
amperometric biosensor constructed by the electropolymerization of a pyrrole amphiphilic monomer-tyrosinase coating.
Measurements were carried out with catechol, dopamiue, L-DOPA or epinephrlne as an enzyme substrate; the enzymatically
generated quinoid products being electroreduced at -200 mV vs. SCE. The detection of these water pollutants was
performed via their inhibiting action on the tyrosinase electrode. The characterization of the inhibition processes (competitive/non-competitive)
and their reversibility were examined. The detection limits are 0.4, 2, 2, 4 and 0.02 ,uM for
3,4-dichlorophenol, chloroisopropylphenylcarbamate, 3-chloroaniline, atrazine and cyanide, respectively.
Keywords:

Biosensors; Enzymatic methods; Tyrosinase electrode; Cyanide; Environmental analysis; Pesticides; Chlorophenols; Water

pollutants

1. Introduction
The release of cyanide, chlorophenols and pesticides into the environment
is of great concern because of their widespread use either in industry or
agriculture and role in drinking water pollution. Owing to their toxicity the pollution of ground, stream
and drinking water with these contaminants
is a
health hazard. It is therefore essential to have a rapid
cheap and yet sensitive method of analysis for these
compounds.

* Corresponding author.

Very suitable and accurate analysis for these pollutants can be performed by chromatographic (TLC,
GC and LC) methods (see for example [l]); nevertheless, analysis time and costs are generally considerable. Moreover the chromatographic methods cannot be used for continuous, on site analysis.
Owing to some advantages over other methods,
including high selectivity and simple use, biosensors
are used in many applications in analytical chemistry. Hence, biosensors for the determination
of
organophosphate
and carbamate pesticides were developed in the recent years, based on the inhibiting
action exerted by pesticides on esterases [il. Recently, the detection of cyanide [3] and the herbicide

0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0003-2670(94)00686-5

J.-L. Besombes et al. /Analytica

256

atrazine [4] based on the inhibition of the amperometric response of tyrosinase electrodes, have been
reported. In addition, Wang et al. [5] have demonstrated, in non-aqueous media, the inhibitory effect
of diethyldithiocarbamate on the tyrosinase activity.
Recently, we reported a novel procedure of enzyme immobilization, exemplified with tyrosinase
[6], which allowed the designing of sensitive and fast
responsive amperometric biosensors [7-91. This
method consists of the adsorption on the electrode
surface of a cationic amphiphilic pyrrole l-enzyme
mixture followed by the electropolymerization of the
amphiphilic monomers [lO,ll]. The structure of 1 is
shown below:

-(cH242-

iJ (C 2H5j3 , BFi

Following this procedure, we describe here the

construction of a tyrosinase-based biosensor and its
application to the detection of cyanide, chlorophenols as well as atrazine and chloroisopropylphenylcarbamate (CIPC) pesticides. The determination of
these pollutants can be accomplished from their inhibitory effects on the tyrosinase activity. The characterization of the inhibition process in terms of
reversibility and competition as well as the detection
limits of the different toxic substances are described.
This tyrosinase electrode is the first example, to
our knowledge, of a bioanalytical device sensitive to
a wide range of water pollutants.

2. Experimental
2. I. Instrumentation
The electrochemical equipment consisted of a
PAR Model 362 potentiostat in conjunction with a
Sefram TRP recorder. All electrochemical experiments were carried out using undivided thermostated
three-electrode cell. The working electrode was a
glassy carbon disk (diameter 5 mm> polished with
diamond paste. A saturated calomel electrode (SCE)
and a platinum counter electrode were also used.

