An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos

An

acetylcholinesterase

biosensor

for

determination

of

low

concentrations

of

Paraoxon

and

Dichlorvos

D.

Di

Tuoro

1

,

M.

Portaccio

1,2

,

M.

Lepore

1,2

,

F.

Arduini

1,3

,

D.

Moscone

1,3

,

U.

Bencivenga

1,4

and

D.G.

Mita

1,2,4,

1_{NationalInstituteofBiostructuresandBiosystems(INBB),Rome,Italy}

2_{DepartmentofExperimentalMedicine,SecondUniversityofNaples,Naples,Italy} 3_{DepartmentofChemicalSciencesandTechnologies,UniversityofTorVergata,Rome,Italy} 4_{InstituteofGeneticsandBiophysicsofCNR,Naples,Italy}

The

characterization

of

an

economic

and

ease-to-use

carbon

paste

acetylcholinesterase

(AChE)

based

biosensor

to

determine

the

concentration

of

pesticides

Paraoxon

and

Dichlorvos

is

discussed.

AChE

hydrolyses

acetylthiocholine

(ATCh)

in

thiocoline

(TC)

and

acetic

acid

(AA).

When

AChE

is

immobilized

into

a

paste

carbon

working

electrode

kept

at

+410

mV

vs.

Ag/AgCl

electrode,

the

enzyme

reaction

rate

using

acetylthiocholine

chloride

(ATCl)

as

substrate

is

monitored

as

a

current

intensity.

Because

Paraoxon

and

Dichlorvos

inhibit

the

AChE

reaction,

the

decrease

of

the

current

intensity,

at

fixed

ATCl

concentration,

is

a

measure

of

their

concentration.

Linear

calibration

curves

for

Paraoxon

and

Dichlorvos

determination

have

been

obtained.

The

detection

limits

resulted

to

be

0.86

ppb

and

4.2

ppb

for

Paraoxon

and

Dichlorvos,

respectively,

while

the

extension

of

the

linear

range

was

up

23

ppb

for

the

former

pesticide

and

up

to

33

ppb

for

the

latter.

Because

the

inhibited

enzyme

can

be

reactivated

when

immediately

treated

with

an

oxime,

the

biosensor

reactivation

has

been

studied

when

1,1

0

-trimethylene

bis

4-formylpyridinium

bromide

dioxime

(TMB-4)

and

pyridine

2-aldoxime

methiodide

(2-PAM)

were

used.

TMB-4

resulted

more

effective.

The

comparison

with

the

behavior

of

similar

AChE

based

biosensors

is

also

presented.

Introduction

Organophosphorus (OPs) pesticides are very toxiccompounds, intensivelyusedinagriculturebecauseoftheirhighinsecticidal activity and their rapidmineralization in the environment [1]. Theirpresenceintheenvironmentisveryharmfulforwildlifeand humanhealthastheyirreversiblyinhibitthecatalyticactivityof acetylcholinesterase (AChE), an enzyme which catalyzes the hydrolysisoftheacetylcholine(ACh),oneofthemostimportant neurotransmittersplayingavitalroleinthecentralandperipheral nervoussystem[2,3].Acetylcholineisbelievedtoaffectmemory, sleep, concentrationabilities.Ashortsupplyofacetylcholine is associatedwithAlzheimer’sdisease[3,4].Duetotheir

toxicologi-calactivity someOPshavebeenusedalsoasChemicalWarfare Agents(CWAs)[5,6].

In presence of these health risks, in recent years a growing attentionhasbeenpaidindevelopingreliable,fastandinexpensive analyticalsystemstomonitorthisfamilyofpesticides.Biosensors basedonacetylcholinesterase,indeed,representanemergingand promisingtechniquefortoxicityanalysis,environmental monitor-ing,foodqualitycontrolandmilitaryinvestigations[6–9].Themain applicationofAChEbiosensorsisthedetectionofOPspesticides basedonenzymeinhibition.Biosensorscansubstitutethecurrent analyticalmethods,suchaschromatographicandcoupled chroma-tographic–spectrometricprocedures,bysimplifyingoreliminating sample preparation,thus decreasingthe analysistime and cost. Themost criticalpoint inthe development ofbiosensorsisthe

