ev food

7/29/2019 ev food

1/19

Biosensors and Bioelectronics 21 (2006) 14051423

Review

Enzyme inhibition-based biosensors for food safetyand environmental monitoring

Aziz Amine a,, Hasna Mohammadi a, Ilhame Bourais a, Giuseppe Palleschi ba Laboratoire des Analyses Chimiques et des Biocapteurs, Facult e 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 2005Available online 25 August 2005

Abstract

Analytical technology based on sensors is an extremely broad eld which impacts on many major industrial sectors such as the pharma-ceutical, healthcare, food, and agriculture industries as well as environmental monitoring. This review will highlight the research carried outduring the last 5 years on biosensors that are based on enzyme inhibition for determination of pollutants and toxic compounds in a widerange of samples. Here the different enzymes implicated in the inhibition, different transducers forming the sensing devices, and the differentcontaminants analyzed are considered.

The general application of the various biosensors developed, with emphasis on food and environmental applications, is reviewed as well asthe 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14062. Theoretical and practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406

2.1. Principle of the enzyme-based biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14062.2. Enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14072.3. Enzymeinhibitor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408

2.3.1. Reversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14082.3.2. Irreversible inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1408

2.4. Limit of detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14092.5. Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14102.6. Parameters generally affecting the performance of enzymatic biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1410

2.6.1. Effect of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14102.7. Effect of substrate concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14112.8. Effect of enzyme concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1411

3. Inhibition determination in organic phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14114. Inhibitor investigation using enzyme-based biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412

4.1. Pesticide inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412

Corresponding author. E-mail addresses: [email protected], [email protected]

(A. Amine).

0956-5663/$ see front matter 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2005.07.012

7/29/2019 ev food

2/19

1406 A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423

4.2. Heavy metal inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14154.3. Other inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1417

5. Food and environmental applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14176. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14197. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1420

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1420References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1420

1. Introduction

Because of their exceptional performance capabilities,which include high specicity and sensitivity, rapidresponse,low cost, relatively compact size anduser-friendly operation,biosensors have become an important tool for detection of chemical and biological components for clinical, food andenvironmental monitoring. While electrochemical transduc-ers combined with an enzyme as the biochemical compo-nent form the largest category, those biosensing systems thatspecically depend on inhibition can be divided into threecategories:

Biosensors based on the immobilization of whole cellsused 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 andrender the regeneration of the enzyme easier. However,such biosensors may suffer from side reactions due to thecoexistence of several enzymes.

Sensor devices coupled with reactors which contain animmobilized enzyme matrix. The inhibitor passes throughthe reactor and inhibits the enzyme ( Lee et al., 2002 ). Theresidual activity of the enzyme is evaluated by measuringthe enzymatic product before and after the inhibition.

Biosensors based on direct enzyme immobilization on atransducer device. The enzyme and transducer elementsare in close contact with each other and incorporated ina single unit. Some biosensors based on enzyme inhi-bition 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 sev-eral recent reviews. Biosensors based on ion sensitive eldeffect transistors (ISFETs) for the determination of somesubstrates and inhibitors were reviewed by Dzyadevychet al. (2003) . Luque de Castro and Herrera (2003 ) havediscussed the inhibition-based biosensorsand biosensingsys-tems. Other authors have reviewed theuseof electrochemicalenzyme-based biosensors for the determination of pesticides(Trojanowicz, 2002; Sol e et al., 2003). Patel reported somespecic applications of inhibition-based biosensors in thearea of chemical and microbiological contaminant analysisimplicated in food safety ( Patel, 2002 ). Finally, some recentdesigns and developments relating to screen-printed carbonelectrochemical sensors and biosensors for biomedical, envi-

ronmental, and industrial analyses have been reviewed byHart et al. (2004) .

In this review, we specically provide an overview of theactivity carried out since 2000 relative to biosensor systemswhich use an enzyme for inhibition-based analysis for foodand environment safety. We also report the results from somestudies in which the inhibited target enzyme is in solutionwhile its product is detected using an enzyme-based biosen-sor.

Biosensors based on the principle of enzyme inhibitionhave by now been applied for a wide range of signi-cant analytes such as organophosphorous pesticide (OP),organochlorine pesticides, derivatives of insecticides, heavymetals and glycoalkaloids. The choice of enzyme/analytesystem is based on the fact that these toxic analytes inhibitnormalenzyme function.In general, thedevelopmentof thesebiosensing systems relies on a quantitative measurement of the enzyme activity before and after exposure to a target ana-lyte. Typically the percentage of inhibited enzyme ( I %) thatresults after exposure to the inhibitor is quantitatively relatedto the inhibitor (i.e. analyte) concentration and the incubation

time (Guerrieri et al., 2002; Ivanov et al., 2003 ). Conse-quently, the residual enzyme activity is inversely related tothe inhibitor concentration.

2. Theoretical and practical considerations

2.1. Principle of the enzyme-based biosensor

Biosensors are analytical devices which tightly combinebiorecognition elements and physical transducers for detec-tion of the target compounds. In enzyme-based biosensors,the biological element is the enzyme which reacts selectivelywith its substrate ( Guilbault et al., 2004 ).

It is well known that the response of a biosensor to theaddition of a substrate is determined by the concentration of theproduct ( P ) of the enzymatic reaction on the surface of thesensor. Thereaction is controlled by therateof twosimultane-ous 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 substrateconcentration is not totally compensated by the transfer fromthe bulk solution due to the diffusion limitation, and becauseof this, only a fractionof theenzyme active centers is involvedin the interaction with a substrate. In this case (diffusion

7/29/2019 ev food

3/19

A. Amine et al. / Biosensors and Bioelectronics 21 (2006) 14051423 1407

control of the response), the sensitivity of the immobilizedenzyme toward inactivation, be it heating or inhibitor effect,is lower than that for the enzyme used in solution.

Given that pollutant compounds selectively inhibit theactivity of certain enzymes, their activity and the resultingproduct concentration is affected. This inhibition is analyti-

cally useful and has been used advantageously in the fabrica-tion of many biosensing devices. First we will summariseimmobilization techniques and their effects on biosensorfunction. Then general schemes for the various possible inhi-bition processes that have been effectively used in biosensorswill be described.

2.2. Enzyme immobilization

The development of biosensors based on immobilizedenzymes came about to solve several problems such as lossof enzyme (especially if expensive), maintenance of enzymestability and shelf life of the biosensor, and additionally toreduce the time of the enzymatic response and offer dispos-able devices which can be easily used in stationary or in owsystems. To do this, several immobilization techniques havebeen investigated. These techniques include physical entrap-ment, microencapsulation, adsorption, covalent binding andcovalent cross-linking, and several different approaches toenzyme immobilization have been reported in the literature.Acetylcholinesterase (AChE) was encapsulated in solgellm on a glass cap that could be xed on an optical ber(Doong and Tsai, 2001 ). Solgel lms have been formedusing enzymatic solutions mixed with different uorescentindicators. The design of such biosensors takes advantage of

the ability to entrap large amounts of enzyme and enhancethermal and chemical stability; the techniques offer simplic-ity of preparationwithout covalentmodication, exibility incontrolling pore size, and geometry and minimal quenchingof uorescent reagents.

In another strategy, AChE was co-immobilised withcholine oxidase (ChO) onto a Pt surface using a solutionof glutaraldehyde. The activity of immobilized enzymeswas evaluated in the presence of dimethyl-2,2-dichlorovynylphosphate pesticide (DDVP). The cross-linking involvingglutaraldehyde signicantly increased the attachment of theenzyme to the transducer and thus, the electron exchangescould occur more directly.

Urease has been entrapped in both polyvinyl chloride(PVC) and cellulose triacetate (CTA) layers on the surface of pH-sensitiveiridium oxideelectrodes andusedfordetermina-tion of mercury. Gulla et al. (2002) reported the immobiliza-tion of AChE in nylon net using glutaraldehyde. The enzymelayer was sandwiched between two cellophane membranesandkeptin closecontact with a gold electrode. This methodof AChE immobilization permits the removal of the enzymaticmembrane, the enzymatic reactivation (by TMB-4), and thereuse of reactivated membrane. The immobilization of theenzyme polyphenol oxidase (PPO) during the anodic elec-tropolymerisation of polypyrrole (PPy) was also reported.

The enzyme was trapped on the electrode surface during anelectrochemical synthesis process ( El Kaoutit et al., 2004 ).This biosensor was used for the evaluation of atrazine andprovidesa rapid andtechnicallysimplesystem fordetermina-tion at concentrations below the ppm level. Sotiropoulou andChaniotakis (2005) have used a nanoporous carbonmatrix for

acetylcholinesterase immobilization and stabilization. Theyreported that the use of this activated carbon matrix providedboth signicant enzyme stabilization and a lowering of thedetection limit. Using this biosensor the monitoring of theorganophosphorus pesticide dichlorvos at picomolar levelswas achieved; calculated on the basis of 20% inhibition, theycould detect 10 12 moll 1 pesticide which was a level 1000times lower than for other systems reported so far. Recently,using nanoporous conductive carbon for immobilization of 0.02pmol of very sensitive acetylcholinesterase from thedouble mutant E69Y, Y71D of Drosophila melanogaster ,Sotiropoulou et al. (2005) were able to detect dichlorvos atattomolar levels, 10 17 M with 40% inhibition. It was shownthat in comparison with AChE from E. electricus (electriceel), theuseof doublemutantAChE (E69Y, Y71D) produceda drastic increase of the inhibition constant, K i , value for thedichlorvos pesticide.

