Enzyme Inhibition-Based Biosensors For Food Safety and Environmental Monitoring

Biosensors and Bioelectronics 21 (2006) 14051423
Review
Enzyme inhibition-based biosensors for food safety

and environmental monitoring
Aziz Amine a, , Hasna Mohammadi a , Ilhame Bourais a , Giuseppe Palleschi b
a
Laboratoire des Analyses Chimiques et des Biocapteurs, Faculte des Sciences et Techniques, Mohammadia, Morocco
b Dipartimento di Scienze e Tecnologie Chimiche, Universit`
a di Roma Tor Vergata, Rome, Italy
Received 25 March 2005; received in revised form 23 June 2005; accepted 11 July 2005
Available online 25 August 2005
Abstract
Analytical technology based on sensors is an extremely broad field which impacts on many major industrial sectors such as the pharmaceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried out
during the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a wide
range of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the different
contaminants analyzed are considered.
The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well as
the general approaches that have been used for enzyme immobilization, the enzyme catalysis, and the inhibition mechanism.
2005 Elsevier B.V. All rights reserved.
Keywords: Enzyme; Biosensors; Environment; Food; Inhibitors
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Theoretical and practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Principle of the enzyme-based biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Enzymeinhibitor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1. Reversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Irreversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Limit of detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Parameters generally affecting the performance of enzymatic biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.1. Effect of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7. Effect of substrate concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8. Effect of enzyme concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inhibition determination in organic phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inhibitor investigation using enzyme-based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Pesticide inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author.
E-mail addresses: a.amine@univh2m.ac.ma, aziz-amine@uh2m.ac.ma
(A. Amine).
0956-5663/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2005.07.012
1406
1406
1406
1407
1408
1408
1408
1409
1410
1410
1410
1411
1411
1411
1412
1412
A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423
1406
5.
6.
7.
4.2. Heavy metal inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3. Other inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Food and environmental applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Because of their exceptional performance capabilities,
which include high specificity and sensitivity, rapid response,
low cost, relatively compact size and user-friendly operation,
biosensors have become an important tool for detection of
chemical and biological components for clinical, food and
environmental monitoring. While electrochemical transducers combined with an enzyme as the biochemical component form the largest category, those biosensing systems that
specifically depend on inhibition can be divided into three
categories:
Biosensors based on the immobilization of whole cells
used as the biochemical component (Rekha et al., 2000;
Durrieu et al., 2004; Chouteau et al., 2005). The use of
this type of biosensor can increase the sensor stability and
render the regeneration of the enzyme easier. However,
such biosensors may suffer from side reactions due to the
coexistence of several enzymes.
Sensor devices coupled with reactors which contain an
immobilized enzyme matrix. The inhibitor passes through
the reactor and inhibits the enzyme (Lee et al., 2002). The
residual activity of the enzyme is evaluated by measuring
the enzymatic product before and after the inhibition.
Biosensors based on direct enzyme immobilization on a
transducer device. The enzyme and transducer elements
are in close contact with each other and incorporated in
a single unit. Some biosensors based on enzyme inhibition have been reported in the literature (Tran-Minh,
1985; Evtugyn et al., 1999; Luque de Castro and Herrera,
2003).
Inhibition-based biosensors have been the subject of several recent reviews. Biosensors based on ion sensitive field
effect transistors (ISFETs) for the determination of some
substrates and inhibitors were reviewed by Dzyadevych
et al. (2003). Luque de Castro and Herrera (2003) have
discussed the inhibition-based biosensors and biosensing systems. Other authors have reviewed the use of electrochemical
enzyme-based biosensors for the determination of pesticides
(Trojanowicz, 2002; Sole et al., 2003). Patel reported some
specific applications of inhibition-based biosensors in the
area of chemical and microbiological contaminant analysis
implicated in food safety (Patel, 2002). Finally, some recent
designs and developments relating to screen-printed carbon
electrochemical sensors and biosensors for biomedical, envi-
1415
1417
1417
1419
1420
1420
1420
ronmental, and industrial analyses have been reviewed by

Hart et al. (2004).
In this review, we specifically provide an overview of the
activity carried out since 2000 relative to biosensor systems
which use an enzyme for inhibition-based analysis for food
and environment safety. We also report the results from some
studies in which the inhibited target enzyme is in solution
while its product is detected using an enzyme-based biosensor.
Biosensors based on the principle of enzyme inhibition
have by now been applied for a wide range of significant analytes such as organophosphorous pesticide (OP),
organochlorine pesticides, derivatives of insecticides, heavy
metals and glycoalkaloids. The choice of enzyme/analyte
system is based on the fact that these toxic analytes inhibit
normal enzyme function. In general, the development of these
biosensing systems relies on a quantitative measurement of
the enzyme activity before and after exposure to a target analyte. Typically the percentage of inhibited enzyme (I%) that
results after exposure to the inhibitor is quantitatively related
to the inhibitor (i.e. analyte) concentration and the incubation
time (Guerrieri et al., 2002; Ivanov et al., 2003). Consequently, the residual enzyme activity is inversely related to
the inhibitor concentration.
2. Theoretical and practical considerations

2.1. Principle of the enzyme-based biosensor
Biosensors are analytical devices which tightly combine
biorecognition elements and physical transducers for detection of the target compounds. In enzyme-based biosensors,
the biological element is the enzyme which reacts selectively
with its substrate (Guilbault et al., 2004).
It is well known that the response of a biosensor to the
addition of a substrate is determined by the concentration of
the product (P) of the enzymatic reaction on the surface of the
sensor. The reaction is controlled by the rate of two simultaneous processes, i.e. the enzymatic conversion of the substrate
(S) and the diffusion of the product from the enzyme layer. If
there is a high enzyme activity, the decrease of the substrate
concentration is not totally compensated by the transfer from
the bulk solution due to the diffusion limitation, and because
of this, only a fraction of the enzyme active centers is involved
in the interaction with a substrate. In this case (diffusion
control of the response), the sensitivity of the immobilized

enzyme toward inactivation, be it heating or inhibitor effect,
is lower than that for the enzyme used in solution.
Given that pollutant compounds selectively inhibit the
activity of certain enzymes, their activity and the resulting
product concentration is affected. This inhibition is analytically useful and has been used advantageously in the fabrication of many biosensing devices. First we will summarise
immobilization techniques and their effects on biosensor
function. Then general schemes for the various possible inhibition processes that have been effectively used in biosensors
will be described.
2.2. Enzyme immobilization
The development of biosensors based on immobilized
enzymes came about to solve several problems such as loss
of enzyme (especially if expensive), maintenance of enzyme
stability and shelf life of the biosensor, and additionally to
reduce the time of the enzymatic response and offer disposable devices which can be easily used in stationary or in flow
systems. To do this, several immobilization techniques have
been investigated. These techniques include physical entrapment, microencapsulation, adsorption, covalent binding and
covalent cross-linking, and several different approaches to
enzyme immobilization have been reported in the literature.
Acetylcholinesterase (AChE) was encapsulated in solgel
film on a glass cap that could be fixed on an optical fiber
(Doong and Tsai, 2001). Solgel films have been formed
using enzymatic solutions mixed with different fluorescent
indicators. The design of such biosensors takes advantage of
the ability to entrap large amounts of enzyme and enhance
thermal and chemical stability; the techniques offer simplicity of preparation without covalent modification, flexibility in
controlling pore size, and geometry and minimal quenching
of fluorescent reagents.
In another strategy, AChE was co-immobilised with
choline oxidase (ChO) onto a Pt surface using a solution
of glutaraldehyde. The activity of immobilized enzymes
was evaluated in the presence of dimethyl-2,2-dichlorovynyl
phosphate pesticide (DDVP). The cross-linking involving
glutaraldehyde significantly increased the attachment of the
enzyme to the transducer and thus, the electron exchanges
could occur more directly.
Urease has been entrapped in both polyvinyl chloride
(PVC) and cellulose triacetate (CTA) layers on the surface of
pH-sensitive iridium oxide electrodes and used for determination of mercury. Gulla et al. (2002) reported the immobilization of AChE in nylon net using glutaraldehyde. The enzyme
layer was sandwiched between two cellophane membranes
and kept in close contact with a gold electrode. This method of
AChE immobilization permits the removal of the enzymatic
membrane, the enzymatic reactivation (by TMB-4), and the
reuse of reactivated membrane. The immobilization of the
enzyme polyphenol oxidase (PPO) during the anodic electropolymerisation of polypyrrole (PPy) was also reported.
1407
The enzyme was trapped on the electrode surface during an

electrochemical synthesis process (El Kaoutit et al., 2004).
This biosensor was used for the evaluation of atrazine and
provides a rapid and technically simple system for determination at concentrations below the ppm level. Sotiropoulou and
Chaniotakis (2005) have used a nanoporous carbon matrix for
acetylcholinesterase immobilization and stabilization. They
reported that the use of this activated carbon matrix provided
both significant enzyme stabilization and a lowering of the
detection limit. Using this biosensor the monitoring of the
organophosphorus pesticide dichlorvos at picomolar levels
was achieved; calculated on the basis of 20% inhibition, they
could detect 1012 mol l1 pesticide which was a level 1000
times lower than for other systems reported so far. Recently,
using nanoporous conductive carbon for immobilization of
0.02 pmol of very sensitive acetylcholinesterase from the
double mutant E69Y, Y71D of Drosophila melanogaster,
Sotiropoulou et al. (2005) were able to detect dichlorvos at
attomolar levels, 1017 M with 40% inhibition. It was shown
that in comparison with AChE from E. electricus (electric
eel), the use of double mutant AChE (E69Y, Y71D) produced
a drastic increase of the inhibition constant, Ki , value for the
dichlorvos pesticide.
Malitesta and Guascito (2005) have described the application of biosensors based on glucose oxidase immobilized by electropolymerisation for heavy metal determination; the investigated enzymatic inhibition appears
reversible and in agreement with the data reported for
the enzyme in solution. A comparison of several acetylcholinesterase immobilization procedures carried out on the
7,7,8,8-tetracyanoquinonediaminomethane (TCNQ) modified graphite working electrode was presented by Nunes
et al. (2004). The enzyme immobilization through photopolymerization with polyvinyl alcohol bearing styrylpyridinium
groups (PVA-SbQ) produced good results, fast response,
good reproducibility, wide working range for pesticides and
excellent sensitivity to N-methylcarbamates. Ivanov et al.
(2003) evaluated the detection limit and the sensitivity toward
some pesticides using different modified sccreen-printed
electrodes. In comparison with the use of unmodified transducers, they concluded that the modification of the sensor
surface with 7,7,8,8-tetracyanoquinodimethane (TCNQ) is a
powerful tool for the improvement of biosensor performance.
According to reports in the literature, the performance of
a biosensor device is strongly dependent on its configuration.
In a paper that reported the immobilization of enzymes with
clay, the influence of the enzyme/clay ratio and the amount
of adsorbed coating on cyanide sensing by polyphenol oxidase was investigated (Shan et al., 2004). It was reported that,
when the enzyme/clay ratio was decreased from 1 to 0.125,
the sensitivity of the biosensor decreased sharply from 9130
to 0.5 mA M1 cm2 and the detection limit increased from
0.1 nM to 50 M. In the same paper, the influence of the
amount of deposited coating and hence the thickness of the
clay film on the biosensor performance was studied. Higher
sensitivity to cyanide and a lower detection limit are observed
1408
with thinner coatings. In another paper, Ciucu et al. (2003)