Chimica Acta 311 (1995) 255-263

Experiments were carried out in a 5 ml electrochemical cell at 30 C. The detection limit was defined for
a signal value corresponding to threefold the noise
value. The amperometric detection was performed by
applying a potential of - 0.2 V vs. SCE. Spectrophotometric measurements were performed with a Shimadzu UV-VIS-2101PC spectrophotometer.
2.2. Solutions and reagents
The synthesis of monomer 1 has been described
previously [lo]. Tyrosinase (E.C. 1.14.18.1, from
mushroom, 2600 Sigma units mg-I), dopamine and
L-DOPA were purchased from Sigma. Catechol,
epinephrine, 3,4-dichlorophenol and cyanide were
obtained from Aldrich. Atrazine was from Riedel-de
Haen and 3chloroaniline was purchased from Merck.
Sodium diethylthiocarbamate and CIPC were obtained respectively from Prolabo and TCI.
Supporting electrolytes were 0.1 M LiClO, (from
Janssen) and 0.1 M phosphate buffer (pH 6.5) prepared by mixing NaH,PO, * 2H,O and Na,HPO, .
2H,O from Fluka in deionised water.
Since cyanide is a dangerous toxic compound,
precautions must be taken to prevent any accidental
release. Fresh solutions were prepared in pH 6.5
phosphate buffer. All the cyanide solutions were
disposed of by adding an excess of sodium
hypochlorite.
2.3. Enzyme electrode preparation
The poly 1-tyrosinase electrodes were prepared
as previously reported [6]. Tyrosinase was dissolved
in an aqueous dispersion of 1. 30 ~1 of this mixture
containing 0.06 mg of enzyme and 18 nmol of
monomer were spread onto a glassy carbon disk
electrode (diameter 5 mm) and water was removed
under vacuum. The polymerization of the adsorbed
monomer l-enzyme film was carried out by controlled potential electrolysis for 30 min at +0.75 V
vs. SCE in deaerated aqueous 0.1 M LiClO, solution.
Before electrochemical experiments, the electrode
was potentiostated at -0.2 V for 30 min allowing
the background current to decay to a steady state
value.

J.-L. Besombes et al, /Analytica

Chimica Acta 311 (1995) 255-263

257

2.4. Estimation of native tyrosinase activity

The activity of native enzyme in the presence and
in the absence of inhibitors was evaluated as follows:
different amounts of an inhibitor solution were added
to a catechol phosphate buffer solution (pH 6.5)
followed by the addition of 2 pg of tyrosinase. In a
total volume of 2 ml, the final concentrations were
0.33 mM catechol and 49 mM sodium phosphate
buffer. Immediately after addition of tyrosinase, the
increase in concentration of 1,2-benzoquinone enzymatically produced was monitored at 380 nm by
measuring the increase in absorbance vs. time. The
enzymatic activity was evaluated from the slope of
the rectilinear part of the absorbance vs. time dependence. By comparison with the slope obtained under
the same conditions, but without inhibitor in the
reaction mixture, the inhibitory effect was quantified.

3. Results and discussion

The concept of a biosensor warning device for the
detection of a wide range of aqueous pollutants is
illustrated in the following sections for monitoring
various inhibitors of tyrosinase. This enzyme catalyses the orthohydroxylation of monophenols and the
oxidation of o&o-diphenols to ortho-quinones while
molecular oxygen is reduced to water. Tyrosinase is
a bifunctional copper protein which presents at least
two distinct binding sites [12] of which one has
affinity for aromatic compounds (substrate site) and
the other has affinity for metal binding agent (oxygen
site). As a consequence the activity of tyrosinase
could be affected by a large variety of inhibitors. As
reported previously, the analytical capabilities of the
poly 1-tyrosinase electrodes have been investigated
in air saturated 0.1 M phosphate buffer by potentiostating the electrode at -0.2 V vs. SCE [6]. Fig. 1
presents schematically the principle of functioning of
the tyrosinase based biosensor.
Most of the experiments were done using catechol
as a substrate, although several other substrates,
characterized by a lower affinity for the active site,
were also tested. The sensitivity of the bioelectrode
taken as the slope of the linear part of the calibration
curve, varies over a wide range depending on the
nature of the substrate: catechol (136.1 mA M-l),

#
50

\
phenol
or cati

o*

Fig. 1. Schematic description of the electroenzymatic cycle for the

detection of phenol or catechol as substrate.