Malitesta and Guascito (2005) have described the appli-cation of biosensors based on glucose oxidase immo-bilized by electropolymerisation for heavy metal deter-mination; the investigated enzymatic inhibition appearsreversible and in agreement with the data reported forthe enzyme in solution. A comparison of several acetyl-cholinesterase immobilization procedures carried out on the7,7,8,8-tetracyanoquinonediaminomethane (TCNQ) modi-

ed graphite working electrode was presented by Nuneset al. (2004) . The enzyme immobilization through photopoly-merization with polyvinyl alcohol bearing styrylpyridiniumgroups (PVA-SbQ) produced good results, fast response,good reproducibility, wide working range for pesticides andexcellent sensitivity to N -methylcarbamates. Ivanov et al.(2003) evaluated thedetection limitand the sensitivity towardsome pesticides using different modied sccreen-printedelectrodes. In comparison with the use of unmodied trans-ducers, they concluded that the modication of the sensorsurface with 7,7,8,8-tetracyanoquinodimethane (TCNQ) is apowerful tool for the improvement of biosensorperformance.

According to reports in the literature, the performance of a biosensor device is strongly dependent on its conguration.In a paper that reported the immobilization of enzymes withclay, the inuence of the enzyme/clay ratio and the amountof adsorbed coating on cyanide sensing by polyphenol oxi-dase was investigated ( Shan et al., 2004 ). It wasreported that,when the enzyme/clay ratio was decreased from 1 to 0.125,the sensitivity of the biosensor decreased sharply from 9130to 0.5mA M 1 cm 2 and the detection limit increased from0.1 nM to 50 M. In the same paper, the inuence of theamount of deposited coating and hence the thickness of theclay lm on the biosensor performance was studied. Highersensitivity to cyanide anda lowerdetection limit areobserved

7/29/2019 ev food

4/19


with thinner coatings. In another paper, Ciucu et al. (2003)have conrmed that a lower concentration of paraoxon couldbemeasuredusinga membranewith a loweramountof immo-bilized AChE.

It has been shown that the sensitivity of enzymes towardheavy metal ions is a function of enzyme loading ( Soldatkin

et al., 2000). These authors have demonstrated that ureaseimmobilization under a negatively charged polymer inducesan increase of the inhibition effect of the heavy metal ionsdue to cation accumulation in the polymeric matrix. A lowerconcentration of invertase (2 U) in a tri-enzymatic biosen-sor matrix resulted in a signicant increase of sensitivity,as well as in a decrease of the detection limit of mer-cury (Mohammadi et al., 2005 ). These results conrm thoseobtained with the free enzyme, i.e. that a higherpercentage of inhibition is oftenobservedwith lower enzyme concentration(Evtugyn et al., 1998; Mohammadi et al., 2002 , Sotiropoulouet al., 2005) and thus a low enzyme concentration should ingeneral enhance sensitivity to the inhibitor.

On the basic of the numerous studies reported before, theenzyme immobilization is one of the most important stepsinvolved in the biosensor design. The choice of the techniqueusedfor connectingthebiologicalcomponent(enzyme)to thetransducer is crucial, since the stability, the longevity and thesensitivity largely depend on enzymatic layer conguration.

2.3. Enzymeinhibitor system

The enzymeinhibitor reaction is often complex. Theparagraphbelow reports different inhibitionmechanismsthatresult from the interaction between the enzyme and the toxic

compoundinvolved, reversible andirreversible inhibitionandtheir respective mechanisms are considered. For each mech-anism of inhibition, an example of a biosensor based on thistype interaction is also reported.

2.3.1. Reversible inhibitionThe 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 irre-versible inactivation of the enzyme. Inhibitors structurallyrelated to the substrate may be bound to the enzyme activecenter and compete with the substrate (competitive inhibi-

tion). Dzyadevych et al. (2004b) reported that the glucoal-kaloids are competitive inhibitors of BChE. Also Moraleset al. (2002) showed a competitive inhibition of tyrosinaseby benzoic acid:

(1)

If the inhibitor is not only bound to the enzyme but also tothe enzymesubstrate complex, the active center is usually

deformed and its function is thus impaired. In this case thesubstrate and the inhibitor do not compete with each other(non-competitive inhibition). The inhibition of horseradishperoxidase was apparently reversible and non-competitive inthe presence of HgCl 2 for less than 8 s incubation time ( Hanet al., 2001 ). Cyanide showednon-competitive inhibitionver-

sus polyphenol oxidase ( Shan et al., 2004 ). The inhibitionof immobilized acetylcholinesterase with metal ions (Cu 2+ ,Cd2+ , Fe3+ , Mn2+) has a reversible and a non-competitivecharacter ( Stoytcheva, 2002 ):

(2)

Competitive and non-competitive inhibitions affect theenzyme kinetics differently ( Segel, 1976 ). A competitive

inhibitor does not change V max but increases the K M ; onthe contrary a , non-competitive inhibition results in anunchanged K M and in a decrease of V max .

In the case of mixed inhibition, the inhibitor binds theenzyme and the enzymesubstrate complex with a differentafnity. Malitesta and Guascito (2005) demonstrated that theinhibition mechanism of glucose oxidase by heavy metals isreversible and mixed:

(3)

For uncompetitive inhibition, the inhibitor binds only whenthe 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 analo-gous to that usually considered for uncompetitive inhibition:

(4)Dixons plot ( Segel, 1976 ) is often used for the evaluationof the inhibition constant and for the differentiation betweendifferent types of inhibition.

2.3.2. Irreversible inhibitionFor irreversible inhibitors, the enzymeinhibitor interac-

tion results in the formation of a covalent bond between theenzyme active center and the inhibitor. The term irreversiblemeans that the decomposition of the enzymeinhibitor com-plex results in the destruction of enzyme, e.g. its hydrolysis,oxidation, etc. This process usually proceeds stepwise, as for

7/29/2019 ev food

5/19


phosphorylated cholinesterases, and can be accelerated byparticular reagents:

(5)

The kinetics of the inhibition depend strongly on the biosen-sor conguration. In the case of a thin enzymatic layer, thekinetics observed are similar to that of the enzyme in solu-tion. For native enzymes the inhibition is related directly tothe 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 investigatedan interesting casecon-

cerning the inhibition of peroxidase. There is an early phaseof reversible inhibition (5 s) followed by irreversible inhibi-tion. However, since the reversible inhibition lasted for just afew seconds, it was difcult to carry out the measurement of residual activity within that interval. Therefore, irreversibleinhibition has to be dealt with in cases where longer incuba-tion times must be used. In these cases the measurement of the residual enzyme activity resulted in a log linear with theincubation time. Neufeld et al. (2000) , Guerrieri et al. (2002)have reported that the degree of inhibition also depends onthe inhibitor concentration and exposure time (at a dened

pH value and at inhibitor concentration which is in excesswith respect to enzyme).There have been some initial attempts at the develop-

ment and experimental verication of theoretical modelsfor the inhibition of immobilized enzymes used for biosen-sors. When diffusion phenomenon are taken into account, themodel predicts that the percentage of enzyme inhibition (%),after exposure to an inhibitor, is linearly related to both theinhibitor concentration [ I ] and the square root of incubationtime (t 1/2) (Zhang et al., 2001 ).

2.4. Limit of detection

Thedetermination of the inhibitory effect includes the fol-lowing steps: the determination of initial enzymatic activity,the incubation of a biosensor in a solution that contains aninhibitor, and nally the measurement of the residual activity(activity after exposure of the biosensor to the inhibitor). Thelimit of detection (LOD) has been dened as the concentra-tion of the species being measured which gives a minimumdetectable difference signal (reduction in activity) that isequal to2 or3 standarddeviations(S.D.)of themeanresponseof the blank samples (zero concentration of the inhibitor).This simple approach, although widely reported in the liter-ature (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 thecondenceinterval of theinhibitor. Thetruevalue ofLODcanbe denedas theconcentration of the inhibitorwhere thecon-denceinterval does notoverlapthatof thezeroconcentrationof the inhibitor standard. This is shown diagrammatically inFig. 1. Any concentration above the LOD value has a 95%(2S.D.) or 99% (3S.D.) probability of being a true positiveresult. The LOD value generally corresponds to 9080% of residual activity, that is 1020% inhibition.