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aper

immobilizationofthebiologicalrecognitionelementinanactive formontotheelectrodesurface.Becausetheanalyticalperformance ofanelectrodeisstronglyaffectedbythisprocess,intensiveefforts have to bemade to develop effective immobilization methods, allowingimprovedoperationalandstoragestability,responsetime, linearrangeandsensitivity,andpreservingatthesametimethe enzymeaffinityforthesubstratesor/andinhibitors.Physical entrap-mentoftheenzymehasbeenusedtoimmobilizeenzymesbecause theabsenceofchemicalbondsformationhelpstopreserve enzy-maticactivityduring immobilization.However, the main draw-backsaretheenzymeleakingandthepossiblediffusion barriers, imposedbytheentrappinglayer.ImmobilizationofAChEbysimple incorporation into thegraphite paste and by cross-linkingwith glutaraldehyde ina single or multi-layerdepositionwas accom-plished[10,11].Several biosensorsfunctioningaccording tothe principleofAChEinhibitionhavealsobeenproposed[12]. Numer-ousprototypesbasedonpotentiometric[13,14],amperometric[15– 17] and piezoelectric [18,19] transducers have been developed. Because the enzymatic activity in AChE-based biosensors is destroyeduponcalibrationorinthepresenceoftheanalyte,the sensormustbediscardedafterdetermination[20,21],or, alterna-tively,theenzymemustbereplaced[22–25]orreactivated[26,27]. Inthispaperweproposeaneconomicandeaseofusecarbon paste AChE biosensor to detect OPs concentration in aqueous solutions.Oncecharacterized, thebiosensorhasbeen employed inaqueoussolutiontodetecttheconcentrationofthepesticides paraoxonanddichlorvos,onthebasisoftheirinhibitoryeffecton AChEactivity.TheconditionsfortheAChEreactivationhavebeen reported,togetherwithexperimentsofrecoverystudiesofspilled realsamples.

Material

and

methods

Materials

Acetylcholinesterase(AChE,EC3.1.1.7,TypeVI–S,10kUmg1, from electric eels), acetylthiocholine chloride (ATCl) and the oximes pyridine-2-aldoxime methoiodide (2-PAM) and 1,10

-tri-methylene bis4-formylpyridinium bromide (TMB-4) were pur-chased from Sigma (USA) and used as received. Paraoxon and DichlorvoswerealsopurchasedfromSigma(USA),andtheirstock solutions were prepared in ethanol. Phosphate buffer solution (PBS) 0.1M at pH 7.5, containing 0.1M KCl, wasprepared by mixing stocksolutions of0.1M NaH2PO4 and 0.1M Na2HPO4.

Carbon powder (1.2mM in size) and mineral oil (employed as pasting material) were purchased from Sigma–Aldrich (Milan, Italy).Allchemicalswereofanalyticalreagentgradeandallthe aqueoussolutionswerepreparedwithredistilleddeionizedwater.

Biosensor

design

and

fabrication

Carbon paste wasused to buildup the working electrode.The carbon paste was prepared by hand mixing graphite powder, mineraloiland,whenitwasthecase,acetylcholinesteraseinan appropriateweightratio.Theresultingpasteswerepackedintothe well of a Teflon tube (3mm internal diameter and 1.2mm in thickness)withanelectricalcontactprovidedbyacopperwire.For economicreasonstheTeflontubewaspackedwithadoublelayer ofcarbonpaste.Theinnerlayercontainedonlygraphitepowder mixed to mineral oil,and the outer layercomposed of carbon paste,mineraloilandacetylcholinesterase.Theinnerlayer,about

2/3ofthetotalvolume,waspreparedbymixing60%ofgraphite powderand40%ofmineraloil,foratotalweightof4.8mg.The outerlayer,about1/3ofthetotalvolumeandwithatotalweight of2.5mg,waspreparedbymixinggraphitepowderwithmineral oilandacetylcholinesteraseintheratio(w/w)of50:40:10, respec-tively.Foreachelectrodeweusedabout0.25mgof acetylcholi-nesterase which corresponds to about 2500U. The electrode surfacewaspolishedonweighingpapertogiveasmoothfinish beforeuse.Amperometricmeasurementswerecarriedoutusinga PalmSens instrument (Palm Instruments, the Netherlands) coupled to a PC. All experiments were carried out in a three electrode electrochemical cell with a volume of 10mL (0.1M sodium phosphate buffer, pH 7.5, containing 0.1M KCl) with theenzymemodifiedCPEasworkingelectrode,anAg/AgCl elec-trode as reference electrode and a platinum wire as auxiliary electrode (seeFigure 1).The workingelectrode wasoperatedat +410mVvs.Ag/AgCl.Amagneticstirreranda stirringbar pro-videdtheconvectivetransportintheelectrolyticcell.All measure-mentswereperformedatroomtemperature,thatis25.00.58C.