dopamine (19.2 mA M-l>, L-DOPA (10.3 mA M- 1

and epinephrine (0.7 mA M-l).
The storage stability of the bioelectrode stored dry
at 4 C was examined by checking periodically its
amperometric response to 5 PM catechol. The sensitivity decreases to 85% and 20% of the initial value
after 7 and 54 days, respectively. The operational
stability at pH 6.5 and 25 C was satisfactory since
more than 200 determinations can be carried out
with the same electrode. Reproducibility of biomaterial elaboration was checked on ten poly I-tyrosinase
electrodes by testing their analytical capacities. The
mean sensitivity for catechol was 144 mA M-l with
only a 23% maximum deviation. This variability
results from the precision of the enzyme weighting
and from the reproducibility of the enzyme-monomer mixture deposition onto the electrode surface as
well as from the use of different enzyme-monomer
mixtures on different days. The reproducibility of the
biosensor construction could be greatly improved by
using the same enzyme-monomer mixture for the
simultaneous elaboration of several bioelectrodes.
Nevertheless, this small deviation is not really a
problem, since with our measurements it is the inhibition percentage of the electrode response in the
presence of inhibitor and not the absolute electrode
response which is important. Therefore, inhibitor
determination can be accomplished with an electrode
having variable enzyme activity.
3.1. Chlorophenol inhibition of the tyrosinase electrode
Phenol, 3-chlorophenol and 4-chlorophenol are
tyrosinase substrates and thus can be monitored by
the direct amperometric response of the bioelectrode

258

J.-L. Besombes et al. /Analytica

SonA

2mh
-

Time

Fig. 2. (A) Amperometric

response of a poly l-tyrosinase
electrode to 5 PM of catechol and (B) inhibition of this response
induced by the successive additions of 3&dichlorophenol.
(C)
Amperometric
response of the completely inhibited bioelectrode
for 5 PM of catechol after washing and transfer to clean phosphate buffer. Applied potential
-0.2
V vs. SCE, 30 C air
saturated 0.1 M phosphate buffer (pH 6.5) kept under vigourous
stirring.

[13]. Work in this laboratory has demonstrated that

2-chlorophenol and several di-and trichlorophenols
are efficient inhibitors of tyrosinase 1131.This property can be exploited for their detection by monitoring the inhibition process of the bioelectrode functioning. The 3,4-dichlorophenol pollutant (DCP) is
used to illustrate this concept. In order to determine
the influence of DCP on tyrosinase, the activity of
the free enzyme towards catechol was assayed spectrophotometrically in the presence and absence of
DCP. The slope of the rectilinear part of the absorbance versus time dependence decreases as the
DCP concentration increases. 50% of inhibition was
achieved with 34 PM of DCP.
The effect of DCP on the amperometric response
of the poly 1-tyrosinase electrode was investigated
with catechol as substrate. Fig. 2 presents the
steady-state current (I) of the bioelectrode upon
successive additions of DCP illustrating the inhibition exerted by this compound as well as the relatively fast response time (2-7 min> of the electrode.
The addition of DCP was carried on yielding a
completely inhibited bioelectrode. The bioelectrode
was then transferred to clean phosphate buffer and
its tyrosinase activity was tested again with 5 /IM
catechol as the substrate (Fig. 2C). It was found that

Chimica Acta 311 (I 995) 255-263

77% of the inhibition caused by DCP was reversed

(i.e., 77% of the initial amperometric current, I,
was recovered) indicating that the tyrosinase-DCP
complex is dissociable and that the inhibition of
tyrosinase is quasi-reversible. It should be noted that
in the case of partial inhibition processes, the reversibility is quantitative. The sensor response to this
compound was quantified as the ratio of the current
decrease (I -I) to the original current (I 1 (no
inhibition) in the steady state (Fig. 3A). A detection
limit of 0.4 PM (based on a signal-to-noise ratio of
3) was obtained. A linear calibration curve could be
obtained up to 500 PM of DCP by using a reciprocal
plot (Fig. 3B).
To obtain further information on the type of
inhibition exerted by DCP on poly 1-tyrosinase, the
amperometric responses of the bioelectrode were
measured as a function of catechol concentration for
several concentrations of DCP. The electrochemical
Lineweaver-Burk plots (l/Z vs. l/C where Z is the
steady-state current and C is the catechol concentration [7,14]) corresponding to 0, 10, 20 and 39 Z_LM
of
DCP demonstrate that the reaction was governed by
Michaelis-Menten kinetics. In absence of DCP, the
K$r (200 PM) is of the same order of magnitude as
that for free soluble tyrosinase (240 PM) [15] illustrating the good permeability of the biopolymer. In
the presence of 10, 20 and 39 Z_LM
of DCP, the K$P

0.2

0.6 0.6
PA-PlmM

0.6

1.0

l.2

Fig. 3. Inhibition of the amperometric

response of a poly ltyrosinase electrode to 5 PM of catechol induced by the increasing concentrations of 3,4_dichlorophenol.
(A) The inhibition process is quantified by the (I - 1)/P
ratio where P corresponds
to the current intensity after substrate injection in the absence of
inhibitor and I represents this intensity upon addition of the
inhibitor, other experimental conditions as in Fig. 1. (B) Reciprocal plots as l/I vs. 3,4-dichlorophenol
concentration.