Kuswandi (2003) has developed a simple optical biosen-sor based on immobilized urease for the monitoring of heavy

metals. He conrmed that the detection limit depends on theincubation time of the enzyme with the inhibitor; the opti-mum time of inhibition selected was 6 min. In other work,the residual enzymatic activity was also studied using differ-ent incubation times (5, 15, 30 min) with the AChE and ChObienzymatic system ( Kok et al., 2002 ). The degree of theenzyme inhibition increased with increase of the incubationperiod until reaching a plateau in 1530 min. The decrease inthe enzyme activity could be detected after 5 min, and there-fore theincubation time selectedwas 5 min, andthe biosensorcould detect 10 ppb aldicarb which gave a 10% inhibition of the initial acethylcholinesterase activity. In another work thesame inhibition study has been performed using an incuba-tion time of 30 min ( Ciucu et al., 2003 ). In this case, thedetection of paraoxon at 10 nM has been achieved. Shanet al. (2004) have highlighted specic electrostatic interac-tion of the hostmatrix thatmay induceanaccumulationof theinhibitor within the anionic clay. Thisphenomenonimprovedthe sensitivity of the amperometric biosensor toward cyanide(0.1 nM).

According to the literature data, it has shown that thelimit of the detection of the different developed biosensorsdepends on the several parameters such as pH, temperature,theenzymeloading (in thecase of irreversible inhibition), thesubstrate concentration (in the case of competitive reversible

7/29/2019 ev food

6/19


inhibition), the immobilization matrix and the time of reac-tion between the enzyme and the inhibitor. Thus, direct com-parison of the sensitivity between the different biosensorsbased on enzyme immobilization is not easy and should takeinto consideration the cited parameters.

2.5. Regeneration

Understanding the mechanisms of inhibition and regen-eration of enzymes is a general problem of great impor-tance for many biochemists and biotechnologists, especiallywhen using immobilized enzymes. The mode of analyteinhibition of enzymes such as peroxidase, tyrosinase andcatalase can occur through blocking of the active sites of these enzymes due to complex formation with copper cofac-tors and blocking of the electron transfer chain. OPs inhibitacetylcholinesterase (AChE) by blocking the serine in theactive site through nucleophilic attack to produce a ser-ine phosphoester (via phosphorylation) ( Simonian et al.,2001). In any case, the strong inhibition of the enzymes canpresent a serious problem for practical applications by lim-iting the reuse of biosensors. To overcome the problem of irreversible enzyme inhibition in the application of AChE-based biosensors, reactivation by oximes was investigated(Gulla et al., 2002 ). Further,another phenomenon observed isthat of permanent inhibition when the phoshorylated (inhib-ited) enzyme is left for a period of time without exposingit 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 hydrolysisand reactivation with oxime so that it becomes permanently

inhibited.Reactivation of inhibited AChE was investigatedusing pyridine-2-aldoxime methyliodide (2-PAM) and 4-formylpyridinium bromide dioxime (TMB-4). TMB-4 wasfound to be a more efcient reactivator with repeated use,retaining more than 60% of initial activity after 11 reuses,whereas in the case of 2-PAM, the activity retention droppedto less than 50% after only six reuses. The effect of ageingon the enzyme activity retained has been studied by Gullaet al. (2002) . It was found that this effect sets in after 15 minwhen the inhibited enzyme is left without reactivation andincreases with time elapsed before exposure of the biosensorto the reactivator (2-PAM and TMB-4). Thus, it was recom-mended that the pesticide-treated enzyme should be reacti-vatedwithin10 minto achieve themaximum reactivation andalso to ensure a maximum number of reuses of the immobi-lized enzyme membrane. This study has demonstrated thatTMB-4 is much more effective agent for reactivation of theimmobilized AChE enzyme for biosensor applications. Inanother case of acetylcholinesterase inhibition, 0.4mmol l 1

sodium uoride (NaF) was successfully used for 10 minfor the reactivation of an inhibited biosensor ( Kok et al.,2002).

Heavy metals act by the binding of the metal salts to pro-tein thiol groups. Depending on the sensing enzyme used,

some biosensors canbe regenerated after inhibition by use of a metal chelating agent, such as EDTA or thiols ( Evtugynet al., 1998 ). Mohammadi et al. (2005) tried to regener-ate a 50% mercury-inhibited invertase biosensor by soakingin a cysteine solution; the recovery was 30% of the initialbiosensor signal. They also tried to regenerate this biosensor

with EDTA solution. Unfortunately, no reactivation has beennoted. However, a full and rapid restoration of response hasbeen recently obtained by treatment of Hg 2+-inhibited glu-cose oxidase biosensor with EDTA solution ( Malitesta andGuascito, 2005 ).

Itwas also seen that if exposure timewas short enough, theenzymaticactivitycouldbe recovered without usingreactiva-tors even in the case of inhibition by a high concentration of pollutant ( Okazaki et al., 2000 ). Such effectively reversibleinhibition can prove an advantage in the analysis by owinjectionwhere the inhibitor is elutedby simpleushing withan electrolyte or a buffer solution ( Shan et al., 2004 ). In sum-mary, for irreversible inhibition, the damaged enzyme couldoften be reactivated using specic regenerating reagents. If the reactivation achieved is not sufcient, the biosensorswould have to be assembled for single use, in which casescreen-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. Itis well known that the enzyme activity is highly pH depen-dent and the optimum pH for an enzymatic assay must bedetermined empirically. It is best to choose a plateau regionso that the pH should not have any effect on enzyme activ-ity and will not interfere with the results obtained relativeto the inhibition of the enzyme by the inhibitor. The activityof the immobilized acetylcholinesterase as a function of pHhas been studied between pH 2 and 9 by Stoytcheva (2002) .She has reported a decrease in the activity of approximately70% at pH 2 compared to of that at pH 7. Mohammadi et al.(2005) investigated theeffect of pH of a tri-enzymatic biosen-sor in which the optimum pHof the three enzymes isdifferent(invertase pH, 4.5; glucose oxidase, pH 5.5; mutarotase, pH7.4). The pH effect on the biosensor response was analyzedbetween pH 4 and 8 and the highest activity was found at pH6.0. In order to improve the selectivity of the invertase towardmercury and to avoid silver interference, a medium exchangetechniquehasbeen carried out. Thebiosensorwas exposed tomercury in an acetate buffer solution at pH 4 while the resid-ual activity was evaluated with phosphate buffer solution atpH 6. Dzyadevych et al. (2004b) have studied the inuenceof pH on the analytical performance of a BChE modied pH-SFET biosensor for tomatine. In this study the best responsewas obtained for a buffer solution at pH 7.2, whereas the inhi-

7/29/2019 ev food

7/19


bition level did not depend on the pH of the solution studiedin the range 6.08.5.

2.7. Effect of substrate concentration

Thesubstrateconcentration canaffect thedegree of inhibi-

tion. Kok et al. (2002) concluded that the inhibition level (%)increases with increasing of the substrate concentration andthey have worked with a saturating substrate concentrationin the case of pesticide inhibition. Joshi et al. (2005) haveused a concentration of acetylhtiocholine two times higherthan the apparent K M for the determination of the maximumactivity of AChE before and after the inhibition by the OPparaoxon which was selected as model pesticide. In the caseof competitive inhibition, at high substrate concentrations,the inhibition effect is not observed since the substrate com-petes with the inhibitor. Dzyadevych et al. showed that thesensitivity of a BuChE biosensor toward tomatine decreaseswith an increase in the substrate concentration ( Dzyadevychet al., 2004a ).

2.8. Effect of enzyme concentration

The highest sensitivity to inhibitors was found for a mem-brane containing low enzyme loading ( Shan et al., 2004;Mohammadi et al., 2005; Sotiropoulou and Chaniotakis,2005; Sotiropoulou et al., 2005 ). In order to optimize theamperometric biosensor, Ciucu et al. (2003) studied a setof ve membranes with different amounts of AChE; theresponse of the biosensors decreases with a decrease of theenzyme concentration. The lowest concentration of paraoxon

detected (10 8

moll 1

) was achieved by using a membranewith 0.1IU/cm 2 immobilized AChE. The detection limitof pesticide equal to 10 17 M was achieved with very lowconcentration (0.02pmol)of engineeredacethylcholinesterseenzyme ( Sotiropoulou et al., 2005 ).

3. Inhibition determination in organic phases

The detection of inhibitors (pesticides, heavy metals) isgenerally performed in aqueous solution. However, thesecompounds are generally characterized by a low solubility inwater and a high solubility in organic solvent. Extraction andconcentration of pesticides or heavy metal from solid matri-ces (fruits, vegetables, sh, etc.) are thus commonly carriedout in such solvents.