Acethylthiocholine

concentration

measurements

Theacetylthiocolineconcentrationwasdeterminedbymeasuring the oxidationcurrent of thiocholine according to the reaction occurringattheworkingelectrodesurface:

acetylthiocolineþH2O! AChE

thiocholineþaceticacid 2ThiocholineðredÞ!ThiocholineðoxÞðdimericÞ

þ2Hþþ2e

AChE

inhibition

experiments

Experimentsaboutinhibitionmeasurementsandpesticides con-centrationdeterminationwerecarriedoutwith[ATCl]=100mM. To determinethe pesticide concentration, the initial electrode

FIGURE1

Theelectrochemicalsystem:1:Pbcounterelectrode;2:Ag/AgClreference electrode;3:workingelectrode,4:magneticbar;5:magneticstirrer;6: electrolyticcell.

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response (I0) in presence of a known ATCl concentration

and in the absence of the inhibitor is determined. Then the electrode is immersed in an aqueous solution containing the pesticide at a given concentration and incubated for 30min. After washing, thecurrent intensity response(I1) is measured

againundertheinitialstandardconditionsofATCl.The inhibi-tionpercentageI(%)wasdeterminedaccordingto the expres-sion[9]:I(%)=[(I0I1)/I0]100.Itisimportanttoobservethat

thesampleincubationandtheelectrochemicalmeasurementare performed in two separate steps and among them a washing procedureisincluded.

Thedetectionlimit(LOD)correspondstotheamountof inhi-bitorwhichleadstothedecreaseofthesignalequaltothreetimes thestandarddeviationofthesignalmeasurement[28–30].

AChE

reactivation

experiments

Immediately afterthe enzymeinhibition byorganophosphorus pesticides,torestoreitsfunctionalityanoximeshouldbe admi-nistered [26,27].Enzymereactivationprocedure wascarriedout accordingtoGullaetal.[26].Inbrief,afterbiosensorwasexposed topesticide,itwaswashedwithPBS(pH7.5,0.1mM)and reacti-vated for15minwith theoximesTMB-4 or2-PAM, then trans-ferredtoelectrochemicalcellof10mL0.1PBSatpH7.5withKCl 0.1M containing 100mM ATCl to study the electrochemical response. Thereactivation efficiency(R%)wasestimatedas fol-lows:R0(%)=[(IrI0)/(I1I0)]100%,whereIrwasthe

steady-statecurrentofATClatbiosensorafterreactivation,andI1andI0

havethemeaningsasdescribedabove.

Experimental

data

treatment

Eachexperimentalpointinthefigureistheaverageoffive experi-mentscarriedoutunderthesameconditions.Theexperimental errorneverexceeded3%.Theoperationalstabilityoftheelectrode was more than 40 days. In fact,the electrodes were discarded when,after40days oftwo weeklyuse,the ATCldetermination understandardconditionswaschangedof20%.

Results

and

discussion

First, we have studied the electrode response by varying the appliedpotentialdifferenceunderconstantATClconcentration (20mM)andtemperature(258C).

Results reported inFigure 2 show the biosensors response as functionoftheappliedpotentialduetooxidationofthiocholine. It is observed an exponential increase of the current intensity accordingtoalawofthetype:

IðpotÞ¼Aepot=B

withA=2.690.61nAandB=24020mV.