have confirmed that a lower concentration of paraoxon could
be measured using a membrane with a lower amount of immobilized AChE.
It has been shown that the sensitivity of enzymes toward
heavy metal ions is a function of enzyme loading (Soldatkin
et al., 2000). These authors have demonstrated that urease
immobilization under a negatively charged polymer induces
an increase of the inhibition effect of the heavy metal ions
due to cation accumulation in the polymeric matrix. A lower
concentration of invertase (2 U) in a tri-enzymatic biosensor matrix resulted in a significant increase of sensitivity,
as well as in a decrease of the detection limit of mercury (Mohammadi et al., 2005). These results confirm those
obtained with the free enzyme, i.e. that a higher percentage of
inhibition is often observed with lower enzyme concentration
(Evtugyn et al., 1998; Mohammadi et al., 2002, Sotiropoulou
et al., 2005) and thus a low enzyme concentration should in
general enhance sensitivity to the inhibitor.
On the basic of the numerous studies reported before, the
enzyme immobilization is one of the most important steps
involved in the biosensor design. The choice of the technique
used for connecting the biological component (enzyme) to the
transducer is crucial, since the stability, the longevity and the
sensitivity largely depend on enzymatic layer configuration.
2.3. Enzymeinhibitor system
The enzymeinhibitor reaction is often complex. The
paragraph below reports different inhibition mechanisms that
result from the interaction between the enzyme and the toxic
compound involved, reversible and irreversible inhibition and
their respective mechanisms are considered. For each mechanism of inhibition, an example of a biosensor based on this
type interaction is also reported.
2.3.1. Reversible inhibition
The long-term function of enzyme-based biosensors may
be severely restricted by the very inhibitors being measured.
The inhibition can be either reversible or result in an irreversible inactivation of the enzyme. Inhibitors structurally
related to the substrate may be bound to the enzyme active
center and compete with the substrate (competitive inhibition). Dzyadevych et al. (2004b) reported that the glucoalkaloids are competitive inhibitors of BChE. Also Morales
et al. (2002) showed a competitive inhibition of tyrosinase
by benzoic acid:
(1)
If the inhibitor is not only bound to the enzyme but also to
the enzymesubstrate complex, the active center is usually
deformed and its function is thus impaired. In this case the

substrate and the inhibitor do not compete with each other
(non-competitive inhibition). The inhibition of horseradish
peroxidase was apparently reversible and non-competitive in
the presence of HgCl2 for less than 8 s incubation time (Han
et al., 2001). Cyanide showed non-competitive inhibition versus polyphenol oxidase (Shan et al., 2004). The inhibition
of immobilized acetylcholinesterase with metal ions (Cu2+ ,
Cd2+ , Fe3+ , Mn2+ ) has a reversible and a non-competitive
character (Stoytcheva, 2002):
(2)
Competitive and non-competitive inhibitions affect the
enzyme kinetics differently (Segel, 1976). A competitive
inhibitor does not change Vmax but increases the KM ; on
the contrary a, non-competitive inhibition results in an
unchanged KM and in a decrease of Vmax .
In the case of mixed inhibition, the inhibitor binds the
enzyme and the enzymesubstrate complex with a different
affinity. Malitesta and Guascito (2005) demonstrated that the
inhibition mechanism of glucose oxidase by heavy metals is
reversible and mixed:
(3)
For uncompetitive inhibition, the inhibitor binds only when
the enzymesubstrate complex is formed. The inhibition of
tyrosinase by carbaryl was studied by Kuusk and Rinken
(2004); the mechanism of inhibition was found to be analogous to that usually considered for uncompetitive inhibition:
(4)
Dixons plot (Segel, 1976) is often used for the evaluation
of the inhibition constant and for the differentiation between
different types of inhibition.
2.3.2. Irreversible inhibition
For irreversible inhibitors, the enzymeinhibitor interaction results in the formation of a covalent bond between the
enzyme active center and the inhibitor. The term irreversible
means that the decomposition of the enzymeinhibitor complex results in the destruction of enzyme, e.g. its hydrolysis,
oxidation, etc. This process usually proceeds stepwise, as for
1409
phosphorylated cholinesterases, and can be accelerated by

particular reagents:
(5)
The kinetics of the inhibition depend strongly on the biosensor configuration. In the case of a thin enzymatic layer, the
kinetics observed are similar to that of the enzyme in solution. For native enzymes the inhibition is related directly to
the incubation time (Kitz and Wilson, 1962).
ln i is linear with the incubation time:
d[E]/dt = kobs [E],
d[E]/[E] = kobs dt,
ln i = kobs + constant
where i is the remaining activity.
Han et al. (2001) have investigated an interesting case concerning the inhibition of peroxidase. There is an early phase
of reversible inhibition (5 s) followed by irreversible inhibition. However, since the reversible inhibition lasted for just a
few seconds, it was difficult to carry out the measurement of
residual activity within that interval. Therefore, irreversible
inhibition has to be dealt with in cases where longer incubation times must be used. In these cases the measurement of
the residual enzyme activity resulted in a log linear with the
incubation time. Neufeld et al. (2000), Guerrieri et al. (2002)
have reported that the degree of inhibition also depends on
the inhibitor concentration and exposure time (at a defined
pH value and at inhibitor concentration which is in excess
with respect to enzyme).
There have been some initial attempts at the development and experimental verification of theoretical models
for the inhibition of immobilized enzymes used for biosensors. When diffusion phenomenon are taken into account, the
model predicts that the percentage of enzyme inhibition (%),
after exposure to an inhibitor, is linearly related to both the
inhibitor concentration [I] and the square root of incubation
time (t1/2 ) (Zhang et al., 2001).
2.4. Limit of detection
The determination of the inhibitory effect includes the following steps: the determination of initial enzymatic activity,
the incubation of a biosensor in a solution that contains an
inhibitor, and finally the measurement of the residual activity
(activity after exposure of the biosensor to the inhibitor). The
limit of detection (LOD) has been defined as the concentration of the species being measured which gives a minimum
detectable difference signal (reduction in activity) that is
equal to 2 or 3 standard deviations (S.D.) of the mean response
of the blank samples (zero concentration of the inhibitor).
This simple approach, although widely reported in the literature (Kuswandi, 2003; Del Carlo et al., 2004; Suprun et al.,
Fig. 1. General method to establish LOD for enzyme inhibition assays.
2004), is not correct because it does not take into account the
confidence interval of the inhibitor. The true value of LOD can
be defined as the concentration of the inhibitor where the confidence interval does not overlap that of the zero concentration
of the inhibitor standard. This is shown diagrammatically in
Fig. 1. Any concentration above the LOD value has a 95%
(2S.D.) or 99% (3S.D.) probability of being a true positive
result. The LOD value generally corresponds to 9080% of
residual activity, that is 1020% inhibition.
Kuswandi (2003) has developed a simple optical biosensor based on immobilized urease for the monitoring of heavy
metals. He confirmed that the detection limit depends on the
incubation time of the enzyme with the inhibitor; the optimum time of inhibition selected was 6 min. In other work,
the residual enzymatic activity was also studied using different incubation times (5, 15, 30 min) with the AChE and ChO
bienzymatic system (Kok et al., 2002). The degree of the
enzyme inhibition increased with increase of the incubation
period until reaching a plateau in 1530 min. The decrease in
the enzyme activity could be detected after 5 min, and therefore the incubation time selected was 5 min, and the biosensor
could detect 10 ppb aldicarb which gave a 10% inhibition of
the initial acethylcholinesterase activity. In another work the
same inhibition study has been performed using an incubation time of 30 min (Ciucu et al., 2003). In this case, the
detection of paraoxon at 10 nM has been achieved. Shan
et al. (2004) have highlighted specific electrostatic interaction of the host matrix that may induce an accumulation of the
inhibitor within the anionic clay. This phenomenon improved
the sensitivity of the amperometric biosensor toward cyanide
(0.1 nM).
According to the literature data, it has shown that the
limit of the detection of the different developed biosensors
depends on the several parameters such as pH, temperature,
the enzyme loading (in the case of irreversible inhibition), the
substrate concentration (in the case of competitive reversible
1410
inhibition), the immobilization matrix and the time of reaction between the enzyme and the inhibitor. Thus, direct comparison of the sensitivity between the different biosensors
based on enzyme immobilization is not easy and should take
into consideration the cited parameters.
2.5. Regeneration
Understanding the mechanisms of inhibition and regeneration of enzymes is a general problem of great importance for many biochemists and biotechnologists, especially
when using immobilized enzymes. The mode of analyte
inhibition of enzymes such as peroxidase, tyrosinase and
catalase can occur through blocking of the active sites of
these enzymes due to complex formation with copper cofactors and blocking of the electron transfer chain. OPs inhibit
acetylcholinesterase (AChE) by blocking the serine in the
active site through nucleophilic attack to produce a serine phosphoester (via phosphorylation) (Simonian et al.,
2001). In any case, the strong inhibition of the enzymes can
present a serious problem for practical applications by limiting the reuse of biosensors. To overcome the problem of
irreversible enzyme inhibition in the application of AChEbased biosensors, reactivation by oximes was investigated
(Gulla et al., 2002). Further, another phenomenon observed is
that of permanent inhibition when the phoshorylated (inhibited) enzyme is left for a period of time without exposing
it to the reactivator. This phenomenon is called ageing,
and is catalyzed by the enzyme itself. After this ageing,
the inhibited enzyme is even more resistant to hydrolysis
and reactivation with oxime so that it becomes permanently
inhibited.
Reactivation of inhibited AChE was investigated
using pyridine-2-aldoxime methyliodide (2-PAM) and 4formylpyridinium bromide dioxime (TMB-4). TMB-4 was
found to be a more efficient reactivator with repeated use,
retaining more than 60% of initial activity after 11 reuses,
whereas in the case of 2-PAM, the activity retention dropped
to less than 50% after only six reuses. The effect of ageing
on the enzyme activity retained has been studied by Gulla
et al. (2002). It was found that this effect sets in after 15 min
when the inhibited enzyme is left without reactivation and
increases with time elapsed before exposure of the biosensor
to the reactivator (2-PAM and TMB-4). Thus, it was recommended that the pesticide-treated enzyme should be reactivated within 10 min to achieve the maximum reactivation and
also to ensure a maximum number of reuses of the immobilized enzyme membrane. This study has demonstrated that
TMB-4 is much more effective agent for reactivation of the
immobilized AChE enzyme for biosensor applications. In
another case of acetylcholinesterase inhibition, 0.4 mmol l1
sodium fluoride (NaF) was successfully used for 10 min
for the reactivation of an inhibited biosensor (Kok et al.,
2002).
Heavy metals act by the binding of the metal salts to protein thiol groups. Depending on the sensing enzyme used,
some biosensors can be regenerated after inhibition by use of

a metal chelating agent, such as EDTA or thiols (Evtugyn
et al., 1998). Mohammadi et al. (2005) tried to regenerate a 50% mercury-inhibited invertase biosensor by soaking
in a cysteine solution; the recovery was 30% of the initial
biosensor signal. They also tried to regenerate this biosensor
with EDTA solution. Unfortunately, no reactivation has been
noted. However, a full and rapid restoration of response has
been recently obtained by treatment of Hg2+ -inhibited glucose oxidase biosensor with EDTA solution (Malitesta and
Guascito, 2005).
It was also seen that if exposure time was short enough, the
enzymatic activity could be recovered without using reactivators even in the case of inhibition by a high concentration of
pollutant (Okazaki et al., 2000). Such effectively reversible
inhibition can prove an advantage in the analysis by flow
injection where the inhibitor is eluted by simple flushing with
an electrolyte or a buffer solution (Shan et al., 2004). In summary, for irreversible inhibition, the damaged enzyme could
often be reactivated using specific regenerating reagents. If
the reactivation achieved is not sufficient, the biosensors
would have to be assembled for single use, in which case
screen-printed electrodes would be recommended.
2.6. Parameters generally affecting the performance of
enzymatic biosensors
2.6.1. Effect of pH
The pH of the solutions containing substrates can affect
the overall enzymatic activity since, like all natural proteins,
enzymes have a native tertiary structure that is sensitive to
pH; denaturation of enzymes can occur at extreme pHs. It
is well known that the enzyme activity is highly pH dependent and the optimum pH for an enzymatic assay must be
determined empirically. It is best to choose a plateau region
so that the pH should not have any effect on enzyme activity and will not interfere with the results obtained relative
to the inhibition of the enzyme by the inhibitor. The activity
of the immobilized acetylcholinesterase as a function of pH
has been studied between pH 2 and 9 by Stoytcheva (2002).
She has reported a decrease in the activity of approximately
70% at pH 2 compared to of that at pH 7. Mohammadi et al.
(2005) investigated the effect of pH of a tri-enzymatic biosensor in which the optimum pH of the three enzymes is different
(invertase pH, 4.5; glucose oxidase, pH 5.5; mutarotase, pH
7.4). The pH effect on the biosensor response was analyzed
between pH 4 and 8 and the highest activity was found at pH
6.0. In order to improve the selectivity of the invertase toward
mercury and to avoid silver interference, a medium exchange
technique has been carried out. The biosensor was exposed to
mercury in an acetate buffer solution at pH 4 while the residual activity was evaluated with phosphate buffer solution at
pH 6. Dzyadevych et al. (2004b) have studied the influence
of pH on the analytical performance of a BChE modified pHSFET biosensor for tomatine. In this study the best response
was obtained for a buffer solution at pH 7.2, whereas the inhi-
bition level did not depend on the pH of the solution studied

in the range 6.08.5.
2.7. Effect of substrate concentration
The substrate concentration can affect the degree of inhibition. Kok et al. (2002) concluded that the inhibition level (%)
increases with increasing of the substrate concentration and
they have worked with a saturating substrate concentration
in the case of pesticide inhibition. Joshi et al. (2005) have
used a concentration of acetylhtiocholine two times higher
than the apparent KM for the determination of the maximum
activity of AChE before and after the inhibition by the OP
paraoxon which was selected as model pesticide. In the case
of competitive inhibition, at high substrate concentrations,
the inhibition effect is not observed since the substrate competes with the inhibitor. Dzyadevych et al. showed that the
sensitivity of a BuChE biosensor toward tomatine decreases
with an increase in the substrate concentration (Dzyadevych
et al., 2004a).
2.8. Effect of enzyme concentration
The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading (Shan et al., 2004;
Mohammadi et al., 2005; Sotiropoulou and Chaniotakis,
2005; Sotiropoulou et al., 2005). In order to optimize the
amperometric biosensor, Ciucu et al. (2003) studied a set
of five membranes with different amounts of AChE; the
response of the biosensors decreases with a decrease of the
enzyme concentration. The lowest concentration of paraoxon
detected (108 mol l1 ) was achieved by using a membrane
with 0.1 IU/cm2 immobilized AChE. The detection limit
of pesticide equal to 1017 M was achieved with very low
concentration (0.02 pmol) of engineered acethylcholinesterse
enzyme (Sotiropoulou et al., 2005).
3. Inhibition determination in organic phases