Depending on the nature and the amount of organic sol-vent involved, the enzyme can be strongly inactivated whenexperiments are performed in these media ( Amine et al.,2004). Thus, the choice of organic solvent needs to be con-sidered as part of the method development in order to avoidundesirable effects. The effects of organic solvents havebeen shown to be quite variable and depend on the con-guration in which the enzyme is employed. For example,Montesinos et al. (2001) and Andreescu et al. (2002b) have

reported the inuence of acetonitrile, ethanol and DMSOon a cholinesterase sensor using acetylthiocholine as sub-strate. An increase of the output current was noticed whenworking in 5% acetonitrile and 10% ethanol, resulting frompartial deactivation of the enzyme. The detection of dichlor-vos,diazinon andfenthion in thepresenceof ethanol hasbeen

reported using a screen-printed biosensor based on immobi-lizedAChE( Andreescuet al., 2002b ). Thisallowed thedetec-tion of 1.91 10 8 and 1.24 10 9 moll 1 of paraoxonand chlorpyrifos ethyl oxon, respectively, in the presenceof 5% acetonitrile. The presence of 5% acetonitrile neitherenhanced the enzyme activity nor affected the detection limitand the selectivity of the AChE biosensor toward insecti-cides. In another study using organic extracts, Del Carloet al. (2002) have followedthe trend of chloropyrifos in grapesamples.Thegrape solvent extractswere analyzed using bothgas chromatography and electrochemical bioassay (acetyl-cholinesterase immobilised on a chemicallymodied screen-printed electrode with 7,7,8,8-tetracynoquinonedimethane)and the results obtained with the two analytical meth-ods were in agreement and indicated that the bioassaywas able to detect chlorpyrifos-methyl with satisfactoryaccuracy.

In another case, Ciucu et al. reported the use of a biosen-sor for organophosphorus compounds (OPCs) in polar andnon-polar solvents ( Ciucu et al., 2002 ). They co-immobilizedthree enzymes: peroxidase, choline oxidase, and cholineesterases. This multienzyme biosensor has been used forthe determination of trichlorfon. It was found that non-polarorganic solvents inactivated the tri-enzymatic biosensor dur-ing the incubation storage. Among the polar solvents used,

acetone and ethanol demonstrated minimal effects on theenzyme 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 estimatedin the presence of ethanol, propanol, cyclohexanoneandben-zene in the range of 10100%. In this work, it was found thatAChE retained its activity only in 10% of ethanol. The assayallowed the determination of OPs at subnanomolar concen-trations with an overall assay time of 10 min.

Finally, a novel approach to circumvent the problems of enzyme inhibition assays for the determination of pesticidesand methylmercury in organic extracts took advantage of the fact that the enzyme (acethylcholinesterase or invertase)in aqueous phase would selectively extract the irreversibleinhibitordissolved in organic solvent when they came in con-tact at the interface of organic/aqueous phases ( Amine et al.,2004). In terms of the organic solvents tested, the best resultswere obtained with toluene and benzene for invertase andwith hexane for AChE.

Theextraction withorganic solventsseems to be necessaryin thecase of real sampleanalysis in order to avoid interferingspecies insoluble in organic solvent. Therefore, the choice of an appropriate organic solvent is important to circumvent orminimize the enzyme inactivation.

7/29/2019 ev food

8/19


4. Inhibitor investigation using enzyme-basedbiosensors

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 biolog-ical activityanda relatively low persistence.As illustrated bythe literature reports compiled in Table 1 , the developmentof biosensors for pesticides is the subject of considerableinterest, particularly in the areas of food and environmentmonitoring ( Dzyadevych et al., 2005 ).

Several enzymes such as cholinesterase enzymes (AChE,BChE) and urease have been used in the design of directelectrochemical 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; Choiet al., 2001; Zhang et al., 2001; Jeanty et al., 2002; Turdeanet al., 2002; Schulze et al., 2003; Danet et al., 2003; Brazil deOliveira 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 assess-ment of the toxicity of methyl parathion and its photodegra-dation products in water ( Dzyadevych et al., 2002 ). In thisstudy it was shown for the rst time that the inhibition effectof methyl parathion on the AChE activity increases dramati-cally as soon as the photodegradation begins. Such a sensorcould be employed as part of an early warning system for

detecting various cholinesterase inhibitors.Collier et al. (2002) incorporated AChEand BChE into thecarbon-based ink used for printing electrodes. The printedelectrodes elaborated were used for the determination of chlorfenvinphos and diazinon, organophosphate pesticidesknown to be used for the control of insect pests in wool.The BChE-based biosensor was shown to be the most sensi-tive sensor towarda mixture of chlorfenvinphos anddiazinon,with a limit of detection equal to 0.5 g g 1 .

Sccreen-printed electrodes (SPE) have also been chem-ically modied with 7,7,8,8-tetracyanoquinodimethane(TCNQ) and used for the mediated electrochemical detec-tion of acetylcholinesterase activity ( Del Carlo et al., 2004 ).In this study, theAChE inhibition hasbeencarriedouton sam-ples from different food commodities. The AChE biosensorallowed detection of carbaryl at concentration of 10 ng g 1

which corresponds to the limit value set by the Europeanlegislation.

Recently a novel enzyme capable of selective recogni-tion of OP compounds was employed. In particular, a faster,simpler, and direct biosensing protocol suitable for rapidtesting of OP substances can be accomplished utilizing thebiocatalytic action of oraganophosphorus-hydrolase (OPH)(Mulchandani et al., 2001; Wanget al., 2002, 2003; Simonianet al., 2004; Gaberlein et al., 2000 ). However, the detection

limit obtained using OPH was not very low compared towhat is generally obtained by inhibition of cholinesteraseenzymes.

A new strategy for the detection and discrimination of neurotoxins, the anti-acetylcholinesterase activities of mix-tures of different neurotoxins, were investigated ( Simonian

et al., 2001). It was possible to separate the effects of differ-ent inhibitors, using a combined recognition/discriminationstrategy based on the joint action of acetylcholinesterase andorganophosphorus-hydrolase enzymes. They demonstratedthe feasibility of eliminating the organophosphate neurotox-ins from the different multi-component inhibitor combina-tion via sample pre-treatment with immobilized forms of OPH. Because of such manipulation, the inhibiting inuenceof non-OP neurotoxins on the AChE can be separated andtheir true concentration may be determined. In other study,engineered variants of Drosophila melanogaster acetyl-cholinesterase (AChE) were used as biological receptors inAChE-multisensors for simultaneous detection and discrim-ination of binary mixtures of cholinesterase-inhibiting insec-ticides (Bachmann et al., 2000 ). In this method the systemwas based on a combination of amperometricmulti-electrodebiosensors with chemometric data analysis of sensor out-puts using articial neural networks (ANN). The AChEmutants were selected on the basis of displaying an individ-ual sensitivity pattern toward the target analytes ( Bachmannet al., 2000). In this work it was demonstrated that themultisensor was capable of simultaneously detecting anddiscriminating paraoxon and carbofuran with errors of pre-diction of 0.4 and 0.5 g l 1, respectively. In the same vain,Crew et al. (2004) have investigated the determination of

OPs using genetically modied Drosophila melanogaster AChE. In this work, the screen-printed carbon electrodesmodied with cobalt phthalocyanine were used as basetransducers in the construction of amperometric pesticidebiosensors.

Brazil de Oliveira et al. (2004) have reported the compar-ative investigation of biosensors based on different acetyl-cholinesterases. The enzymes used were genetically mod-ied AChE obtained from Drosophila melanogaster andfrom commercial sources: electric eel, bovine erythrocytesand human erythrocytes. The sccreen-printed biosensorsdesignedwereusedfor determinationof methamidophospes-ticides. The inhibition of acetylcholinesterase is measured bydirector indirectmeasurement of itsactivity. In thecase of thedirect method, the assay is based on the spectrophotometricor electrochemical measurement of thiocholine issuing fromthe following reaction:

acetylthiocholine + H2O acetic acid + thiocholine

However, the measurement of thiocholine production doesnot accurately reect the degree of AChE inhibition by pes-ticides since the sample may also contain heavy metals. Aninterference was also observed from copper ions at mg l 1

levels (Danet et al., 2003 ) while Ricci et al. (2003) , haveshown that 5 M Cu2+ , 1 M Ag+, 0.5 M Hg2+ induced

7/29/2019 ev food

9/19

7/29/2019 ev food

10/19

7/29/2019 ev food

11/19


a 10% decrease of the amperometric signal of 10 M thio-choline.Thus, itwouldseem necessaryto usepriorseparationtechniques in thecase of multicomponent samples or developmore specic methods for their analysis.

A variety of combined systems has been explored withoutmuch success to overcome the problems of analyzing com-

plex mixtures. In another multi-enzyme approach, cholineoxidase (ChO) was co-immobilized with AChE and coupledto amperometric sensors for the measurement of hydrogenperoxide produced by the choline oxidation ( Zhang et al.,2001; Guerrieri et al., 2002; Kok et al., 2002 ):

acetylcholine + H2OAChE acetic acid + choline

choline + O2ChO betaine + 2H2O2

The performance of an oxygen electrode combined with abi-enzymatic membrane (AChE and ChO) was tested for thedetection 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 enzymesystem could not differentiate between the different pesti-cides and the inhibition by total pesticides was not additive.

AChE and ChO were also immobilized in a polyurethanepolyethylene oxide (PU-PEO) layer which was cast on thetop of the probe and covered with a dialysis membrane(Zhang et al., 2001 ). In this study, the biosensing devicewas used in either batch or ow analysis mode for paraoxondetection allowing a detection limit down to 10 nM using0.00025 U of AChE. Andreescu et al. (2002a) have pro-

posed the use of the commercially available phenyl acetateas new substrate for AChE biosensor and tested it in a bi-enzymatic AChE/tyrosinase system applied to the detectionof organophosphoruspesticides. Resultsweresimilar to thoseobtained with the mono-enzymatic AChE system using -aminophenyl acetate as substrate.