J.-L. Besombeset al. /Adytica

-20

io

-10
4.5

2i

3il

4il

l-/W

Fig. 4. Plots of l/I

versus 3,4dichlorophenol
concentration in
the presetice of(W) 8 PM, (0) 39 FM, (+I 78 PM and (0)
136 PM catechol for the poly 1-tyrosinase electrode. I corresponds to the current intensity after catechol injection in the
presence of 3,4_dichlorophenol. Other experimental conditions as
in Fig. 1.

increases to 270,450 and 675 PM, respectively,

whereas the Z,, values are independent of the inhibitor concentration. These findings are characteristic of a competitive inhibition and indicate that DCP
and catechol have an affinity for the same active site
of tyrosinase. An apparent inhibitor binding constant,
Ki = 17 PM, was determined from different catechol
concentrations (Fig. 4). Calculations from the relation KGpp = Kxp(l + Z/K,) gave an average value
of Ki of 15.7 PM in good agreement with the
graphic determination. This value compares well with
that obtained for benzoic acid inhibitor of tyrosinase
(Ki = 17 PM) [5] indicating a similar inhibition
specificity. Thus, it appears that the amperometric
signal of the bioelectrode can be used to study the
inhibition processes.
3.2.Carbamate pesticides

inhibition

Chimica Acta 311 (1995) 255-263

259

terases [2]. Recently, it has been reported that a

tyrosinase electrode can be inhibited in organic solvents by diethyldithiocarbamate [5]. Taking this in
mind, we have investigated the effect of chloroisoprop)ilphenylcarbamate (CIPC) and its hydrolysis
product (3-chloroaniline) [16] on the catalytic behaviour of the free tyrosinase in water. It appears
that, in the presence of 0.33 catechol mM as a
substrate, 50% inhibition of tyrosinase activity was
reached with 330 PM of CIPC or 6.5 PM of 3-chloroaniline. CIPC inhibition of the tyrosinase electrode
was then tested using two different substrates, catechol and dopamine, characterized by different affmities for the active enzyme site [17,18]. Fig. 5 presents the response of the tyrosinase electrode following the successive additions of increasing concentrations of CIPC using catechol(6 PM) or dopamine (6
PM) as a substrate. With the former, fast and sensitive responses (detection limit 2 PM) are observed
due to the inhibitory effect of CIPC. However, the
absence of a relatively short stabilization time after
each addition of CIPC is a drawback for a rapid
determination of this inhibitor. More interesting, the
replacement of catechol by dopamine in our detection system allows a rapid response which stabilizes
within 1 min following CIPC injection, the detection
limit being 2 PM. Moreover, the complete inhibition
of the bioelectrode was shown to be reversible as the
electrode response returned to its original level (98%

of the tyrosi-

nase electrode

The systemic, biodegradable carbamate pesticides

have known a greater popularity in agriculture these
recent years. The ability to detect these toxic compounds in aqueous solutions via their inhibiting action on the esterase enzymes has resulted in the
development of bioelectrodes solely based on es-

Fig. 5. Amperometric response of a poly 1-tyrosinase electrode

upon successive additions of CIPC in the presence of (a) dopamine
(6 PM) and (b) catechol(16 PM). Other experimental conditions
as in Fig. 1.

J.-L. Besombes et al. /Analytica

Chimica Acta 311 (1995) 255-263

same binding site of the enzyme 1121. Thus, the

inhibiting properties of CIPC could be due to its
chlorophenyl moiety; this explaining the suggested
competitive nature of the inhibition process.
3.3. Atrazine inhibition of the tyrosinase electrode

Fig. 6. Inhibition of the amperometric response of a poly ltyrosinase electrode to 6 PM of dopamine induced by the increasing concentrations of CIPC. The inhibition process is quantified
by plotting (A) the (P -1)/p
ratio and (B) l/1 vs. CIPC
concentration. The (P -1)/p
ratio is defined as in Fig. 2.
Other experimental conditions as in Fig. 1.