The detection of inhibitors (pesticides, heavy metals) is
generally performed in aqueous solution. However, these
compounds are generally characterized by a low solubility in
water and a high solubility in organic solvent. Extraction and
concentration of pesticides or heavy metal from solid matrices (fruits, vegetables, fish, etc.) are thus commonly carried
out in such solvents.
Depending on the nature and the amount of organic solvent involved, the enzyme can be strongly inactivated when
experiments are performed in these media (Amine et al.,
2004). Thus, the choice of organic solvent needs to be considered as part of the method development in order to avoid
undesirable effects. The effects of organic solvents have
been shown to be quite variable and depend on the configuration in which the enzyme is employed. For example,
Montesinos et al. (2001) and Andreescu et al. (2002b) have
1411
reported the influence of acetonitrile, ethanol and DMSO

on a cholinesterase sensor using acetylthiocholine as substrate. An increase of the output current was noticed when
working in 5% acetonitrile and 10% ethanol, resulting from
partial deactivation of the enzyme. The detection of dichlorvos, diazinon and fenthion in the presence of ethanol has been
reported using a screen-printed biosensor based on immobilized AChE (Andreescu et al., 2002b). This allowed the detection of 1.91 108 and 1.24 109 mol l1 of paraoxon
and chlorpyrifos ethyl oxon, respectively, in the presence
of 5% acetonitrile. The presence of 5% acetonitrile neither
enhanced the enzyme activity nor affected the detection limit
and the selectivity of the AChE biosensor toward insecticides. In another study using organic extracts, Del Carlo
et al. (2002) have followed the trend of chloropyrifos in grape
samples. The grape solvent extracts were analyzed using both
gas chromatography and electrochemical bioassay (acetylcholinesterase immobilised on a chemically modified screenprinted electrode with 7,7,8,8-tetracynoquinonedimethane)
and the results obtained with the two analytical methods were in agreement and indicated that the bioassay
was able to detect chlorpyrifos-methyl with satisfactory
accuracy.
In another case, Ciucu et al. reported the use of a biosensor for organophosphorus compounds (OPCs) in polar and
non-polar solvents (Ciucu et al., 2002). They co-immobilized
three enzymes: peroxidase, choline oxidase, and choline
esterases. This multienzyme biosensor has been used for
the determination of trichlorfon. It was found that non-polar
organic solvents inactivated the tri-enzymatic biosensor during the incubation storage. Among the polar solvents used,
acetone and ethanol demonstrated minimal effects on the
enzyme activity of the three enzyme system.
In the same way, AChE-based biosensor was used for
direct measurement of OP compounds in organic solvents
(Wilkins et al., 2000). The enzymatic activity was estimated
in the presence of ethanol, propanol, cyclohexanone and benzene in the range of 10100%. In this work, it was found that
AChE retained its activity only in 10% of ethanol. The assay
allowed the determination of OPs at subnanomolar concentrations with an overall assay time of 10 min.
Finally, a novel approach to circumvent the problems of
enzyme inhibition assays for the determination of pesticides
and methylmercury in organic extracts took advantage of
the fact that the enzyme (acethylcholinesterase or invertase)
in aqueous phase would selectively extract the irreversible
inhibitor dissolved in organic solvent when they came in contact at the interface of organic/aqueous phases (Amine et al.,
2004). In terms of the organic solvents tested, the best results
were obtained with toluene and benzene for invertase and
with hexane for AChE.
The extraction with organic solvents seems to be necessary
in the case of real sample analysis in order to avoid interfering
species insoluble in organic solvent. Therefore, the choice of
an appropriate organic solvent is important to circumvent or
minimize the enzyme inactivation.
1412
4. Inhibitor investigation using enzyme-based

biosensors
4.1. Pesticide inhibitors
The determination of pesticides has became increasingly
important in recent years because of the widespread use of
these compounds, which is due to their large range of biological activity and a relatively low persistence. As illustrated by
the literature reports compiled in Table 1, the development
of biosensors for pesticides is the subject of considerable
interest, particularly in the areas of food and environment
monitoring (Dzyadevych et al., 2005).
Several enzymes such as cholinesterase enzymes (AChE,
BChE) and urease have been used in the design of direct
electrochemical biosensors for the detection of pesticides.
Analytical devices, based on the inhibition of cholinesterase,
have been widely used for the detection of OP compounds
(Gogol et al., 2000; Andreescu et al., 2001, 2003; Choi
et al., 2001; Zhang et al., 2001; Jeanty et al., 2002; Turdean
et al., 2002; Schulze et al., 2003; Danet et al., 2003; Brazil de
Oliveira et al., 2004; Crew et al., 2004; Pavlov et al., 2005)
and carbamate pesticides (Lee et al., 2001; Kok and Hasirci,
2004; Zhang et al., 2005).
Conductimetric AChE biosensor was used for the assessment of the toxicity of methyl parathion and its photodegradation products in water (Dzyadevych et al., 2002). In this
study it was shown for the first time that the inhibition effect
of methyl parathion on the AChE activity increases dramatically as soon as the photodegradation begins. Such a sensor
could be employed as part of an early warning system for
detecting various cholinesterase inhibitors.
Collier et al. (2002) incorporated AChE and BChE into the
carbon-based ink used for printing electrodes. The printed
electrodes elaborated were used for the determination of
chlorfenvinphos and diazinon, organophosphate pesticides
known to be used for the control of insect pests in wool.
The BChE-based biosensor was shown to be the most sensitive sensor toward a mixture of chlorfenvinphos and diazinon,
with a limit of detection equal to 0.5 g g1 .
Sccreen-printed electrodes (SPE) have also been chemically modified with 7,7,8,8-tetracyanoquinodimethane
(TCNQ) and used for the mediated electrochemical detection of acetylcholinesterase activity (Del Carlo et al., 2004).
In this study, the AChE inhibition has been carried out on samples from different food commodities. The AChE biosensor
allowed detection of carbaryl at concentration of 10 ng g1
which corresponds to the limit value set by the European
legislation.
Recently a novel enzyme capable of selective recognition of OP compounds was employed. In particular, a faster,
simpler, and direct biosensing protocol suitable for rapid
testing of OP substances can be accomplished utilizing the
biocatalytic action of oraganophosphorus-hydrolase (OPH)
(Mulchandani et al., 2001; Wang et al., 2002, 2003; Simonian
et al., 2004; Gaberlein et al., 2000). However, the detection
limit obtained using OPH was not very low compared to

what is generally obtained by inhibition of cholinesterase
enzymes.
A new strategy for the detection and discrimination of
neurotoxins, the anti-acetylcholinesterase activities of mixtures of different neurotoxins, were investigated (Simonian
et al., 2001). It was possible to separate the effects of different inhibitors, using a combined recognition/discrimination
strategy based on the joint action of acetylcholinesterase and
organophosphorus-hydrolase enzymes. They demonstrated
the feasibility of eliminating the organophosphate neurotoxins from the different multi-component inhibitor combination via sample pre-treatment with immobilized forms of
OPH. Because of such manipulation, the inhibiting influence
of non-OP neurotoxins on the AChE can be separated and
their true concentration may be determined. In other study,
engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors in
AChE-multisensors for simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides (Bachmann et al., 2000). In this method the system
was based on a combination of amperometric multi-electrode
biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The AChE
mutants were selected on the basis of displaying an individual sensitivity pattern toward the target analytes (Bachmann
et al., 2000). In this work it was demonstrated that the
multisensor was capable of simultaneously detecting and
discriminating paraoxon and carbofuran with errors of prediction of 0.4 and 0.5 g l1 , respectively. In the same vain,
Crew et al. (2004) have investigated the determination of
OPs using genetically modified Drosophila melanogaster
AChE. In this work, the screen-printed carbon electrodes
modified with cobalt phthalocyanine were used as base
transducers in the construction of amperometric pesticide
biosensors.
Brazil de Oliveira et al. (2004) have reported the comparative investigation of biosensors based on different acetylcholinesterases. The enzymes used were genetically modified AChE obtained from Drosophila melanogaster and
from commercial sources: electric eel, bovine erythrocytes
and human erythrocytes. The sccreen-printed biosensors
designed were used for determination of methamidophos pesticides. The inhibition of acetylcholinesterase is measured by
direct or indirect measurement of its activity. In the case of the
direct method, the assay is based on the spectrophotometric
or electrochemical measurement of thiocholine issuing from
the following reaction:
acetylthiocholine + H2 O acetic acid + thiocholine
However, the measurement of thiocholine production does
not accurately reflect the degree of AChE inhibition by pesticides since the sample may also contain heavy metals. An
interference was also observed from copper ions at mg l1
levels (Danet et al., 2003) while Ricci et al. (2003), have
shown that 5 M Cu2+ , 1 M Ag+ , 0.5 M Hg2+ induced
Table 1
Survey of enzyme inhibition-based biosensors for pesticides
Enzymes
Immobilisation matrix
Techniques
Sample
Working range/LOD
Comments
Nature of inhibition
Paraoxon
OPH
Optical
20240 M
Method based on competitive

inhibition of OPH by specific
fluorophore
Paraoxon
AChE
Amperometric
Real water sample
0.50.7 nmol l1 , LOD = 0.5 nmol l1
Paraoxon
AChE
Amperometric
AChE
Amperometric
Orange juices, peach

pap baby foods
160 g l1 , LOD = 2 g kg1
Paraoxon
Covalent linkage between

gold nanoparticles and
sufhydryl groups of the
enzyme
AChE dropped on the
multiwall carbon
nanotubes
Entrapment in
TCNQ-graphite
Cross-linking using GA
0.31.8 ppm
Paraoxon
AChE
Simonian et al. (2005)
Irreversible
Joshi et al. (2005)
Schulze et al. (2002)
LOD(I10) = 2 109 mol l1
Extraction of inhibitors with

isooctane
Regeneration with 2-PAM and
TMB-4
Amperometric
Andreescu et al. (2003)
Paraoxon
AChE
Amperometric
Grappes
LOD = 0.1 g l1
Boni et al. (2004)
Paraoxon
AChE
Optical
Contaminated water
02.0 ppm
Irreversible
Choi et al. (2001)
Paraoxon
AChE + ChO
Amperometric
Use of microelectrodes
Zhang et al. (2001)
Amperometric
Milk
080 nmol l1 (batch), 015 mol l1