Recent attempts to demonstrate better systems for detec-tion of pesticides have employed different enzymes orenzyme/sensor interfaces. Mazzei et al. (2004) have reportedthe use a bioelectrochemical system for the determinationof pesticides by alkaline phosphatase inhibition. They founda detection limit for malathion (2,4-dochlorophenoxyaceticacid) of 0.5 g l 1 .

A novel optical detection of OP compounds based onreversible inhibition of OPH by copper complexed porphyrin(CuC1TPP) has been developed by Harmon. The absorbancespectrum of the porphyrinenzyme complex is measured viaplanar waveguide evanescent wave absorbance spectroscopy.Addition of OP compounds displaces the porphyrin from theenzyme resulting in reduced absorbance intensity at 412nm.Using this method the OP compounds can be detected at pptlevels (White and Harmon, 2005 ).

Simonian et al. (2005) described a novel strategy for thedirect detection of OP neurotoxins. Instead of using the pHchange associated with enzymatic hydrolysis of the OP sub-

strate as an indicator of the presence of an OP compound, themethod described is based on the change in uorescence of acompetitive inhibitor of the OPH enzyme when the inhibitoris displaced by the OP substrate. The change in uorescenceof the inhibitor is produced by the presence of gold nanopar-ticle attached to the enzyme.

Finally, the inhibition of tyrosinase has been also investi-gated for the determination of carbaryl using amperometricbiosensors ( KuuskandRinken, 2004 ) with thedetection limitfor carbaryl concentration obtained with this amperometricbiosensor being 0.2 mg l 1. This method also utilised a sim-ple approach of data management which can be automatedand used for different biosensors.

4.2. Heavy metal inhibitors

Table 2 summarizes the characteristics of various biosen-sors for heavy metal ion sensing, produced by integratingimmobilized enzymes with different kinds of transducers.Enzymatic methods are commonly used for metal ion deter-mination, as these can be based on the use of a wide rangeof enzymes that are specically inhibited by low concentra-tions of certain metal ions ( Krawczynski vel Krawczyk et al.,2000). For the inhibitive determination of trace mercury, alarge number of enzymes has been used: horseradish perox-idase (Han et al., 2001 ), urease (Krawezyk et al., 2000; DeMelo et al., 2002; Kuswandi, 2003; Rodriguez et al., 2004b )Krawczynski vel Krawczyk et al. (2000) , glucose oxidase(Alexander and Rechnitz, 2000 ), alcohol oxidase ( Pirvutoiuet 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 formsof mercury: phenyl mercury ( Doong and Tsai, 2001 ) usingurease, methyl mercury and phenyl mercury using invertase(Mohammadi et al., 2005 ), and methyl mercury using perox-idase (Han et al., 2001 ).

Cadmium ion could be monitored by enzymatic sen-sors since it was found that it induced inhibition of severalenzymes such as urease ( Lee and Russel, 2003 ) and butyril-cholinesterase (BChE) ( Mourzina et al., 2004 ). For copperdeterminationa cholinesterasesensorhasbeenused( Evtugynet al., 2003 ). It has been reported thatheavy metal ions inducea reversible cholinesterase inhibition.

For heavy metal screening, Rodriguez et al. (2004b) stud-ied different procedures of ureaseglutamic dehydrogenaseimmobilzation. The urease was either cross-linked with glu-taraldehyde, xed by naon lm, entrapped in alginate gel,or immobilized by adsorption. The designed biosensors werethen tested for the detection of metal ions (Cu, Hg, Cd andPb) in soil andwater extracts. Immobilization of ureaseusinga naon lm resulted in the best sensitivity for the studyof metal inhibition ( Rodriguez et al., 2004b ). Recently, anarray-based urease optical biosensor based on the solgelimmobilization 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 afnity for the cys-

7/29/2019 ev food

12/19

Table 2

Survey of enzyme inhibition-based biosensors for heavy metalsInhibitors Enzymes Immobilisation matrix Techniques Sample Working range/LOD Comments Natu

Hg2+ , Cu2+ , Cd2+ Ure ase Entrapment in solgel matrix Optical Tap and r iverwater

LOD= 10nM, 50 M, 500 M

Hg2+ , Cu2+ Glucoseoxidase(Gox)

Electropolymerisation in PPD Amperometric 2.5 moll 1 to 0.2mmol l 1 ,2.5 moll 1 to 0.2mmol l 1

Rapid regeneration withEDTA

Hg(NO 3)2 , HgCl2 , Hg2(NO3)2 ,phenyl mercury

Urease Entrapment in solgel lm Potentiometric Water samples 0.051.0/0.2, 0.051.0/0.2,0.051.0/0.1,0.15.0/0.5 moll 1

Not selective

Cd2+ Urease Self-assembled monolayer on thegold-coated sensor surface

Optical (SPR) 010 mg l 1 (dynamic range) Regeneration with EDTAand thioacetamide

Hg(II), Ag(I), Cu(II), Ni(II),Zn(II), Co(II), Pb(II)

Urease Immobilization in ultrabindmembrane

Optical berbiosensor

1 10 9 to 1 10 5 ,1 10 8 to 1 10 5 ,1 10 7 to 1 10 5 ,1 10 6 to 1 10 5 ,2 10 5 to 1 10 3 ,2 10 5 to 1 10 3 ,

1 10 4

to 1 10 3

moll 1

Regeneration with cysteine I

Mercury(II), mercury(I),methylmercury,mercuryglutathione complex

HRP Entrapment in -cyclodextrinpolymer

Amperometric LOD = 0.1, 0.1, 1.7 ng ml 1

Ag+ , Ni2+ , Cu2+ Urease Deposition onto electrode area andcovering withpoly(4-vinylpyridine and Naon)

PotentiometricpH-SFET

LOD = 3.5 10 8 , 7 10 5 ,2 10 6 moll 1

The use of the negativelychargedpolymer/regeneration withEDTA

Cu2+ AChE Cross-linking with GA vapour Amperometric 0.054.0 mmol l 1 Hg2+ GOx C ross-linking with GA and BSA Amperometric Spiked water 2.512 n g ml 1 ,

LOD=1ngml 1Inhibition of invertase insolution and detection of product with glucosebiosensor

HgCl2 , Hg(NO 3 )2 , Hg2Cl2 ,methylmercury, phenyl mercury

Invertase Cross-linkage with GA anddeposition on laponit modiedelectrode

Amperometric I 50 = 0.27, 0.032, 0.27, 0.34,0.12ppm

Reactivation with cysteine I

Hg2+ GOx Im mobilized in apolyvinylpyridine (PVP) inpresence of 2-aminoethanethiol

mediator

Amperometric 1100 ppb, LOD = 0.2 ppb R

Chromium(VI) GOx Cross-linking with GA andcovering with aniline membrane

Amperometric Soil sa mples 0.49 g l 1 to 8.05mg l 1 ,LOD= 0.49 g l 1

7/29/2019 ev food

13/19


teine residues of urease and could be detected with a lowerlimit 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 otherpollutants and toxic compounds; Table 3 summarizes thebiosensorcharacteristicsused in these cases.Severalenzyme-based biosensors have been constructed in order to mea-sure glycoalkaloids in foods. Consumption of glycoalka-loids, which are secondary metabolites that can show up infood, has been associated with human death and poisoningbecause of their teratogenic and embryotoxic effects. ThepH-sensitive eld effect transistors (pH-SFET) have beendeveloped for this purpose ( Korpan et al., 2002; Arkhypovaet al., 2003; Dzyadevych et al., 2004b ). In one approach,AChE and BChE were immobilized on a pH-SFET by amethod of protein cross-linking in saturated glutaraldehydevapour (Arkhypova et al., 2003; Korpan et al., 2002 ). It hadbeen shown that BChE is more sensitive to potato glycoal-kaloids than acetylcholinesterase. The BChE modied pH-SFET was then employed for determination of -chaonine,-solanine and solanidine in potato samples. Furthermore,AChEand BChE were also employed forthe determination of chaconine( Dzyadevych et al., 2004b ). These authors demon-strated that theBChE-basedbiosensoris much more sensitiveto the inhibitor than the AChE biosensor.

Detection via enzymatic inhibition has also been demon-strated for other potential food contaminants. Noguer etal. developed a disposable biosensor based on aldehyde

dehydrogenase (AlDH) for the detection of MITC (methylisothiocyanate), the main metabolite of metam-sodium. Ben-zoic acid has also been determined in foodstuffs usinga graphiteteontyrosinase composite biosensor ( Moraleset al., 2002). The composite bioelectrode allows a low detec-tion limit of benzoic acid equal to 9 10 7 moll 1 in non-aqueous (mayonnaise) and aqueous (cola soft drinks) mediaand exhibits good analytical characteristics such as renewa-bility, stability and low cost.

Anatoxin-a(s), a cyanotoxin produced by cyanobacteria,was detected in freshwater andlakesamples usinga biosensorbased on AChE ( Devic et al., 2002 ). Different mutants of theenzyme were used and the limit of detection allowed by themost sensitive biosensors was 0.5nmol of toxin per liter.