recovery) after washing with buffer and repeating the

experiment in the absence of CIPC. The calibration
plot (Fig. 6A) for CIPC indicates that the inhibition
percent rises with CIPC concentration and then it
starts to level off reflecting the saturation of tyrosinase sites by CIPC. Reciprocal plots allow to obtain
a linear calibration curve up to 350 PM of CIPC
(Fig. 6B).
We have also examined in aqueous solution the
influence of diethyldithiocarbamate (DTC) on the
biosensor response, this compound being a tyrosinase inhibitor in organic medium [5]. It appears that
the presence of DTC causes a continuous decrease in
current response of the bioelectrode. Thus, the tyrosinase electrode seems to be sensitive to two different kinds of pesticides.
On the other hand, it has been demonstrated that
DTC, which is a well known metal binding agent, is
not competing with phenol substrate for the same
active site [5]. In contrast, the strength of the CIPC
inhibition is strongly dependent on the substrate
nature suggesting a competitive inhibition process.
Finally, the tyrosinase electrode is also inhibited by
3-chloroaniline, the main hydrolysis product of CIPC
[16], the detection limit being 2 PM. Owing to the
aromatic nature of 3-chloroaniline, it is likely that
this compound and the substrate have affinity for the

Many of the photosynthetic inhibiting herbicides

like atrazine are persistent and can remain active for
several years in the environment, causing continuous
water pollution. Currently, these herbicides are determined predominantly by chromatography. Recently
the first example of direct detection of atrazine by
inhibition of a bioelectrode based on tyrosinase has
been reported [4]. Therefore, the influence of atrazine on the amperometric response of our biosensor
has been investigated with catechol and dopamine as
enzyme substrates. It appears first that, in the presence of 5 PM of catechol, the successive additions
of atrazine induce only a slight continuous decrease
of biosensor response without any current stabilizations. In contrast, with 6 PM of dopamine, the
biosensor response displays a rapid decrease followed by a quasi-stabilization after each atrazine
addition, the detection limit being 4 PM (Fig. 7).
With this biosensing system, the same value of the
detection limit, namely 4 PM, was obtained over ten
repetitive assays. As previously described, these results suggest that atrazine is competing with the
enzyme substrates for the same active site [4], catechol having a stronger affinity than dopamine for the

Fig. 7. (A) Steady-state current response of the poly 1-tyrosinase

electrode in the presence of 6 PM of dopamine upon successive
additions of (al isopropanol and (b> atrazine. (B) Amperometric
response of the bioelectrode upon two additions of(c) methanol in
the same conditions. Other experimental conditions as in Fig. 1.

261

J.-L. Besombes et al. /Analytica Chimica Acta 311 (1995) 255-263

active site [17,18]. The better detection limit (4 PM)

as well as the faster response times (2-5 min instead
of 15 min) obtained with our bioelectrode could be
explained by the comparison of the methods used for
the tyrosinase immobilization. Indeed our procedure
of biosensor construction produces biopolymer exhibiting a good permeability without appreciable
change of the enzyme activity [7] whereas the covalent cross-linking immobilization method induced an
inactivation of the cresolase activity of the enzyme
[4]. On the other hand, with this cross-linked configuration of bioelectrode, it has-been reported that the
addition of methanol to render atrazine soluble in
aqueous media did not affect the substrate response
of the tyrosinase enzyme electrode [4]. Contrarily to
these findings, a strong inhibition of our bioelectrode
response is observed when methanol alone is added
to the electrolyte containing dopamine at a concentration of 6 PM (Fig. 7B). This undesirable effect
which could result from an enzyme denaturation by
methanol, has been suppressed by the replacement of
this alcohol by isopropanol (Fig. 7A).

mtFig. 8. (A) Steady-state current response of the poly 1-tyrosinase

electrode upon successive additions of cyanide in the presence of
6 PM epinephrine. (B) Amperometric response of the completely
inhibited bioelectrode to 6 PM epinephrine after washing and
transfer to clean phosphate buffer. Other experimental conditions
as in Fig. 1.

tion of cyanide has been tested in the presence of

catechol, dopamine, L-DOPA and epinephrine substrates characterized by a decreasing affinity for the
active substrate site of tyrosinase (Table 11. The data
in Table 1 reflect the influence of diphenol nature on
the efficiency of the inhibition process of the tyrosinase electrode.
With catechol and dopamine, the cyanide addition
induced a continuous decrease of the biosensor response, thus prohibiting their use for rapid analytical
determinations of cyanide. In contrast, with L-DOPA
or epinephrine, a sharp decrease of the steady-state
current response followed by its rapid stabilization is
observed after each addition. The bioelectrode responded very rapidly to micromolar changes in
cyanide concentration with L-DOPA as a substrate
whereas a longer stabilization time (3-4 min) was
necessary with epinephrine (Fig. 8). With the latter,
the sensor was sensitive to nanomolar cyanide concentrations (Fig. 9). This dependence on the nature
of this diphenolic compound is quite surprising since