(FIA), LOD < 10 nmol l1
LOD = 1, 20 g l1
Paraoxon, carbaryl
AChE variants
Zhang et al. (2005)
Adsorption on activated
silica gel
Entrapment in a
PVA-SbQ polymer
Potentiometric pH
measurements
Amperometric
Potato
Irreversible
reversible
Simonian et al. (2001)
Retention with dialysis

membrane
Entrapment in a
PVA-SbQ polymer
Cross-linking with GA
Amperometrican FIA
Recovery of paraoxon and

carbaryl in milk samples between
89 and 107%
New strategy for discrimination of
neurotoxins
A simultaneous detection and
discrimination of binary mixture
of paraoxon and carbofuran
Paraoxon, carbaryl
AChE + OPH
Paraoxon, carbofuran
AChE variants
Paraoxon, carbofuran,
aldicarb
Paraoxon and chlorpyrifos
ethyl oxon
Paraoxon, diazinon,
malathion, coumaphos
Coumaphos,
chloropyrifos-methyl
Paraoxon-ethyl,
paraoxon-methyl,
diiso-propyl
fluorophosphates,
parathion-methyl,
trichlorfon
Diisopropyl, paraoxon-ethyl,
paroxon-methyl,
trichlorfon,
parathion-methyl
carbofuran
Paraoxon, chlorpyrifos oxon,
malaoxon
Chloropyrifos-ethyl
AChE + ChO
Irreversible
Ciucu et al. (2003)
Amperometric
Andreescu et al. (2002b)
Optical
Water miscible
organic solvent
White and Harmon (2005)
Cross-linking with GA in
polytyramine matrix
Cross-linking with BSA
in a saturated GA vapour
Amperometric
Change of the absorbance of

porphyrinenzyme complex
Bi-enzyme sensor
Suprun et al. (2004)
Conductometric
Soil samples
Dzyadevych et al. (2005)
AChE
Biosensor based on
enzyme field-effect
transistor (ENFET)
Potentiometric
3.0 1011 , 5.0 107 , 5.0 106 ,

2.0 107 , 5.0 106 ,
2.0 106 mol l1
Reactivation with 2-PAM
Irreversible
AChE
Immobilization on PVA
Amperometric and FIA
Spiked river water
0.60.8, 200.8, 0.61.2 mol l1
Reactivation with 2-PAM solution
Jeanty et al. (2002)
Grape and vine

samples
Real water samples
1300 ng ml1
Single use of SPEs, analysis in

organic solvent
Validation of the biosensors with
gas chromatography and mass
spectrometry
Del Carlo et al. (2002)
Suwansa-ard et al. (2005)
Reversible
Xavier et al. (2000)
AChE
OPH
BChE + HRP
AChE
Covalent linkage with

graphite used for the
construction of the SPE
Covalent immobilisation
on a nylon membrane
Adsorption on
LangmuirBlodgett
monolayer film
Retention with dialysis
membrane
Cross-linking using GA
AChE
Immobilization on TCNQ
Amperometric
Carbofuran, carbaryl
AChE
Immobilisation on silica
gel by covalent binding
Potentiometric and FIA
Propoxur, carbaryl
AChE
Linkage onto CPG
Optical and FIA
Spiked river water

samples
I50 = 3.0 107 , 9.1 108 ,

3.0 107 mol l1
LOD = 1.91 108 ,
1.24 109 mol l1
LOD = 7, 800, 1, 250 ppt
0.20.8 mol l1 , LOD = 0.1 mol l1 ;
0.073 mol l1 , LOD = 0.03 mol l1
LOD = 1.0 108 , 5.0 107 ,
5.0 1011 , 2.0 106 ,
3.0 107 mol l1
0.028.0 ppm, LOD = 0.02 ppm,

0.310 ppm
0.030.50 mg l1 , LOD = 0.4 ng l1 ,
0.83.0 mg l1 , LOD = 25 ng l1
Gulla et al. (2002)
Bachmann et al. (2000)
1413
Spiked vegetables
(onion and lettuce)
109 to 105 , 5 108 to

1 105 mol l1
LOD = 0.5 g l1
Reference
Inhibitors
1414
Table 1 (Continued)
Enzymes
Techniques
Sample
Working range/LOD
Comments
Carbaryl, carbofuran
AChE
Potentiometric and FIA
Chloropyrifos-methyl,
coumaphos, carbofuran
BChE
Amperometric
Spiked grape juice
1011 to 104 mol l1 , LOD = 1010 ,

1011 mol l1
LOD = 2 108 , 5 108 ,
1 108 mol l1
Soman, sarin
AChE
Immobilization in CPG in
presence of GA
in a GA vapour (PB-SPE
modified with TCNQ)
Entrapment on the
biotin-embedded
nitrocellulose membrane
Spiked soil
LOD = 2, 8 pg
Alicarb, carbaryl, carbofuran,

methomyl
Atrazine, diuron
AChE
Potentiometric
silicon-based
lightaddressable
potentiometric sensor
(LAPS)
Amperometric
Reactivation with
pyridine-2-aldoxime
For carbofuran the enzyme was
partially reactivated after 20 min
incubation
Regeneration using 2-PAM
Tyrosinase
(PPO)
Amperometric
Dichlorvos
AChE
Entrapment in a poly
3,4-ethylene
dioxythiphene (PEDT)
Adsorption in porous
carbon rod
1.995/1.5, 0.2100/0.9, 0.2166/0.2,

0.4113.5/0.3 ppb
5500 nM/1 mg l1 ,
5500 nM/0.5 mg l1
Amperometric
LOD = 1012 mol l1
Dichlorvos
AChE
mutant
E69Y, Y71D
Amperometric
LOD = 1017 mol l1
Carbofuran
AChE
Adsorption into
nanoporous conductive
carbon
Lipid films and
methylacrylate polymer
Immobilized on to the
PB-SPE surface using
cross-linking method
(GA, nafion and BSA)
PVA-SbQ AlDH coated
SPE
Mixture of enzyme and
PVA-SbQ polymer
deposited on graphite
SPE surface
Adsorbtion onto a
stearylamine monolyer
using the electrostatic
force
Deposition of AChE and
PVA-SbQ polymer onto
Pt
Poly(2-hydroxyethymethacrylate)
menbrane
Immobilisation on a CPE
with polyethylenimine
and GA
Entrapement in
polypyrrol (PPy) film
In gelatine in presence of
GA
Immobilization on SPE
Electrochemical and
FIA
Amperometric
Amperometric
Fruits
107 to 109 mol l1
Wheat and apple

Durum wheat
106
to
251000 ng ml1 , LOD = 38 ng ml1 ,
I50 = 360 ng ml1
Amperometric
Amperometric
Pirimiphos-methyl-oxon
Pirimiphos-methyl
AChE
ChO
Methyl isothiocyanate
(MITC)
Methamidophos
AlDH
dm AChE, ee
AChE
Trichlorfon
BChE
Methylchlor-pyrifos-oxon
AChE
Aldicarb
AChE + ChO
Parathion
Parathion hydrolase
Atrazine
Azide
Polyphenol oxidase (PPO)

Catalase
Trichlorfon
HRP + ChO + ChE
Lee et al. (2001)
Irreversible
Ivanov et al. (2003)
Irreversible
Lee et al. (2000)
Nunes et al. (2004)
PEDT/PPO highly sensitive

comparing to other immobilization
methods
Selective concentration of
dichloros inside the activated
carbon matrix
Selective concentration of
dichlorvos inside the activated
carbon matrix
Good recoveries (96 and 106%)
Vedrine et al. (2003)
Reversible
Sotiropoulou and Chaniotakis (2005)
Reversible
Sotiropoulou et al. (2005)
Nikolelis et al. (2005)
No effect of sample matrix

AChE inhibition in solution. LOD
lower than the regulated level
(5 mg kg1 )
Irreversible
Crew et al. (2004)

Del Carlo et al. (2005)
1001000 ppb, LOD = 100 ppb
Noguer et al. (2001)
0.5100/1, 0.0524/90 ppb
Sensitivity improved with

genetically modified AChE-based
biosensor
Irreversible
Brazil de Oliveira et al. (2004)
Potentiometric ion
sensitive fields effect
transistors (ISFET)
Water
LOD = 107 mol l1
Irreversible
Wan et al. (2000)
Amperometric with
continuous flow system
0.72 106 to 73 109 mol l1
Reactivation with 2-PAM
Irreversible
Jeanty et al. (2001)
Potentiometric
10500 ppb, LOD = 10 ppb
Enzyme stable for 2 months,

regeneration with NaF
Kok et al. (2002)
Amperometric
Spiked river water
10100 ng ml1 , LOD = 10 ng ml1
Reversible
Sacks et al. (2000)
Amperometric
0.050.5 ppm, LOD = 0.1 ppm
El Kaoutit et al. (2004)
Amperometric
Fruit juices
25300 mol l1
Sezginturk et al. (in press)
Potentiometric
010 ppm, LOD = 0.01 ppm
Use of different organic solvents
Irreversible
Ciucu et al. (2002)
108
mol dm3
Reference
PVA-SbQ: polyvinyl alcohol bearing styrylpyridinium groups, TCNQ: 7,7,8,8-tetracyanoquinodimethane, PPD: poly-o-phenylenediamine, AChE: acethylcholinesterase, ChO: choline oxidase, OPH:
organophosphorus-hydrolase, BuChE: butyrylcholinesterase, HRP: horseradich peroxidase, LOD: limit of detection, GA: glutaraldehyde, BSA: bovine serum albumin, 2-PAM: pyridine-2-aldoxime methyliodide,
TMB-4: 4-formylpyridinium bromide dioxime, PB-SPE: Prussian Blue-sccreen-printed electrode. I50 : 50% the biosensor inhibition.
Inhibitors
a 10% decrease of the amperometric signal of 10 M thiocholine. Thus, it would seem necessary to use prior separation
techniques in the case of multicomponent samples or develop
more specific methods for their analysis.
A variety of combined systems has been explored without
much success to overcome the problems of analyzing complex mixtures. In another multi-enzyme approach, choline
oxidase (ChO) was co-immobilized with AChE and coupled
to amperometric sensors for the measurement of hydrogen
peroxide produced by the choline oxidation (Zhang et al.,
2001; Guerrieri et al., 2002; Kok et al., 2002):
AChE
acetylcholine + H2 O acetic acid + choline
1415
strate as an indicator of the presence of an OP compound, the

method described is based on the change in fluorescence of a
competitive inhibitor of the OPH enzyme when the inhibitor
is displaced by the OP substrate. The change in fluorescence
of the inhibitor is produced by the presence of gold nanoparticle attached to the enzyme.
Finally, the inhibition of tyrosinase has been also investigated for the determination of carbaryl using amperometric
biosensors (Kuusk and Rinken, 2004) with the detection limit
for carbaryl concentration obtained with this amperometric
biosensor being 0.2 mg l1 . This method also utilised a simple approach of data management which can be automated
and used for different biosensors.
4.2. Heavy metal inhibitors
ChO
choline + O2 betaine + 2H2 O2