Different enzymatic biosensors have been constructed forthe determination of nitric oxide (NO) ( Kilinc et al., 2000 )and superoxide radicals ( Campanella et al., 2000 ). Thesebiosensors are based on the immobilization of different oxi-dases.

Lactate dehydrogenase (LDH) was competitively inhib-ited by pentachlorophenol (PCP). The limit of detectionallowedbythis biosensorwas270 g l 1 (Youngetal.,2001 ).Thesensitivity could be improved after co-immobilization of LDHwith lactate oxidase (LOD) andglucose dehydrogenase(GDH).

Fig. 2. Distribution of enzymes used for the design of biosensors used fordetection of inhibitors.

5. Food and environmental applications

In the specic context of food andenvironmental analysis,a largenumberof biosensors basedonenzymeinhibitionhavebeen developed for the detection of a variety of compounds.The most commonly used enzymes for the design of biosen-sors areAChEs(50%), followedby BChEs (11%), see Fig. 2 .However, HRP, tyrosinase and urease represent separately7% of the total enzymes used for the construction of biosen-sors 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 usedfor the determination of pesticides including carbamates andorganophosphorus compounds ( Fig. 3) while heavy metalsrepresent only 21% of the total application of these biosen-sors. Other applications of these sensing devices include the

determination of glycoalkaloids,benzoic acid,cyanide,nitricoxide and neurotoxins (anatoxin-a(s)) and other inhibitors.

Despite thevery large numberof publicationsdemonstrat-ing enzyme inhibition, the majority of systems were unfor-tunately not challenged by real samples. Still, the researchpublishedduring the last 5 years showing applicationsto foodand environmental samples was not negligible.

Numerous biosensors were designed for the analysis of inhibitorsin tapand river water samples:acetylcholinesteraseused 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.

7/29/2019 ev food

14/19

Table 3Survey of biosensors for other chemical contaminantsInhibitors Enzymes Immobilization matrix Techniques Sample Working range/LOD Comments

-Chaonine, -solanine,solanidine

BChE Cross-linking with GAvapour

PotentiometricpH-SFET

Potatoes 0.5100 moll 1 /0.5 moll 1 ,2.0 moll 1 , 2.0 moll 1

Overall time for one analysis is10min

-Chaconine, -solanine BChE Cross-linking with BSAand GA vapour

Potentiometric Agriculture 0.2100 moll 1 ,LOD=0.2 moll 1 ,0.2100 moll 1 ,LOD=0.5 moll 1

-Chaonine, -solanine,solanidine

BChE Cross-linking with GAvapour

Potentiometric Potato LOD = 0.2, 0.5, 1 moll 1

-Chaconin, -solanine,tomatine

BChE Deposition of enzyme-loaded gel onthe pH-selective layer

Potentiometric Potatoes LOD = 0.2, 0.5, 0.5 moll 1 Reliable tool for estimation of the overall toxicity level in foodsamples.

Tomatine BChE Cross-linking with BSA

and GA

Potentiometric Tomatoes 0.550 moll 1 ,

LOD=0.2 moll 1

Oxalic acide AChE Cross-linking with GAvapour

Amperometric 0.520 mmol l 1

Nitric oxide Xanthine oxidase(XOD)

Entrapment in poly( o-phenylenediamine)

Amperometric 02 mmol l 1

Nitric oxide HRP Cross-linking BSA andGA

Amperometric 2.7 10 6 to1.1 10 5 moll 1 ,LOD=2.0 10 6 moll 1

Benzoic acid Tyrosinase Mixture of graphite,tyrosinase and teon

Amperometric Mayonnaise sauceand cola soft drinks

9 10 7 moll 1 Use in non-aqueous medium

Cyanide Tyrosinase Immobilization in toanionic clay

Amperometric LOD = 0.1 nmol l 1 Regeneration by washing withelectrolyte.

Methyl isothiocyanate AlDH Entrapment in PVA-SbQ Amperometric 1001000 ppb, LOD = 100 ppb Anatoxin-a(s) AChE Entrapment in PVA-SbQ Amperometric Fresh water Use of different AChE mutants Anatoxin-a(s) AChE Entrapment in

TCNQ-graphiteAmperometric Spiked water

samples110 g l 1 , LOD= 1 g l 1 No reactivation was observed

using oximeCaptan Glutathione- S -

transferase (GST)Entrapment in the gelsodium alginate

Optical Contaminated water 02 ppm

7/29/2019 ev food

15/19


(Young et al., 2001 ), cellobiose dehydrogenase for phenoliccompound investigations ( Nistor et al., 2002 ), glutathione- S -transferase for determination of captan ( Choi et al., 2003 ),urease for mercury analysis ( Krawczynski vel Krawczyk et al., 2000). In addition to mercury analysis, urease wasused for copper and cadmium determination in tap and

river water ( Tsai and Doong, 2005 ), carboxyl esterase forselenium detection ( Saritha and Kumar, 2001 ). Trojanowiczet al. (2004) examined a batch system with dissolved inver-tase and a membrane based amperometric glucose biosensorfor the determination Hg(II) in natural and wastes water.They evaluated the inhibition of invertase by other macro-and micro-components which can be potentially present inenvironmental samples. Choi et al. (2001) developed a AChEoptical biosensorfor paraoxon andcaptan analysis incontam-inated water.

Other biosensors based on enzymatic inhibition wereused for the determination of pesticides in soil extracts,including an AChE-based biosensor developed by Guerrieriet al. (2002) , a carboxyl esterase biosensor employed bySaritha and Kumar (2001) , an aldehyde dehydrogenase-based biosensor designed by Noguer et al. (2001) ,and ureaseglutamic dehydrogenase-based biosensor byRodriguez et al. (2004a) . Collier et al. (2002) developed aurease biosensor for the determination of organophosphoruspesticides in extracts of sheep wool.

Overall, for foodanalysis, themostwidely biosensorsusedare based on the AChE enzyme for the determination of pes-ticides (Del Carlo et al., 2002, 2005; Xavier et al., 200 0;Pogacnik and Franko, 2003; Crew et al., 200 4). The rstapplication of an AChE-basedbiosensor in food produced by

animals, such as eggs,honey andmilk, was developed by DelCarlo et al. (2004) . Dzyadevych et al. (2004b) developed aBChE biosensor for determination of glycoalkaloids (toma-tine) in tomato samples. During the last year, Zhang et al.(2005) have developed a rapid biosensor based on disposablescreen-printed electrodes, which is suitable for monitoringorganophosphate and carbamate residues in milk. In thiswork, threeengineered variantsof Nippostrongylus brasilien-sis acethylcholinesterase were used to obtain enhanced sen-sitivity.

Ivanov et al. (2003) compared the features of variouscholinesterase sensors based on screen-printed carbon elec-trodes differing in the detection mode and modier to estab-lishfactors affectingthesensitivity of pesticidedeterminationin grape juice. The work of Crew et al. (2004) was directedat developing a rapid system capable of identifying and mea-suring specic OPs pesticide residues in wheat and appleextracts. Their system is based on the inhibition of AChEimmobilized on screen-printed carbon electrodes modiedwith cobalt phthalocyanine. They have demonstrated thatthere was no matrix effect on the biosensor response fromwheat or apple extracts. It is envisaged that the proposedamperometricbiosensorwill be integrated into an automated,commercially available, analytical system for rapid qualitycontrol analysis in the food industry. A composite ampero-

metric tyrosinase electrode was designed by Morales et al.(2002) f or benzoic acid determination in mayonnaise andcola soft drinks.

As can be seen, the application of the various developedbiosensors in food and environmental analysis is still limited.A main reason seems to be that the most used enzymes are

notsufciently selective or discriminating,as forexample thecase of cholinesterase-based biosensors where the enzyme isinhibited by both pesticides and heavy metals. Some authorshave developed novel strategies to solve these problems byoffering new methods of data analysis, by using engineeredenzymes instead of the classical ones, or by combining dif-ferent enzymes.

6. Summary and conclusions

Despite the considerable research activity devoted to thedevelopment of biosensors based on enzyme inhibition, ana-lytical applications are still limited since these sensor tech-nologies are not usually able to discriminate various toxiccompounds in the same sample. In particular, the simultane-ous presence of heavy metals and pesticides in contaminatedsamples provides a challenge for their use for purely regu-latory purposes where a specic analyte must be determinedwitha prescribedaccuracy. Nevertheless, thepropertiesof thedevices under development suggest that these biosensors canbe used as alarm systems; they would provide either quanti-cation of one contaminant when this analyte is present aloneor an indication of total contamination of particular samples.

In many cases, the biosensor assays based on inhibitory

effects of pesticides or other chemical contaminants showhigh sensitivity and could be the basis for relatively sim-ple and cost-effective procedures. A particular advantage of these biosensing systems is that they offer the possibility of analysis in both batch and ow mode, allowing the use of these sensors for analysis of a large number of samples in areasonable time interval. These methods can also be recom-mended for development of single use test strips, especiallywith screen-printed electrodes, to avoid problems related tofouling of the electrode which generally involves a chemicalor electrochemical deactivation of the working electrode sur-face. SPEs offer several additional advantages including lowcost, simple handling, being amenable to both mass produc-tion andinstrument miniaturizationfor in loco analysis.Also,thesingleuseof these sensors avoids theneed of reactivation.