Electrochemicaloxidationofthiocholinemayleadto dimeriza-tion products[31] causingfouling effectson the working elec-trode.Thiswasobservedathighpotentials(from500to700mV) afterfewminutesofoperation,whereasatanappliedpotentialof 410mV, the thiocholine response decreased significantly only after several hours of operation (data not shown). Due to the improvedlong-termapplicabilityoftheelectrode,apotentialof 410mV was used in the following investigation. Moreover, at 410mV,theinterferenceofotherelectro-oxidablechemicals pre-sentin realmatricesto beanalyzed isreduced.InFigure 3 the

electroderesponseatdifferentacetylthiocholineconcentrationsis reported. Data show a characteristic Michaelis–Menten kinetic mechanism. The apparent Michaelis–Menten constant (KM,app)

is the result of both the enzymatic affinity and the ratio of microscopic kinetic constants. The data of Figure 3 can be re-plottedinformofLineweaver–Burkequation:

1 I ¼ KM;app Imax 1 Cþ 1 Imax

whereIisthesteady-statecurrentafteradditionofsubstrate,Cis itsbulkconcentration,andImaxisthemaximumcurrentmeasured

undersaturatedsubstrateconditions.TheKM,appvaluefor

AChE-biosensorwascalculatedtobe1.120.14mM,whilethevalueof 1.030.07mAforImaxwasobtained.InsertinFigure3showsthe

linearcalibrationcurveofourbiosensorundertheadopted

experi-2000 1500 1000 500 0 0 100 200 300 400 500 600 700 500 400 300 200 100 0 0 50 100 150 200 250 300 350 I (nA) [ATCh] (μM) I (nA) [ATCh] (μM) FIGURE3

CurrentintensityresponseofAChEbasedbiosensor:saturationcurrentsasa functionofATClconcentration.Insert:thelinearpartofthecurveisreported ascalibrationcurve. 700 600 500 400 300 200 100 0 0 10 20 30 40 50 I (nA) Potential (mV) FIGURE2

Oxidationcurrentsasfunctionoftheappliedpotential.Experimental condition:T=258C,[ATCl]=20mMinphosphatebuffer0.1MpH7.5 containing0.1MKCl.

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mentalconditions.Fromthedataitispossibletoobtainavalueof 0.690.01mA/mMforthebiosensorsensitivity.

Havingcharacterizedourbiosensor,wecarriedoutexperiments todeterminethelinearcalibrationcurvesforthepesticidesofour interest.

TheirreversiblephosphorylationoftheAChEactivesitewhen biosensorsareincubatedwithpesticidesleadstoadecreaseinthe electroderesponse.Thedetectionofthepesticidesisquantifiedby measuringtheAChEinhibitionresponseaspercentdecreaseofthe currentbeforeandafterinhibition,whichisdirectlyproportional totheconcentrationofpesticideinthetestedsolution.

Figure4showsthe percentageinhibitionofthecurrent mea-suredbythebiosensorasafunctionofthepesticideconcentration accordingtothereportedmethodology.

The acetylthiocoline concentration and the temperature were100mMand250.58C,respectively.Inparticular,resultsin

Figure4refertoParaoxon(Figure4a)andtoDichlorvos(Figure4b). Forsakeofclarity,doublex-axisisintroduced.Inparticular,lower axisreports concentrations in ppm,whilein the upperaxisthe concentrationisreportedinmM.

In both figures, the data of percentage of inhibition of the currentintensityversusthepesticideconcentrationarewellfitted byanhyperbolicfunctionwithabehaviorsimilartoaMichaelis– Mentendependence.Thecorrespondingequations(R2=0.90for ParaoxonandR2=0.96forDichlorvos)allowedustocalculatethe

Km,Inhibandthemaximuminhibitionpercentage,IPmax(%).For

paraoxonthesevaluesresultedtobeequalto0.0120.004ppm and 80.46.7%, respectively. Similarly, the values of K m,In-hib=0.080.02ppmandIPmax(%)=79.83.8%wereobtained

fordichlorvos.Theinsertsinbothfiguresshowthelinearcalibration lines(S=257547ppm1_{forParaoxonandS=8849ppm}1_for

Dichlorvos)fromwhich,knowingthepercentageofinhibition,itis possibletodeterminetheconcentrationofpesticidepresentinthe aqueoussolution.

HavingascertainedthefunctionalityofourAChEcarbonpaste modifiedbiosensorindetectingthepesticidesofinterest,wehave studiedtheenzymereactivationafterinhibition.

Theoximesusedareseveralandinclude,amongother,TMB-4 and 2-PAM. These substances, when the phosphoester bond betweenthe enzymeand the inhibitoris not tooold, areable toremovetheinhibitorfromtheenzymeactivesitebymeansofa transesterificationreaction.Onthecontrary,iftheenzymeisleft inhibited forlong time,in the absence of refill ofany kindof oxime,itundergoestheso-called‘agingeffect’[32,33],resultingin apermanentenzymeinhibition.