The performance of an oxygen electrode combined with a
bi-enzymatic membrane (AChE and ChO) was tested for the
detection of alicarb (AS), carbofuran (CF) and carbaryl (CL)
as well as with two mixtures (AS + CF) and (AS + CL) (Kok
and Hasirci, 2004). The results indicated that this bi enzyme
system could not differentiate between the different pesticides and the inhibition by total pesticides was not additive.
AChE and ChO were also immobilized in a polyurethane
polyethylene oxide (PU-PEO) layer which was cast on the
top of the probe and covered with a dialysis membrane
(Zhang et al., 2001). In this study, the biosensing device
was used in either batch or flow analysis mode for paraoxon
detection allowing a detection limit down to 10 nM using
0.00025 U of AChE. Andreescu et al. (2002a) have proposed the use of the commercially available phenyl acetate
as new substrate for AChE biosensor and tested it in a bienzymatic AChE/tyrosinase system applied to the detection
of organophosphorus pesticides. Results were similar to those
obtained with the mono-enzymatic AChE system using aminophenyl acetate as substrate.
Recent attempts to demonstrate better systems for detection of pesticides have employed different enzymes or
enzyme/sensor interfaces. Mazzei et al. (2004) have reported
the use a bioelectrochemical system for the determination
of pesticides by alkaline phosphatase inhibition. They found
a detection limit for malathion (2,4-dochlorophenoxyacetic
acid) of 0.5 g l1 .
A novel optical detection of OP compounds based on
reversible inhibition of OPH by copper complexed porphyrin
(CuC1TPP) has been developed by Harmon. The absorbance
spectrum of the porphyrinenzyme complex is measured via
planar waveguide evanescent wave absorbance spectroscopy.
Addition of OP compounds displaces the porphyrin from the
enzyme resulting in reduced absorbance intensity at 412 nm.
Using this method the OP compounds can be detected at ppt
levels (White and Harmon, 2005).
Simonian et al. (2005) described a novel strategy for the
direct detection of OP neurotoxins. Instead of using the pH
change associated with enzymatic hydrolysis of the OP sub-
Table 2 summarizes the characteristics of various biosensors for heavy metal ion sensing, produced by integrating
immobilized enzymes with different kinds of transducers.
Enzymatic methods are commonly used for metal ion determination, as these can be based on the use of a wide range
of enzymes that are specifically inhibited by low concentrations of certain metal ions (Krawczynski vel Krawczyk et al.,
2000). For the inhibitive determination of trace mercury, a
large number of enzymes has been used: horseradish peroxidase (Han et al., 2001), urease (Krawezyk et al., 2000; De
Melo et al., 2002; Kuswandi, 2003; Rodriguez et al., 2004b)
Krawczynski vel Krawczyk et al. (2000), glucose oxidase
(Alexander and Rechnitz, 2000), alcohol oxidase (Pirvutoiu
et al., 2002) and glycerol 3-phosphate oxidase (Ciucu et al.,
2001), and invertase (Pirvutoiu et al., 2001). Some studies
have also focused on the analysis of different organic forms
of mercury: phenyl mercury (Doong and Tsai, 2001) using
urease, methyl mercury and phenyl mercury using invertase
(Mohammadi et al., 2005), and methyl mercury using peroxidase (Han et al., 2001).
Cadmium ion could be monitored by enzymatic sensors since it was found that it induced inhibition of several
enzymes such as urease (Lee and Russel, 2003) and butyrilcholinesterase (BChE) (Mourzina et al., 2004). For copper
determination a cholinesterase sensor has been used (Evtugyn
et al., 2003). It has been reported that heavy metal ions induce
a reversible cholinesterase inhibition.
For heavy metal screening, Rodriguez et al. (2004b) studied different procedures of ureaseglutamic dehydrogenase
immobilzation. The urease was either cross-linked with glutaraldehyde, fixed by nafion film, entrapped in alginate gel,
or immobilized by adsorption. The designed biosensors were
then tested for the detection of metal ions (Cu, Hg, Cd and
Pb) in soil and water extracts. Immobilization of urease using
a nafion film resulted in the best sensitivity for the study
of metal inhibition (Rodriguez et al., 2004b). Recently, an
array-based urease optical biosensor based on the solgel
immobilization has been used for Hg(II), Cu(II) and Cd(II)
determination in tap and river water (Tsai and Doong, 2005).
It was reported that mercury had a higher affinity for the cys-
1416
Table 2
Survey of enzyme inhibition-based biosensors for heavy metals
Enzymes
Techniques
Sample
Working range/LOD
Comments
Reference
Hg2+ , Cu2+ , Cd2+
Urease
Entrapment in solgel matrix
Optical
LOD = 10 nM, 50 M, 500 M
Tsai and Doong (2005)
2.5 mol l1
to
2.5 mol l1 to 0.2 mmol l1
Rapid regeneration with

EDTA
Reversible
Malitesta and Guascito (2005)
Not selective
Doong and Tsai (2001)
Regeneration with EDTA

and thioacetamide
Regeneration with cysteine
Lee and Russel (2003)
Irreversible
Kuswandi (2003)
Reversible in less than

8 s and irreversible in
18 min
Irreversible
Han et al. (2001)
Hg2+ ,
Cu2+
Hg(NO3 )2 , HgCl2 , Hg2 (NO3 )2 ,

phenyl mercury
Cd2+
Glucose
oxidase
(Gox)
Urease
Urease
Electropolymerisation in PPD
Amperometric
Tap and river

water
Entrapment in solgel film
Potentiometric
Water samples
Self-assembled monolayer on the

gold-coated sensor surface
Immobilization in ultrabind
membrane
Optical (SPR)
0.051.0/0.2, 0.051.0/0.2,
0.051.0/0.1,
0.15.0/0.5 mol l1
010 mg l1 (dynamic range)
Optical fiber
biosensor
1 109
0.2 mmol l1 ,
1 105 ,
Hg(II), Ag(I), Cu(II), Ni(II),

Zn(II), Co(II), Pb(II)
Urease
Mercury(II), mercury(I),
methylmercury,
mercuryglutathione complex
Ag+ , Ni2+ , Cu2+
HRP
Entrapment in -cyclodextrin
polymer
Amperometric
Urease
Deposition onto electrode area and

covering with
poly(4-vinylpyridine and Nafion)
Potentiometric
pH-SFET
LOD = 3.5 108 , 7 105 ,

2 106 mol l1
Cu2+
Hg2+
AChE
GOx
Cross-linking with GA vapour

Cross-linking with GA and BSA
Amperometric
Amperometric
Spiked water
0.054.0 mmol l1
2.512 ng ml1 ,
LOD = 1 ng ml1
HgCl2 , Hg(NO3 )2 , Hg2 Cl2 ,

methylmercury, phenyl mercury
Invertase
Amperometric
I50 = 0.27, 0.032, 0.27, 0.34,

0.12 ppm
Hg2+
GOx
Amperometric
Chromium(VI)
GOx
Cross-linkage with GA and

deposition on laponit modified
electrode
Immobilized in a
polyvinylpyridine (PVP) in
presence of 2-aminoethanethiol
mediator
Cross-linking with GA and
covering with aniline membrane
Amperometric
Soil samples
to
1 108 to 1 105 ,
7
1 10 to 1 105 ,
1 106 to 1 105 ,
2 105 to 1 103 ,
2 105 to 1 103 ,
1 104 to 1 103 mol l1
LOD = 0.1, 0.1, 1.7 ng ml1
Soldatkin et al. (2000)
The use of the negatively

charged
polymer/regeneration with
EDTA
Inhibition of invertase in
solution and detection of
product with glucose
biosensor
Reactivation with cysteine
Reversible inhibition
Evtugyn et al. (2003)

Mohammadi et al. (2002)
Irreversible
Mohammadi et al. (2005)
1100 ppb, LOD = 0.2 ppb
Reversible
Alexander and Rechnitz (2000)
0.49 g l1 to 8.05 mg l1 ,
LOD = 0.49 g l1
Guang-Ming et al. (2004)
Inhibitors
1417
teine residues of urease and could be detected with a lower

limit of 10 nM.
4.3. Other inhibitors
In addition to the determination of pesticides and heavy
metals, the biosensors were used for the analysis of other
pollutants and toxic compounds; Table 3 summarizes the
biosensor characteristics used in these cases. Several enzymebased biosensors have been constructed in order to measure glycoalkaloids in foods. Consumption of glycoalkaloids, which are secondary metabolites that can show up in
food, has been associated with human death and poisoning
because of their teratogenic and embryotoxic effects. The
pH-sensitive field effect transistors (pH-SFET) have been
developed for this purpose (Korpan et al., 2002; Arkhypova
et al., 2003; Dzyadevych et al., 2004b). In one approach,
AChE and BChE were immobilized on a pH-SFET by a
method of protein cross-linking in saturated glutaraldehyde
vapour (Arkhypova et al., 2003; Korpan et al., 2002). It had
been shown that BChE is more sensitive to potato glycoalkaloids than acetylcholinesterase. The BChE modified pHSFET was then employed for determination of -chaonine,
-solanine and solanidine in potato samples. Furthermore,
AChE and BChE were also employed for the determination of
chaconine (Dzyadevych et al., 2004b). These authors demonstrated that the BChE-based biosensor is much more sensitive
to the inhibitor than the AChE biosensor.
Detection via enzymatic inhibition has also been demonstrated for other potential food contaminants. Noguer et
al. developed a disposable biosensor based on aldehyde
dehydrogenase (AlDH) for the detection of MITC (methyl
isothiocyanate), the main metabolite of metam-sodium. Benzoic acid has also been determined in foodstuffs using
a graphiteteflontyrosinase composite biosensor (Morales
et al., 2002). The composite bioelectrode allows a low detection limit of benzoic acid equal to 9 107 mol l1 in nonaqueous (mayonnaise) and aqueous (cola soft drinks) media
and exhibits good analytical characteristics such as renewability, stability and low cost.
Anatoxin-a(s), a cyanotoxin produced by cyanobacteria,
was detected in freshwater and lake samples using a biosensor
based on AChE (Devic et al., 2002). Different mutants of the
enzyme were used and the limit of detection allowed by the
most sensitive biosensors was 0.5 nmol of toxin per liter.
Different enzymatic biosensors have been constructed for
the determination of nitric oxide (NO) (Kilinc et al., 2000)
and superoxide radicals (Campanella et al., 2000). These
biosensors are based on the immobilization of different oxidases.
Lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP). The limit of detection
allowed by this biosensor was 270 g l1 (Young et al., 2001).
The sensitivity could be improved after co-immobilization of
LDH with lactate oxidase (LOD) and glucose dehydrogenase
(GDH).
Fig. 2. Distribution of enzymes used for the design of biosensors used for
detection of inhibitors.
5. Food and environmental applications

In the specific context of food and environmental analysis,
a large number of biosensors based on enzyme inhibition have
been developed for the detection of a variety of compounds.
The most commonly used enzymes for the design of biosensors are AChEs (50%), followed by BChEs (11%), see Fig. 2.
However, HRP, tyrosinase and urease represent separately
7% of the total enzymes used for the construction of biosensors applied for investigations based on inhibition. Others,
such as AlDH, CDH, OPH and GST are used in limited cases
(Fig. 2). About 71% of these enzymatic biosensors were used
for the determination of pesticides including carbamates and
organophosphorus compounds (Fig. 3) while heavy metals
represent only 21% of the total application of these biosensors. Other applications of these sensing devices include the
determination of glycoalkaloids, benzoic acid, cyanide, nitric
oxide and neurotoxins (anatoxin-a(s)) and other inhibitors.
Despite the very large number of publications demonstrating enzyme inhibition, the majority of systems were unfortunately not challenged by real samples. Still, the research
published during the last 5 years showing applications to food
and environmental samples was not negligible.
Numerous biosensors were designed for the analysis of
inhibitors in tap and river water samples: acetylcholinesterase
used for determination of pesticides (Bachmann et al., 2000;
Dzyadevych et al., 2003; Jeanty et al., 2002; Joshi et al.,
2005) and anatoxin-a(s) (Devic et al., 2002; Villatte et al.,
2002), lactate dehydrogenase for pentachlorophenol analysis
Fig. 3. Inhibitors distribution in enzymatic biosensors investigations.
1418
Table 3
Survey of biosensors for other chemical contaminants
Enzymes
Immobilization matrix
Techniques
Sample
Working range/LOD
Comments
-Chaonine, -solanine,
solanidine
-Chaconine, -solanine
BChE
vapour
and GA vapour
Potentiometric
pH-SFET
Potentiometric
Potatoes
Agriculture
Overall time for one analysis is

10 min
vapour
Deposition of
enzyme-loaded gel on
the pH-selective layer
and GA
vapour
Entrapment in poly(ophenylenediamine)
Cross-linking BSA and
GA
Potentiometric
Potato
0.5100 mol l1 /0.5 mol l1 ,

2.0 mol l1 , 2.0 mol l1
0.2100 mol l1 ,
LOD = 0.2 mol l1 ,
0.2100 mol l1 ,
LOD = 0.5 mol l1
LOD = 0.2, 0.5, 1 mol l1
Lee et al. (2002)
Potentiometric
Potatoes
LOD = 0.2, 0.5, 0.5 mol l1
Reversible
Potentiometric
Tomatoes
Reversible
Dzyadevych et al. (2004b)
Amperometric
0.550 mol l1 ,
LOD = 0.2 mol l1
0.520 mmol l1
Reliable tool for estimation of

the overall toxicity level in food
samples.
Reversible
Evtugyn et al. (2003)
02 mmol l1
Kilinc et al. (2000)
Amperometric
2.7 106
Casero et al. (2003)
Mixture of graphite,
tyrosinase and teflon
Immobilization in to
anionic clay
Entrapment in PVA-SbQ
Entrapment in PVA-SbQ
Entrapment in
TCNQ-graphite
Entrapment in the gel
sodium alginate
Amperometric
Mayonnaise sauce
and cola soft drinks
to
1.1 105 mol l1 ,
LOD = 2.0 106 mol l1
9 107 mol l1
Use in non-aqueous medium
Reversible
Morales et al. (2002)
LOD = 0.1 nmol l1
Regeneration by washing with

electrolyte.
Use of different AChE mutants

No reactivation was observed
using oxime
Reversible
Shan et al. (2004)
Irreversible
Noguer et al. (2001)