Current research studies involve numerous efforts atimproving the analytical performance of the biosensing sys-tems in order to be able to monitor a wide range of pollutantsin environmental andfoodsamples.Effectively, articialneu-ral networks (ANN) have already been shown to be usefuladjunct for interpretation of experimental data in analysis of binarymixtures of insecticideswith theuseof severalbiosen-sors exhibiting various responses to different analytes. Theuseof genetically modied enzymes for thedesignof biosen-sors can be an effective way to improve sensor sensitivity.

7/29/2019 ev food

16/19


Indeed, the combination of such engineered enzymes (dou-ble and triple mutants) with microporous-activated carbontechnology could improved the efciency of enzyme-basedbiosensors.

7. Future perspectives

Engineered variants of enzymes could be anotherapproach in biosensor design for the discrimination anddetection of various enzyme-inhibiting compounds whenused in combination with chemometric data analysis usingarticial neural networks. The useof transducers constructedfrom nano-structured material might also improve the ef-ciency of these biosensors. The crucial issues that should beaddressed in the development of new analytical methods isrst to enable the possibility of simultaneousand discrimina-tive monitoring of several contaminants in a multicomponentsample and then the conversion of the biosensing systems to

marketable devicessuitable for large scale environmental andfood applications.

Acknowledgements

The authors would like to thank the Novtech and Craft EUprojects for research funding leading to some of the publica-tions cited in this review.

References

Alexander, P.W., Rechnitz, G.A., 2000. Enzyme inhibition assays withan amperometric glucose biosensor based on thiolate self-assembledmonolayer. Electroanalysis 12, 343350.

Amine, A., Mohammadi, H., Arduini, F., Ricci, F., Moscone, D., Palleschi,G., 2004. Extraction of enzyme inhibitors using a mixture of organicsolvent and aqueous solution and their detection with electrochemicalbiosensors. In: Proceedings of the Eighth World Congress on Biosen-sors, 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 anhistidine tail. Application to the detection of pesticides using modiedcholinesterase. Anal. Lett. 34 (4), 529540.

Andreescu, S., Avramescu, A., Bala, C., Magearu, V., Marty, J.-L.,2002a. Detection of organophosphorus insecticides with immobilizedacetylcholinesterase-competitive study of two enzyme sensors. Anal.

Bioanal. Chem. 374, 3945.Andreescu, S., Noguer, T., Magearu, V., Marty, J.-L., 2002b. Screen-printed electrode based on AChE for the detection of pesticides inpresence of organic solvents. Talanta 57, 169176.

Andreescu, S., Fournier, D., Marty, J.-L., 2003. Development of highlysensitive sensor based on bioengineered acetylcholinesterase immobi-lized by afnity 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 opti-misation of biosensors based on pH-sensitive eld effect transistorsand cholinesterases for sensitive detection of solanaceous glycoalka-loids. 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 andcarbamates with disposable multielectrode biosensors using recombi-

nat mutants of Drosophila acethylcholinesterase and articial neuralnetworks. Biosens. Bioelectron. 15, 193201.

Boni, A., Cremisini, C., Magano, E., Tosi, M., Vastarella, W., Pilloton,2004. Optimized biosensors based on puried enzymes and engineeredyeasts: 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 acetyl-cholinesterase obtained from commercial sources and geneticallymodied Drosophila melanogaster . Application in amperometricbiosensors for methamidophos pesticide detection. Biosens. Bioelec-tron. 20, 825832.

Campanella, L., Persi, L., Tomasseti, M., 2000. A new tool for superox-ide and nitric oxide radicals determination using suitable enzymaticsensors. Sens. Actuators B 68, 351359.

Casero, E., Losada, J., Pariente, F., Lorenzo, E., 2003. Modied electrodeapproaches for nitric oxide sensing. Talanta 61, 6170.

Choi, J.-W., Kim, Y.-K., Lee, I.-H., Min, J., Lee, W.H., 2001. Opticalorganophosphorus biosensor consisting acetyl cholinesterase/viologenhetero LangmuirBlodjett lm. Biosens. Bioelectron. 16, 937943.

Choi, J.-W., Kim, Y.-K., Song, S.-Y., Lee, I.-H., Lee, W.H., 2003. Opti-cal 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 bi-enzymatic whole cell conductometric biosensor for heavy metal ionsand pesticides detection in water samples. Biosens. Bioelectron. 21,273281.

Ciucu, A., Negulescu, C., Baldwin, R.P., 2003. Detection of pesticidesusing an amperometric biosensor based on ferophthalocyanine chem-ically modied carbon paste electrode and immobilized bienzymaticsystem. Biosens. Bioelectron. 18, 303310.

Ciucu, A., Ciucu, C., Baldwin, R.B., 2002. Organic phase potentiomet-ric biosensor for detection of pesticides. Roum. Biotechnol. Lett. 7,625630.

Ciucu, A., Lupu, A., Pirvutoiu, S., Palleschi, G., 2001. Biosensors forheavy 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 detec-tion of pesticides of extracts of sheep wool. Biosens. Bioelectron. 17,815819.

Crew, A., Hart, J.P., Wedge, R., Marty, J.-L., Fournier, D., 2004. Ascreen-printed, amperometric, biosensor array for the detection of organophosphate pesticides based on inhibition of wild type, andmutant 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 inFIA 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 twooppositely charged clays (laponite and ZnAl layered double hydrox-ides). Anal. Chem. 74, 40374043.

Del Carlo, M., Mascini, M., Pepe, A., Compagnone, D., Mascini, M.,2002. Electrochemical bioassay for the investigation of chlorpyrifos-methyl 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 organophos-phate pesticides using electrochemical bioassay. Food Chem. 84, 651656.

Del Carlo, M., Pepe, A., Mascini, M., De Gregorio, M., Visconti, A.,Compagnone, D., 2005. Determining pirimiphos-methyl in durumwheat 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 environmentalsamples of cyanobacteria by using a biosensor with engineered acetyl-cholinesterases. Appl. Environ. Microbiol. 68 (8), 41024106.

7/29/2019 ev food

17/19


Doong, R.-A., Tsai, H.-C., 2001. Immobilization and characterization of solgel encapsulated acetylcholinesterase ber-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 pol-luants. Anal. Lett. 37, 15891599.

Dzyadevych, S., Soldatkin, A.P., Arkhypova, V.N., Elskaya, A.V., 2005.Early-warning electrochemical biosensor system for environmentalmonitoring 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 toma-tine 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. Potentio-metric 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 eld-effect transistors for determi-nation 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 inwater 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 determi-nation of atrazine. Anal. Lett. 37, 16711681.

Evtugyn, G.A., Stoikov, I.I., Budnikov, H.C., Stoikova, E.E., 2003. Acholinesterase sensor based on a graphite electrode modied with1,3-disubstituted calixarenes. J. Anal. Chem. 58, 11511156.

Evtugyn, G.A., Budnikov, H.C., Nikolskaya, E.B., 1999. Biosensorsfor the determination of environmental inhibitors of enzymes. Russ.Chem. Rev. 68, 10411064.

Evtugyn, G.A., Budnikov, H.C., Nikolskaya, E.B., 1998. Sensitivity andselectivity of electrochemical enzyme sensors for inhibitor determina-

tion. Talanta 46, 465484.Gaberlein, S., Knoll, M., Spener, F., Zaborosch, C., 2000. Disposablepotentiometric enzyme senor for direct determination of organophos-phorus insecticides. Analyst 125, 22742279.

Gogol, E.V., Evtugyn, G.A., Marty, J.-L., Budnikov, H.C., Winter, V.G.,2000. Amperometric biosensor based on naon coated screen printedelectrodes 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 enzymebiosensor incorporating an electro polymerized aniline membrane andferrocene as electron transfer mediator. Int. J. Environ. Sci. Eng. 84(10), 761774.

Guerrieri, A., Monaci, L., Quinto, M., Palmisano, F., 2002. A dispos-able 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 andcounting. Anal. Lett. 37, 1448114496.

Gulla, K.C., Gouda, M.D., Thakur, M.S., Karanth, N.G., 2002. Reactiva-tion of immobilized acetylcholinesterase in an amperometric biosen-sor for organophosphorus pesticide. Biochim. Biophys. Acta 1597,133139.

Han, S., Zhu, M., Yuan, Z., Li, X., 2001. A methylene blue-mediatedenzyme electrode for the determination of trace mercury(II), mer-cury(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 carbonelectrochemical sensors/biosensors for biomedical, environmental, andindustrial analyses. Anal. Lett. 37 (5), 789830.

Ivanov, A., Evtugyn, G., Budnikov, H.C., Ricci, F., Moscone, D.,Palleschi, G., 2003. Cholinesterase sensors based on screen-printedelectrode for detection of organophosphorus and carbamic pesticides.Anal. Bioanal. Chem. 377, 624631.