3.4. Cyanide inhibition of the tyrosinase electrode

It has been reported that the tyrosinase activity is
inhibited by cyanide in a manner that is competitive
with dioxygen and non competitive with o-diphenol
substrates [12]. This is probably due to the coordination of cyanide to the copper binding site of dioxygen [3,19]. Recently, this property has been exploited
to elaborate a bioelectrocatalytic device sensitive to
cyanide [3]. However, the described system did not
exhibit an extremely sensitive detection limit (2 X
lo- M). With our bioelectrode, the inhibiting ac-

Table 1
Influence

of the type of catechol

substitution

on the cyanide inhibition

Catechol

Amperometric response a (nA)

Cyanide detection limit ( PM)
kc ( PM)
Response time for cvanide (s)

816
1
_b
_b

process of the tyrosinase

electrode

Dopamine

L-DOPA

Epinephrine

113
0.5
_b
_b

62
0.5
24
10

6
0.02
0.35
200

Bioelectrode response at 6 /.LM substrate; other conditions as in Fig. 1.

b Undetermined, the addition of cyanide inducing a continuous decrease of the biosensor response.
i,, represents the molar concentration of cyanide needed to inhibit the amperometric response of the bioelectrode

by 50%.

J.-L. Besombes et al. /Analytica

262

Chimica Acta 311 (1995) 255-263

4. Conclusion

0.2

0.4

0.6

0.8

1.0

IKCNII llM
Fig. 9. Inhibition of the amperometric
response of a poly ltyrosinase electrode to 6 FM of epineph~ne induced by the
increasing concentrations
of cyanide. The inbibition process is
quantified by plotting (A) the (I - Ii/P
ratio and (B) l/1 vs.
cyanide concentrations.
The (P - 1)/I
ratio is defined as in
Fig. 2. Other experimental conditions as in Fig. 1.

it is generally agreed that cyanide inhibition is noncompetitive with catechol substrate but competitive
with dioxygen 13,181. However, Duckworth and
Coleman [12] have demonstrated that the functioning
principle of tyrosinase cannot be simply restricted to
two independent binding sites, one for dioxygen and
one for phenolic or diphenolic substrates. They have
reported that the affinity of dioxygen for its binding
site can be modulated by the aromatic substrate
affinity. Therefore, the weaker affinity revealed by
epinephrine compared to those exhibited by L-DOPA,
dopamine and catechol, for the substrate site, could
induce a decrease of dioxygen affinity. As a consequence cyanide could dislodge dioxygen more efficiently explaining thus the extremely sensitive detection of cyanide obtained with epinephrine as a substrate.
The very low detection limit (20 nM) of this
bioelectrode and its operative concentration range of
cyanide detection (0.02-l PM) as well as the reversibility of the inhibition process (Fig. 81, make
this device particularly interesting for monitoring
toxic cyanide. Indeed, the maximum acceptable
cyanide concentration for drinking water or pretreated water is 1.9 FM. It should be also noted that
20 repetitive cycles of complete inhibition and regeneration of the bioelectrode have been accomplished without any significant loss of activity.

It has been demonstrated for the first time, that

the tyrosinase electrode can be employed for monitoring chlorophenols and carbamate pesticides in
aqueous environments. Furthermore, this bioelectrode provides, to our knowledge, the lowest detection limit for cyanide described for an electroenzymatic sensor.
Owing to the insensitiveness of the biosensor
functioning towards ionic species which are currently present in ground- and streamwater (e.g., Ca2+,
Na+, CO:-, Cl- and SO;-, etc.) as well as heavy
metal cations eventually present such as Cu*+, Pb2+
or Hgzf, this biosensor could be used in real water
samples 1131.Furthermore this unique property of the
tyrosinase enzyme to be sensitive to a wide range of
possible water pollutants like ~~orophenols, cyanide,
the photosynthetic herbicide atrazine, dithiocarbamate and carbamate pesticides, could be exploited to
use the tyrosinase electrode as a detective warning
device for accidental water pollution.

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