Devic et al. (2002)
Villatte et al. (2002)
Biosensor stable for

30 days
Choi et al. (2003)
BChE
-Chaonine, -solanine,
solanidine
-Chaconin, -solanine,
tomatine
BChE
Tomatine
BChE
Oxalic acide
AChE
Nitric oxide
BChE
Nitric oxide
Xanthine
(XOD)
HRP
Benzoic acid
Tyrosinase
oxidase
Cyanide
Tyrosinase
Methyl isothiocyanate
Anatoxin-a(s)
Anatoxin-a(s)
AlDH
AChE
AChE
Captan
Glutathione-Stransferase (GST)
Amperometric
Amperometric
Amperometric
Amperometric
Amperometric
Optical
Fresh water
Spiked water
samples
Contaminated water
1001000 ppb, LOD = 100 ppb

110 g l1 ,
02 ppm
LOD = 1 g l1
Reference
Korpan et al. (2002)
Inhibitors
(Young et al., 2001), cellobiose dehydrogenase for phenolic

compound investigations (Nistor et al., 2002), glutathione-Stransferase for determination of captan (Choi et al., 2003),
urease for mercury analysis (Krawczynski vel Krawczyk
et al., 2000). In addition to mercury analysis, urease was
used for copper and cadmium determination in tap and
river water (Tsai and Doong, 2005), carboxyl esterase for
selenium detection (Saritha and Kumar, 2001). Trojanowicz
et al. (2004) examined a batch system with dissolved invertase and a membrane based amperometric glucose biosensor
for the determination Hg(II) in natural and wastes water.
They evaluated the inhibition of invertase by other macroand micro-components which can be potentially present in
environmental samples. Choi et al. (2001) developed a AChE
optical biosensor for paraoxon and captan analysis in contaminated water.
Other biosensors based on enzymatic inhibition were
used for the determination of pesticides in soil extracts,
including an AChE-based biosensor developed by Guerrieri
et al. (2002), a carboxyl esterase biosensor employed by
Saritha and Kumar (2001), an aldehyde dehydrogenasebased biosensor designed by Noguer et al. (2001),
and ureaseglutamic dehydrogenase-based biosensor by
Rodriguez et al. (2004a). Collier et al. (2002) developed a
urease biosensor for the determination of organophosphorus
pesticides in extracts of sheep wool.
Overall, for food analysis, the most widely biosensors used
are based on the AChE enzyme for the determination of pesticides (Del Carlo et al., 2002, 2005; Xavier et al., 2000;
Pogacnik and Franko, 2003; Crew et al., 2004). The first
application of an AChE-based biosensor in food produced by
animals, such as eggs, honey and milk, was developed by Del
Carlo et al. (2004). Dzyadevych et al. (2004b) developed a
BChE biosensor for determination of glycoalkaloids (tomatine) in tomato samples. During the last year, Zhang et al.
(2005) have developed a rapid biosensor based on disposable
screen-printed electrodes, which is suitable for monitoring
organophosphate and carbamate residues in milk. In this
work, three engineered variants of Nippostrongylus brasiliensis acethylcholinesterase were used to obtain enhanced sensitivity.
Ivanov et al. (2003) compared the features of various
cholinesterase sensors based on screen-printed carbon electrodes differing in the detection mode and modifier to establish factors affecting the sensitivity of pesticide determination
in grape juice. The work of Crew et al. (2004) was directed
at developing a rapid system capable of identifying and measuring specific OPs pesticide residues in wheat and apple
extracts. Their system is based on the inhibition of AChE
immobilized on screen-printed carbon electrodes modified
with cobalt phthalocyanine. They have demonstrated that
there was no matrix effect on the biosensor response from
wheat or apple extracts. It is envisaged that the proposed
amperometric biosensor will be integrated into an automated,
commercially available, analytical system for rapid quality
control analysis in the food industry. A composite ampero-
1419
metric tyrosinase electrode was designed by Morales et al.

(2002) for benzoic acid determination in mayonnaise and
cola soft drinks.
As can be seen, the application of the various developed
biosensors in food and environmental analysis is still limited.
A main reason seems to be that the most used enzymes are
not sufficiently selective or discriminating, as for example the
case of cholinesterase-based biosensors where the enzyme is
inhibited by both pesticides and heavy metals. Some authors
have developed novel strategies to solve these problems by
offering new methods of data analysis, by using engineered
enzymes instead of the classical ones, or by combining different enzymes.
6. Summary and conclusions

Despite the considerable research activity devoted to the
development of biosensors based on enzyme inhibition, analytical applications are still limited since these sensor technologies are not usually able to discriminate various toxic
compounds in the same sample. In particular, the simultaneous presence of heavy metals and pesticides in contaminated
samples provides a challenge for their use for purely regulatory purposes where a specific analyte must be determined
with a prescribed accuracy. Nevertheless, the properties of the
devices under development suggest that these biosensors can
be used as alarm systems; they would provide either quantification of one contaminant when this analyte is present alone
or an indication of total contamination of particular samples.
In many cases, the biosensor assays based on inhibitory
effects of pesticides or other chemical contaminants show
high sensitivity and could be the basis for relatively simple and cost-effective procedures. A particular advantage of
these biosensing systems is that they offer the possibility of
analysis in both batch and flow mode, allowing the use of
these sensors for analysis of a large number of samples in a
reasonable time interval. These methods can also be recommended for development of single use test strips, especially
with screen-printed electrodes, to avoid problems related to
fouling of the electrode which generally involves a chemical
or electrochemical deactivation of the working electrode surface. SPEs offer several additional advantages including low
cost, simple handling, being amenable to both mass production and instrument miniaturization for in loco analysis. Also,
the single use of these sensors avoids the need of reactivation.
Current research studies involve numerous efforts at
improving the analytical performance of the biosensing systems in order to be able to monitor a wide range of pollutants
in environmental and food samples. Effectively, artificial neural networks (ANN) have already been shown to be useful
adjunct for interpretation of experimental data in analysis of
binary mixtures of insecticides with the use of several biosensors exhibiting various responses to different analytes. The
use of genetically modified enzymes for the design of biosensors can be an effective way to improve sensor sensitivity.
1420
Indeed, the combination of such engineered enzymes (double and triple mutants) with microporous-activated carbon
technology could improved the efficiency of enzyme-based
biosensors.
7. Future perspectives
Engineered variants of enzymes could be another
approach in biosensor design for the discrimination and
detection of various enzyme-inhibiting compounds when
used in combination with chemometric data analysis using
artificial neural networks. The use of transducers constructed
from nano-structured material might also improve the efficiency of these biosensors. The crucial issues that should be
addressed in the development of new analytical methods is
first to enable the possibility of simultaneous and discriminative monitoring of several contaminants in a multicomponent
sample and then the conversion of the biosensing systems to
marketable devices suitable for large scale environmental and
food applications.
Acknowledgements
The authors would like to thank the Novtech and Craft EU
projects for research funding leading to some of the publications cited in this review.
References
Alexander, P.W., Rechnitz, G.A., 2000. Enzyme inhibition assays with
an amperometric glucose biosensor based on thiolate self-assembled
monolayer. Electroanalysis 12, 343350.
Amine, A., Mohammadi, H., Arduini, F., Ricci, F., Moscone, D., Palleschi,
G., 2004. Extraction of enzyme inhibitors using a mixture of organic
solvent and aqueous solution and their detection with electrochemical
biosensors. In: Proceedings of the Eighth World Congress on Biosensors, Granada, Spain, p. 3.7.64 (abstract book).
Andreescu, S., Magearu, V., Lougarre, A., Fournier, D., Marty, J.-L.,
2001. Immobilization of enzymes on screen printed sensors via an
histidine tail. Application to the detection of pesticides using modified
cholinesterase. Anal. Lett. 34 (4), 529540.
Andreescu, S., Avramescu, A., Bala, C., Magearu, V., Marty, J.-L.,
2002a. Detection of organophosphorus insecticides with immobilized
acetylcholinesterase-competitive study of two enzyme sensors. Anal.
Bioanal. Chem. 374, 3945.
Andreescu, S., Noguer, T., Magearu, V., Marty, J.-L., 2002b. Screenprinted electrode based on AChE for the detection of pesticides in
presence of organic solvents. Talanta 57, 169176.
Andreescu, S., Fournier, D., Marty, J.-L., 2003. Development of highly
sensitive sensor based on bioengineered acetylcholinesterase immobilized by affinity method. Anal. Lett. 36 (9), 18651885.
Arkhypova, V.N., Dzyadevych, S.V., Soldatkin, A.P., Elskaya, A.V.,
Martelet, C., Jaffrezic-Renault, N., 2003. Developpement and optimisation of biosensors based on pH-sensitive field effect transistors
and cholinesterases for sensitive detection of solanaceous glycoalkaloids. Biosens. Bioelectron. 18, 10471053.
Bachmann, T.T., Leaca, B., Vilatte, F., Marty, J.-L., Fournier, D., Schmid,
D.R., 2000. Improved multianalyte detection of organophosphates and
carbamates with disposable multielectrode biosensors using recombi-
nat mutants of Drosophila acethylcholinesterase and artificial neural

networks. Biosens. Bioelectron. 15, 193201.
Boni, A., Cremisini, C., Magano, E., Tosi, M., Vastarella, W., Pilloton,
2004. Optimized biosensors based on purified enzymes and engineered
yeasts: detection of inhibitors of cholinesterases on grapes. Anal. Lett.
37, 16831699.
Brazil de Oliveira, P.R., Nunes, G.S., Santos, T.C.R., Andreescu,
S., Marty, J.-L., 2004. Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically
modified Drosophila melanogaster. Application in amperometric
biosensors for methamidophos pesticide detection. Biosens. Bioelectron. 20, 825832.
Campanella, L., Persi, L., Tomasseti, M., 2000. A new tool for superoxide and nitric oxide radicals determination using suitable enzymatic
sensors. Sens. Actuators B 68, 351359.
Casero, E., Losada, J., Pariente, F., Lorenzo, E., 2003. Modified electrode
approaches for nitric oxide sensing. Talanta 61, 6170.
Choi, J.-W., Kim, Y.-K., Lee, I.-H., Min, J., Lee, W.H., 2001. Optical
organophosphorus biosensor consisting acetyl cholinesterase/viologen
hetero LangmuirBlodjett film. Biosens. Bioelectron. 16, 937943.
Choi, J.-W., Kim, Y.-K., Song, S.-Y., Lee, I.-H., Lee, W.H., 2003. Optical biosensor consisting of glutathione-S-transferase for detection of
captan. Biosens. Bioelectron. 18, 14611466.
Chouteau, C., Dzyadevych, S., Durrieu, C., Chovelon, J.-M., 2005. A bienzymatic whole cell conductometric biosensor for heavy metal ions
and pesticides detection in water samples. Biosens. Bioelectron. 21,
273281.
Ciucu, A., Negulescu, C., Baldwin, R.P., 2003. Detection of pesticides
using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic
system. Biosens. Bioelectron. 18, 303310.
Ciucu, A., Ciucu, C., Baldwin, R.B., 2002. Organic phase potentiometric biosensor for detection of pesticides. Roum. Biotechnol. Lett. 7,
625630.
Ciucu, A., Lupu, A., Pirvutoiu, S., Palleschi, G., 2001. Biosensors for
heavy metals determination based on enzyme inhibition. U.P.B. Sci.
Bull. Ser. B 63, 3344.
Collier, W.A., Clear, M.A., Hart, A.L., 2002. Convenient and rapid detection of pesticides of extracts of sheep wool. Biosens. Bioelectron. 17,
815819.
Crew, A., Hart, J.P., Wedge, R., Marty, J.-L., Fournier, D., 2004. A
screen-printed, amperometric, biosensor array for the detection of
organophosphate pesticides based on inhibition of wild type, and
mutant acetylcholinesterases, from Drosophila melanogaster. Anal.
Lett. 37, 16011610.
Danet, A.F., Bucur, B., Cheregi, M.-C., Badea, M., Serban, S., 2003.
Spectrophotometric determination of organophosphoric insecticides in
FIA system based on AChE inhibition. Anal. Lett. 36 (1), 5973.
De Melo, J.V., Cosnier, S., Mousty, S., Martelet, C., Jaffrezic-Renault,
N., 2002. Urea biosensors based on immobilization of urease into two
oppositely charged clays (laponite and ZnAl layered double hydroxides). Anal. Chem. 74, 40374043.
Del Carlo, M., Mascini, M., Pepe, A., Compagnone, D., Mascini, M.,
2002. Electrochemical bioassay for the investigation of chlorpyrifosmethyl in vine samples. J. Agric. Food Chem. 50, 72067210.
Del Carlo, M., Mascini, M., Pepe, A., Diletti, G., Compagnone, D.,
2004. Screening of food samples for carbamate and organophosphate pesticides using electrochemical bioassay. Food Chem. 84, 651
656.
Del Carlo, M., Pepe, A., Mascini, M., De Gregorio, M., Visconti, A.,
Compagnone, D., 2005. Determining pirimiphos-methyl in durum
wheat samples using an acethylcholinesterase inhibition essay. Anal.
Bioanal. Chem. 381, 13671372.
Devic, E., Li, D., Dauta, A., Henriksen, P., Codd, G.A., Marty, J.L., Fournier, D., 2002. Detection of anatoxin-a(s) in environmental
samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases. Appl. Environ. Microbiol. 68 (8), 41024106.