Jeanty, G., Wojiciechowska, A., Marty, J.-L., 2002. Flow-injection amper-ometric 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 ow system-basedenzyme 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 agentsbased on carbon nanotubes modied thick lm strip electrode. Elec-troanalysis 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 irreversibleinhibitors of acetylcholinesterase. J. Biol. Chem. 237, 32453249.

Kok, F.N., Hasirci, V., 2004. Determination of binary pesticide mixturesby an acetylcholinesterase-choline oxidase biosensor. Biosens. Bio-electron. 19, 661665.

Kok, F.N., Bozoglu, F., Hasirci, V., 2002. Construction of anacethylcholinesterase-choline oxidase biosensopr for aldicarb deter-mination. 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 novelenzyme biosensor for steroidal glycoalkaloidsdetection based on pH-sensitive eld effect eld transistors. Bioelectrochemistry 55, 911.

Krawczynski vel Krawczyk, T., Mosszczynska, M., Trojanowicz, M.,2000. Inhibitive determination of mercury and other metal ionsby potentiolmetric urea biosensor. Biosens. Bioelectron. 15, 681691.

Krawezyk, T.K., Moszezynska, M., Trojanowiez, M., 2000. Inhibitivedetermination of mercury and other metal ions by potentiometric ure-ase biosensor. Biosens. Bioelectron. 15, 681691.

Kuswandi, B., 2003. Simple optical bre biosensor based on immobi-

lized enzyme for monitoring of trace heavy metal ions. Anal. Bioanal.Chem. 376, 11041110.Kuusk, E., Rinken, T., 2004. Transient phase calibration of tyrosinase-

based carbaryl biosensor. Enzyme Microb. Technol. 234, 657661.Lee, W.E., Thompson, H.G., Hall, J.G., Bader, D.E., 2000. Rapid

detection and identication of biological and chemical agents byimmunoassay, gene probe assay and enzyme inhibition using silicon-based biosensor. Biosens. Bioelectron. 14, 784804.

Lee, H.-S., Kim, Y., Chi, Y., Lee, T.Y., 2002. Oxidation of organophos-phorus pesticides for the sensitive detection by cholinesterase-basedbiosensor. Chemosphere 46, 571576.

Lee, H.-S., Kim, Y.-A., Chung, D.H., Lee, T.Y., 2001. Determination of carbamate pesticides by a cholinesterase-based ow injection biosen-sor. 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 plasmonresonance. 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 bybiosensors based on enzyme immobilised by electropolymerisation.Biosens. Bioelectron. 20, 16431647.

Mazzei, F., Botr e, F., Montilla, S., Pilloton, R., Podest a, E., Botr e, C.,2004. Alkaline phosphatase inhibition based electrochemical sensorsfor the detection of pesticides. J. Electroanal. Chem. 574, 95100.

Mohammadi, H., Amine, A., Cosnier, S., Mousty, C., 2005.Mercuryenzyme inhibition assays with an amperometric sucrosebiosensor based on a trienzymaticclay matrix. Anal. Chim. Acta 543,143149.

7/29/2019 ev food

18/19


Mohammadi, H., El Rhazi, M., Amine, A., Brett, A.M.O., Brett, C.M.A.,2002. Determination of mercury(II) by invertase enzyme inhibitioncoupled with batch injection analysis. Analyst 127, 10881093.

Montesinos, T., P erez-Munguia, S., Valdez, F., Marty, J.-L., 2001. Dis-posable cholinesterase biosensor for the detection of pesticides inwater-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 enzymeelectrode for the control of the benzoic acid content in food. Talanta57, 11891198.

Mourzina, I.G., Yoshinobu, T., Ermolenko, Y.E., Vlasov, Y.G., Schoning,M.J., Iwasaki, H., 2004. Immobilization of urease and cholinesteraseon 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 amper-ometric enzyme bioensor for direct dtermination of organophosphatenerve agents. Environ. Sci. Technol. 35, 25622565.

Neufeld, T., Eshkenazi, I., Cohen, E., Rishpon, J., 2000. A micro owinjection electrochemical biosenor for organophosphorus pesticides.Biosens. Bioelectron. 15, 323329.

Nikolelis, D.P., Simantiraki, M.G., Siontorou, C.G., Toth, K., 2005. Flowinjection analysis of carbofuran in foods using air stable lipid lm

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-eldmonitoring of cleaning efciency in water treatment plants using twophenol sensitive biosensors. Anal. Chim. Acta 456, 317.

Noguer, T., Balasoiu, A-M., Avramescu, A., Marty, J.-L., 2001. Develop-ment of disposable biosensor for the detection of metam-sodium andits metabolite MITC. Anal. Lett. 34 (4), 513528.

Nunes, G.S., Jeanty, G., Marty, J.-L., 2004. Enzyme immobilizationprocedures on screen printed electrodes used for the detection of anti-cholinesterase 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 ampero-metric organophosphate pesticide biosensor by 2-pyridinealdoxime

methochloride. Sens. Actuators B 66, 131134.Patel, P.D., 2002. (Bio)sensor for measurement of analytes implicated infood safety: a review. Trends Anal. Chem. 21, 96115.

Pavlov, V., Xiao, Yi., Willner, I., 2005. Inhibition of the acetylcholineesterase-stimulated growth of au nanoparticles: nanotechnology-basedsensing 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 enzymeinhibition 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 alcoholoxidase. Roum. Biotechnol. Lett. 7, 975.

Pogacnik, L., Franko, M., 2003. Detection of organophosphate and car-bamate pesticides in vegetable samples by a photothermal biosensor.

Biosens. Bioelectron. 18, 19.Rekha, K., Gouda, M.D., Thakur, M.S., Karanth, N.G., 2000. Ascorbateoxidase based biosensor for organophosphorus pesticide monitoring.Biosens. Bioelectron. 15, 499502.

Ricci, F., Arduini, F., Bourais, I., Moscone, D., Palleschi, G., Amine,A., 2003. Thiocholine mediated oxidation at prussian blue modiedelectrodes for pesticide and heavy metals detection. In: Proceedingsof the First International Workshop on Biosensors for food safetyEnvironmental Monitoring, Marrakech, Morocco (O-4, abstract book).

Rodriguez, B.B., Bolbot, J.A., Tothill, I.E., 2004a. Development of ureaseand glutamic dehydrogenase amperometric assay for heavy metalsscreening in polluted samples. Biosens. Bioelectron. 19, 11571167.

Rodriguez, B.B., Bolbot, J.A., Tothill, I.E., 2004b. Ureaseglutamic dehy-drogenase biosensor for screening heavy metals in water and soilsamples. Anal. Bioanal. Chem. 380, 284292.

Sacks, V., Eshkenazi, I., Neufeld, T., Dosoretz, C., Rishpon, J.,2000. Immobilized parathion hydrolase: an amperometric sensor forparathion. Anal. Chem. 72, 20552058.

Saritha, K., Kumar, N.V.N., 2001. Qualitative detection of selenium in for-tied soil and water samples by a paper chromatographiccarboxylesterase enzyme inhibition technique. J. Chromatogr. A 919, 223228.

Shan, D., Mousty, C., Cosnier, S., 2004. Subnanomolar cyanide detec-tion at polyphenol oxidase/clay biosensors. Anal. Chem. 76, 178183.

Schulze, H., Vorlova, S., Villatte, F., Bachmmann, T.T., Schmid, R.D.,2003. Design of acetylcholinesterases for biosensor applications.Biosens. Bioelectron. 18, 201209.

Schulze, H., Schmid, R.D., Bachmann, T.T., 2002. Rapid detection of neurotoxic insecticides in food using disposable acetylcholinesterase-biosensors and simple solvent extraction. Anal. Bioanal. Chem. 372,268272.

Segel, I.H., 1976. In Biochemical Calculation: How to Solve Mathemati-cal Problems in General Biochemistry, 2nd ed. Wiley, New York, pp.208223.

Sezgint urk, M.K., G oktu g, T., Dincka, E., in press. A biosensor basedon catalase for determination of highly toxic chemical azide in fruit

juices. Biosens. Bioelectron., in press (corrected proof). Availableonline: February 12, 2005.Simonian, A.L., Good, T.A., Wang, S.-S., Wild, J.R., 2005. Nanoparticle-

based optical biosensors for the direct detection of organophosphatechemical warfare agent and pesticides. Anal. Chim. Acta 534, 6977.

Simonian, A.L., Flounders, A.W., Wild, J.R., 2004. FET-based biosensorsfor the direct detection of organophosphate neurotoxins. Electroanal-ysis 16, 18961906.

Simonian, A.L., Efremenko, E.N., Wild, J.R., 2001. Discriminative detec-tion of neurotoxins in multi-component samples. Anal. Chim. Acta444, 179186.

Sole, L.S., Merkoci, A.I., Alegret, S., 2003. Determination of toxic sub-stance based on enzyme inhibition. Part I. Electrochemical biosensorsfor 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 character-istics using additiona

Date post:	04-Apr-2018
Category:	Documents
Upload:	adoniscool86
View:	215 times
Download:	0 times

ev food

Documents