Doong, R.-A., Tsai, H.-C., 2001. Immobilization and characterization of
solgel encapsulated acetylcholinesterase fiber-optic biosensor. Anal.
Chim. Acta 434, 239246.
Durrieu, C., Chouteau, C., Barthet, Chovelon, J.-M., Tran-Minh, C., 2004.
A bi-enzymatic whol-cell algal biosensor for monitoring waster polluants. Anal. Lett. 37, 15891599.
Dzyadevych, S., Soldatkin, A.P., Arkhypova, V.N., Elskaya, A.V., 2005.
Early-warning electrochemical biosensor system for environmental
monitoring based on enzyme inhibition. Sens. Actuators B: Chem.
105, 8187.
Dzyadevych, S.V., Arkhypova, V.N., Soldatkin, A.P., Elskaya, A.V.,
Martelet, C., Jaffrezic-Renault, N., 2004a. Enzyme biosensor for tomatine detection in tomatoes. Anal. Lett. 37, 16111624.
Dzyadevych, S.V., Arkhypova, V.N., Martelet, C., Jaffrezic-Renault, N.,
Chovelon, J.-M., Elskaya, A.V., Soldatkin, A.P., 2004b. Potentiometric biosensors based on ISFETs and immobilized cholinesterases.
Electroanalysis 16, 18731882.
Dzyadevych, S.V., Soldatkin, A.P., Korpan, Y.I., Arkhypova, V.N.,
Elskaya, A.V., Chovelon, J.-M., Martelet, C., Jaffrezic-Renault, N.,
2003. Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors. Anal. Bioanal. Chem. 377,
496506.
Dzyadevych, S.V., Soldatkin, A.P., Chovelon, J.-M., 2002. Assessement of
the toxicity of methyl parathion and its photodegradation products in
water samples using conductometric enzyme biosensors. Anal. Chim.
Acta 459, 3341.
El Kaoutit, M., Bouchta, B., Zejli, H., Izaoumen, N., Temsamani, K.R.,
2004. A simple conducting polymer-based biosensor for the determination of atrazine. Anal. Lett. 37, 16711681.
Evtugyn, G.A., Stoikov, I.I., Budnikov, H.C., Stoikova, E.E., 2003. A
cholinesterase sensor based on a graphite electrode modified with
1,3-disubstituted calixarenes. J. Anal. Chem. 58, 11511156.
Evtugyn, G.A., Budnikov, H.C., Nikolskaya, E.B., 1999. Biosensors
for the determination of environmental inhibitors of enzymes. Russ.
Chem. Rev. 68, 10411064.
Evtugyn, G.A., Budnikov, H.C., Nikolskaya, E.B., 1998. Sensitivity and
selectivity of electrochemical enzyme sensors for inhibitor determination. Talanta 46, 465484.
Gaberlein, S., Knoll, M., Spener, F., Zaborosch, C., 2000. Disposable
potentiometric enzyme senor for direct determination of organophosphorus insecticides. Analyst 125, 22742279.
Gogol, E.V., Evtugyn, G.A., Marty, J.-L., Budnikov, H.C., Winter, V.G.,
2000. Amperometric biosensor based on nafion coated screen printed
electrodes for determination of cholinesterase inhibitors. Talanta 53,
379389.
Guang-Ming, Z., Lin, T., Guo-Li, S., Guo-He, H., Cheng-Gang, N., 2004.
Determination of trace chromium(VI) by an inhibition-based enzyme
biosensor incorporating an electro polymerized aniline membrane and
ferrocene as electron transfer mediator. Int. J. Environ. Sci. Eng. 84
(10), 761774.
Guerrieri, A., Monaci, L., Quinto, M., Palmisano, F., 2002. A disposable amperometric biosensor for rapid screening of anticholinesterase
activity in soil extracts. Analyst 127, 57.
Guilbault, G.G., Pravda, M., Kreuzer, M., 2004. Biosensors-42 years and
counting. Anal. Lett. 37, 1448114496.
Gulla, K.C., Gouda, M.D., Thakur, M.S., Karanth, N.G., 2002. Reactivation of immobilized acetylcholinesterase in an amperometric biosensor for organophosphorus pesticide. Biochim. Biophys. Acta 1597,
133139.
Han, S., Zhu, M., Yuan, Z., Li, X., 2001. A methylene blue-mediated
enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercuryglutathione complex. Biosens.
Bioelectron. 16, 916.
Hart, J.P., Crew, A., Crouch, E., Honeychurch, K.C., Pemberton, R.M.,
2004. Some recent designs and developments of screen-printed carbon
electrochemical sensors/biosensors for biomedical, environmental, and
industrial analyses. Anal. Lett. 37 (5), 789830.
1421
Ivanov, A., Evtugyn, G., Budnikov, H.C., Ricci, F., Moscone, D.,
Palleschi, G., 2003. Cholinesterase sensors based on screen-printed
electrode for detection of organophosphorus and carbamic pesticides.
Anal. Bioanal. Chem. 377, 624631.
Jeanty, G., Wojiciechowska, A., Marty, J.-L., 2002. Flow-injection amperometric determination of pesticides on the basis of their inhibition of
immobilized acetylcholinesterases of different origin. Anal. Bioanal.
Chem. 373, 691695.
Jeanty, G., Ghommidh, Gh., Marty, J.-L., 2001. Automated detection of
chlorpyrifos and its metabolites by a continuous flow system-based
enzyme sensor. Anal. Chim. Acta 436, 119128.
Joshi, K.A., Tang, J., Haddon, R., Wang, J., Chen, W., Mulchandani,
A., 2005. A disposaple biosensor for organophosphorus nerve agents
based on carbon nanotubes modified thick film strip electrode. Electroanalysis 17, 5458.
Kilinc, E., Ozsoz, M., Sadik, O.A., 2000. Electrochemical detection of
NO by inhibition on oxidase activity. Electroanalysis 12, 14671471.
Kitz, R., Wilson, P., 1962. Esters of methanesulfonic acid as irreversible
inhibitors of acetylcholinesterase. J. Biol. Chem. 237, 32453249.
Kok, F.N., Hasirci, V., 2004. Determination of binary pesticide mixtures
by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661665.
Kok, F.N., Bozoglu, F., Hasirci, V., 2002. Construction of an
acethylcholinesterase-choline oxidase biosensopr for aldicarb determination. Biosens. Bioelectron. 17, 531539.
Korpan, Y.I., Volotovsky, V.V., Martelet, C., Jaffrezic-Renault, N.,
Nazarenko, E.A., Elskaya, A.V., Soldatkin, A.P., 2002. A novel
enzyme biosensor for steroidal glycoalkaloidsdetection based on pHsensitive field effect field transistors. Bioelectrochemistry 55, 911.
Krawczynski vel Krawczyk, T., Mosszczynska, M., Trojanowicz, M.,
2000. Inhibitive determination of mercury and other metal ions
by potentiolmetric urea biosensor. Biosens. Bioelectron. 15, 681
691.
Krawezyk, T.K., Moszezynska, M., Trojanowiez, M., 2000. Inhibitive
determination of mercury and other metal ions by potentiometric urease biosensor. Biosens. Bioelectron. 15, 681691.
Kuswandi, B., 2003. Simple optical fibre biosensor based on immobilized enzyme for monitoring of trace heavy metal ions. Anal. Bioanal.
Chem. 376, 11041110.
Kuusk, E., Rinken, T., 2004. Transient phase calibration of tyrosinasebased carbaryl biosensor. Enzyme Microb. Technol. 234, 657661.
Lee, W.E., Thompson, H.G., Hall, J.G., Bader, D.E., 2000. Rapid
detection and identification of biological and chemical agents by
immunoassay, gene probe assay and enzyme inhibition using siliconbased biosensor. Biosens. Bioelectron. 14, 784804.
Lee, H.-S., Kim, Y., Chi, Y., Lee, T.Y., 2002. Oxidation of organophosphorus pesticides for the sensitive detection by cholinesterase-based
biosensor. Chemosphere 46, 571576.
Lee, H.-S., Kim, Y.-A., Chung, D.H., Lee, T.Y., 2001. Determination of
carbamate pesticides by a cholinesterase-based flow injection biosensor. Int. J. Food Sci. Technol. 36, 263269.
Lee, M.M., Russel, D.A., 2003. Novel determination of cadmium ions
using an enzyme self assembled monolayer with surface plasmon
resonance. Anal. Chim. Acta 500, 119125.
Luque de Castro, M.D., Herrera, M.C., 2003. Enzyme inhibition-based
biosensors and biosensing systems: questionable analytical devices.
Biosens. Bioelectron. 18, 279294.
Malitesta, C., Guascito, M.R., 2005. Heavy metal determination by
biosensors based on enzyme immobilised by electropolymerisation.
Mazzei, F., Botrè, F., Montilla, S., Pilloton, R., Podestà, E., Botrè, C.,
2004. Alkaline phosphatase inhibition based electrochemical sensors
for the detection of pesticides. J. Electroanal. Chem. 574, 95100.
Mohammadi, H., Amine, A., Cosnier, S., Mousty, C., 2005.
Mercuryenzyme inhibition assays with an amperometric sucrose
biosensor based on a trienzymaticclay matrix. Anal. Chim. Acta 543,
143149.
1422
Mohammadi, H., El Rhazi, M., Amine, A., Brett, A.M.O., Brett, C.M.A.,
2002. Determination of mercury(II) by invertase enzyme inhibition
coupled with batch injection analysis. Analyst 127, 10881093.
Montesinos, T., Perez-Munguia, S., Valdez, F., Marty, J.-L., 2001. Disposable cholinesterase biosensor for the detection of pesticides in
water-miscible organic solvents. Anal. Chim. Acta 431, 231237.
Morales, M.D., Morante, S., Escarpa, A., Gonzalez, M.C., Reviejo, A.J.,
Pingarron, J.M., 2002. Design of a composite amperometric enzyme
electrode for the control of the benzoic acid content in food. Talanta
57, 11891198.
Mourzina, I.G., Yoshinobu, T., Ermolenko, Y.E., Vlasov, Y.G., Schoning,
M.J., Iwasaki, H., 2004. Immobilization of urease and cholinesterase
on the surface of semiconductor transducer for the development of
light-addressable potentiometric sensors. Microchim. Acta 144, 4150.
Mulchandani, P., Chen, W., Mulchandani, A., 2001. Flow injection amperometric enzyme bioensor for direct dtermination of organophosphate
nerve agents. Environ. Sci. Technol. 35, 25622565.
Neufeld, T., Eshkenazi, I., Cohen, E., Rishpon, J., 2000. A micro flow
injection electrochemical biosenor for organophosphorus pesticides.
Nikolelis, D.P., Simantiraki, M.G., Siontorou, C.G., Toth, K., 2005. Flow
injection analysis of carbofuran in foods using air stable lipid film
based acetylcholinesterase biosensor. Anal. Chim. Acta 537, 169177.
Nistor, C., Rose, A., Farre, M., Stoica, L., Wollenberger, U., Ruzgas,
T., Pfeiffer, D., Barcelo, D., Gorton, L., Emneus, J., 2002. In-field
monitoring of cleaning efficiency in water treatment plants using two
phenol sensitive biosensors. Anal. Chim. Acta 456, 317.
Noguer, T., Balasoiu, A-M., Avramescu, A., Marty, J.-L., 2001. Development of disposable biosensor for the detection of metam-sodium and
its metabolite MITC. Anal. Lett. 34 (4), 513528.
Nunes, G.S., Jeanty, G., Marty, J.-L., 2004. Enzyme immobilization
procedures on screen printed electrodes used for the detection of anticholinesterase pesticides Comparative study. Anal. Chim. Acta 523,
107115.
Okazaki, S., Nakagawa, H., Fukuda, K., Asakura, S., Kiuchi, H.,
Shigemori, T., Tahakashi, S., 2000. Re-activation of an amperometric organophosphate pesticide biosensor by 2-pyridinealdoxime
methochloride. Sens. Actuators B 66, 131134.
Patel, P.D., 2002. (Bio)sensor for measurement of analytes implicated in
food safety: a review. Trends Anal. Chem. 21, 96115.
Pavlov, V., Xiao, Yi., Willner, I., 2005. Inhibition of the acetylcholine
esterase-stimulated growth of au nanoparticles: nanotechnology-based
sensing of nerve gases. Nano Lett. 5, 649653.
Pirvutoiu, S., Surugiu, I., Dey, E.S., Ciucu, A., Magearu, V., Danielsson,
B., 2001. Flow injection analysis of mercury(II) based on enzyme
inhibition and thermometric detection. Analyst 126, 16121616.
Pirvutoiu, S., Estera, D., Bhand, S., Ciucu, A., Magearu, V., Danielsson,
B., 2002. Application of the enzyme thermistor for determination of
mercury and other heavy metals using free and immobilised alcohol
oxidase. Roum. Biotechnol. Lett. 7, 975.
Pogacnik, L., Franko, M., 2003. Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor.
Rekha, K., Gouda, M.D., Thakur, M.S., Karanth, N.G., 2000. Ascorbate
oxidase based biosensor for organophosphorus pesticide monitoring.
Ricci, F., Arduini, F., Bourais, I., Moscone, D., Palleschi, G., Amine,
A., 2003. Thiocholine mediated oxidation at prussian blue modified
electrodes for pesticide and heavy metals detection. In: Proceedings
of the First International Workshop on Biosensors for food safety
Environmental Monitoring, Marrakech, Morocco (O-4, abstract book).
Rodriguez, B.B., Bolbot, J.A., Tothill, I.E., 2004a. Development of urease
and glutamic dehydrogenase amperometric assay for heavy metals
screening in polluted samples. Biosens. Bioelectron. 19, 11571167.
Rodriguez, B.B., Bolbot, J.A., Tothill, I.E., 2004b. Ureaseglutamic dehydrogenase biosensor for screening heavy metals in water and soil
samples. Anal. Bioanal. Chem. 380, 284292.
Sacks, V., Eshkenazi, I., Neufeld, T., Dosoretz, C., Rishpon, J.,
2000. Immobilized parathion hydrolase: an amperometric sensor for
parathion. Anal. Chem. 72, 20552058.
Saritha, K., Kumar, N.V.N., 2001. Qualitative detection of selenium in fortified soil and water samples by a paper chromatographiccarboxyl
esterase enzyme inhibition technique. J. Chromatogr. A 919, 223
228.
Shan, D., Mousty, C., Cosnier, S., 2004. Subnanomolar cyanide detection at polyphenol oxidase/clay biosensors. Anal. Chem. 76, 178
183.
Schulze, H., Vorlova, S., Villatte, F., Bachmmann, T.T., Schmid, R.D.,
2003. Design of acetylcholinesterases for biosensor applications.
Schulze, H., Schmid, R.D., Bachmann, T.T., 2002. Rapid detection of
neurotoxic insecticides in food using disposable acetylcholinesterasebiosensors and simple solvent extraction. Anal. Bioanal. Chem. 372,
268272.
Segel, I.H., 1976. In Biochemical Calculation: How to Solve Mathematical Problems in General Biochemistry, 2nd ed. Wiley, New York, pp.
208223.
Sezginturk, M.K., Goktug, T., Dincka, E., in press. A biosensor based
on catalase for determination of highly toxic chemical azide in fruit
juices. Biosens. Bioelectron., in press (corrected proof). Available
online: February 12, 2005.
Simonian, A.L., Good, T.A., Wang, S.-S., Wild, J.R., 2005. Nanoparticlebased optical biosensors for the direct detection of organophosphate
chemical warfare agent and pesticides. Anal. Chim. Acta 534, 69
77.
Simonian, A.L., Flounders, A.W., Wild, J.R., 2004. FET-based biosensors
for the direct detection of organophosphate neurotoxins. Electroanalysis 16, 18961906.
Simonian, A.L., Efremenko, E.N., Wild, J.R., 2001. Discriminative detection of neurotoxins in multi-component samples. Anal. Chim. Acta
444, 179186.
Sole, L.S., Merkoci, A.I., Alegret, S., 2003. Determination of toxic substance based on enzyme inhibition. Part I. Electrochemical biosensors
for the determination of pesticides using batch procedures. Anal.
Chem. 33, 89126.
Soldatkin, A.P., Volotovsky, V., Elskaya, A.V., Jaffrezic-Renault, N.,
Martelet, C., 2000. Improvement of urease based biosensor characteristics using additional layers of charged polymers. Anal. Chim. Acta
403, 2529.
Sotiropoulou, S., Chaniotakis, N.A., 2005. Lowering the detection limit of
the acethylcholinesterase biosensor using a nanoporous carbon matrix.
Anal. Chim. Acta 530, 199204.
Sotiropoulou, S., Fournier, D., Chaniotakis, N.A., 2005. Genetically engineered acethylcholinesterase-based biosensor for attomolar detection
of dichlorvos. Biosens. Bioelectron. 20, 23472352.
Stoytcheva, M., 2002. Electrochemical evaluation of the kinetic parameters of heterogeneous enzyme reaction in presence of metal ions.
Electroanalysis 14, 923927.
Suprun, E.V., Budnikov, H.C., Evtugyn, G.A., Brainina, Kh.Z., 2004. Bienzyme sensor based on thick-film carbon electrode modified with
electropolymerized tyramine. Bioelectrochemistry 63, 281284.
Suwansa-ard, S., Kanatharana, P., Asawatreratanakul, P., Limsakul, C.,
Wongkittisuksa, B., Thavarungkul, P., 2005. Semi disposable reactor biosensors for detecting carbamate pesticides in water. Biosens.
Tsai, H.-C., Doong, R.-A., 2005. Simultaneous determination of pH, urea,
acetylcholine and heavy metals using array-based enzymatic optical
biosensor. Biosens. Bioelectron. 20, 17961804.
Tran-Minh, C., 1985. Immobilized enzyme probes for determining
inhibitors. Ion Select. Electr. Rev. 7, 4175.
Trojanowicz, M., Compagnone, D., Goncalves, C., Joca, Z., Palleschi, G.,
2004. Limitations in the analytical use of invertase inhibition for the
screening of trace mercury content in environmental samples. Anal.
Sci. 20, 899904.

Trojanowicz, M., 2002. Determination of pesticides using electrochemical
enzymatic biosensors. Electroanalysis 14, 13111328.
Turdean, G.L., Popescu, I.C., Oniciu, L., Thevenot, D.R., 2002. Sensitive detection of organophosphorus pesticides using a needle type
amperometric acetuylcholinesterase-based bioerlectrode. Thiocholine
electrochemistry and immobilised enzyme inhibition. J. Enzyme Inhib.
Med. Chem. 17 (2), 107115.
Vedrine, C., Fabiano, S., Canh, T.-M., 2003. Amperometric tyrosinase
based biosensor using an electrogenerated polythiophene film as an
entrapment support. Talanta 59, 535544.
Villatte, F., Schulze, H., Schmid, R.D., Bachmann, T.T., 2002. A disposable acetylcholinesterase-based electrode biosensor to detect anatoxina(s) in water. Anal. Bioanal. Chem. 372, 322326.
Wan, K., Chovelon, J.M., Jaffrezic-Renault, N., 2000. Enzymeoctadecylamine LangmirBlodgett membranes for ENFET biosensors.
Talanta 52, 663670.
Wang, J., Krause, R., Block, K., Musameh, M., Mulchandani,
A., Mulchandani, P., Chen, W., Schoning, M.J., 2002. Dual
amperometricpotentiometric biosensor detection system for monitoring organophosphorus neurotoxins. Anal. Chim. Acta 469, 197203.
Wang, J., Krause, R., Block, K., Musameh, M., Mulchandani, A., Schoning, M.J., 2003. Flow injection amperometric detection of OP nerve
1423
agents based on an organophosphorus-hydrolase biosensor detector.

White, B.J., Harmon, H.J., 2005. Optical solid-state detection of
organophosphates using organophosphorus hydrolase. Biosens. Bioelectron. 20, 19771983.
Wilkins, E., Carter, M., Voss, J., Ivnitski, D., 2000. A quantitative determination of organophosphate pesticides in organic solvents. Electrochem.
Commun. 2, 786790.
Xavier, M.P., Vallejo, B., Marazuela, M.D., Moreno-Bondi, M.C., Baldini,
F., Falia, A., 2000. Fiber optic monitoring of carbamate pesticides
using porous glass with covalently bound chlorophenol red. Biosens.
Young, S.J., Hart, J.P., Dowman, A.A., Cowell, D.C., 2001. The nonspecific inhibition of enzymes by environmental pollutants: a study
of model system toward the development of electrochemical biosensor
arrays. Biosens. Bioelectron. 16, 887894.
Zhang, Y., Muench, S.B., Schulze, H., Perz, R., Yang, B., Schmid, R.D.,
Bachmann, T.T., 2005. Disposable biosensor test for organophosphate
and carbamate insecticides in milk. Agric. Food Chem. (available on
line).
Zhang, S., Zhao, H., John, R., 2001. A theoretical model for immobilized
enzyme inhibition biosensors. Electroanalysis 13, 15281534.

Enzyme Inhibition-Based Biosensors For Food Safety and Environmental Monitoring

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Enzyme Inhibition-Based Biosensors For Food Safety and Environmental Monitoring

Uploaded by

Copyright:

Available Formats

Biosensors and Bioelectronics 21 (2006) 14051423

Enzyme inhibition-based biosensors for food safety

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

4.2. Heavy metal inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ronmental, and industrial analyses have been reviewed by

2. Theoretical and practical considerations

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

control of the response), the sensitivity of the immobilized

The enzyme was trapped on the electrode surface during an

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

with thinner coatings. In another paper, Ciucu et al. (2003)

deformed and its function is thus impaired. In this case the

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

phosphorylated cholinesterases, and can be accelerated by

d[E]/[E] = kobs dt,

Fig. 1. General method to establish LOD for enzyme inhibition assays.

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

some biosensors can be regenerated after inhibition by use of

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

bition level did not depend on the pH of the solution studied

3. Inhibition determination in organic phases

reported the influence of acetonitrile, ethanol and DMSO

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

4. Inhibitor investigation using enzyme-based

limit obtained using OPH was not very low compared to

Method based on competitive

Real water sample

0.50.7 nmol l1 , LOD = 0.5 nmol l1

Orange juices, peach

160 g l1 , LOD = 2 g kg1

Covalent linkage between

Simonian et al. (2005)

Joshi et al. (2005)

Schulze et al. (2002)

LOD(I10) = 2 109 mol l1

Extraction of inhibitors with

Andreescu et al. (2003)

Boni et al. (2004)

Choi et al. (2001)

Zhang et al. (2001)

080 nmol l1 (batch), 015 mol l1

Zhang et al. (2005)

Simonian et al. (2001)

Retention with dialysis

Recovery of paraoxon and

Ciucu et al. (2003)

Andreescu et al. (2002b)

White and Harmon (2005)

Change of the absorbance of

Suprun et al. (2004)

Dzyadevych et al. (2005)

3.0 1011 , 5.0 107 , 5.0 106 ,

Reactivation with 2-PAM

Dzyadevych et al. (2003)

Amperometric and FIA

Spiked river water

0.60.8, 200.8, 0.61.2 mol l1

Reactivation with 2-PAM solution

Jeanty et al. (2002)

Grape and vine

Single use of SPEs, analysis in

Del Carlo et al. (2002)

Suwansa-ard et al. (2005)