Wehavetestedtwooximes,TMB-4and2-PAM,findingthat,at eachconcentrationused,theformerwasmoreeffectiveandthatthe optimalincubationtimewas15min(datanotshown).Inparticular, with a Paraoxon concentration of 28.6ppb (corresponding to 0.1mM),we havefoundthatat theconcentrationof3.5mMof eachoxime,the2-PAMexhibitsa76%ofreactivation,whilethe AChEtreatedwithTMB-4showsareactivationpercentagehigher than95%.

Justtogiveanexample,inFigure5itispossibletoobservethe current intensity response of the biosensor in the absence of inhibition(trace 1), inthe presence ofinhibitionby 15minof expositionto0.25mMparaoxon(trace2)andafterreactivationby 3.5mMTMB-4 (trace3). Ineachcase thefinalacetylthiocoline concentrationinthereactionmediumwas100mM.Itis interest-ing to observe that after reactivation, the biosensor response reachesquitethe valueobtained beforethe inhibitionby para-oxon.

InFigure6thereactivationpercentagesarereportedasa func-tionofthepesticideconcentration.TheTMB-4concentrationwas 3.5mM.Asexpected,thereactivationpercentagesdecreasewith thepesticideconcentration,beingtotalatlowestconcentrations. ResultsinFigure6showthatthecomplexpesticide/AChEismore resistant to the reactivation in the case of Dichlorvos. As for Paraoxon, the highest percentage of reactivation (90–100%) is obtained with inhibitor concentrations rangingfrom 0.003mM and 0.5mM;thispercentagedropstoa valueof60% forhigher pesticideconcentrations.Instead,inthecaseofDichlorvos,itcan beseenthatthereactivationpowerislower,withvaluesof30%for highpesticideconcentrations.

0,0 0,5 1,0 1,5 2,0 2,5 3,0 0 10 20 30 40 50 60 70 80 90 1000,0 1,8 3,6 5,4 7,2 9,0 10,8 0,0000 0,005 0,010 0,015 0,020 10 20 30 40 50 60 Inhibition (%) [Paraoxon] (ppm)

a

b

[Paraoxon] (μM) Inhibition (%) [Paraoxon] (ppm) 0 1 2 3 4 0 20 40 60 80 1000,0 4,5 9,1 13,6 18,2 0,00 0,01 0,02 0,03 0,04 0 5 10 15 20 25 30 35 Inhibition (%) [Dichlorvos] (ppm) [Dichlorvos] (μM) Inhibition (%) [Dichlorvos] (ppm) FIGURE4

Percentageofinhibitionasafunctionofpesticideconcentration:(a) Paraoxon;(b)Dichlorvos.Inserts:thelinearpartofthecurveisreportedas calibrationcurve.Experimentalcondition:T=258C,[ATCl]=100mMin phosphatebuffer0.1MpH7.5containing0.1MKCl,appliedpotential: +410mVvs.Ag/AgCl;pesticideincubationtime=30min.

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Tochallengethebiosensorwithrealsamples,thebiosensorwas thentestedusingriverwatersamplesfromtwodifferentsitesin AgriRiver(Basilicata,Italy).Thesites,identifiedas1and2,were distantrespectivelyabout1or2kmfromtheartificiallake‘Pietra delPertusillo’.Noinhibitionwasobservedforourbiosensor.To evaluatethe accuracyofthebiosensor,thesampleswerespiked with the two pesticides according to Arduini et al. [34]. Two different concentrations of Paraoxon or Dichlorvos were sepa-rately used. Table 1 shows the obtained results. A recovery of 87% and 140% was observed for Paraoxon, while a recovery varying between 83% and 120% was observed for Dichlorvos. Therelativestandarddeviation(R.S.D.)ofthreesuccessive mea-surementsofthe samesamplewasaroundof5%. Theseresults demonstrate goodrecoveryvaluesand lowmatrix effectonthe

biosensorsignal. The lowmatrix effect isdue tothe procedure adoptedforthepesticidesmeasurement(seeexperimentalpart).In fact,usingthismethod,itispossibletoavoidelectrochemicaland enzymaticinterferences[12].Briefly,theelectrochemical interfer-ences, which can be present in the real sample tested, were eliminatedbecausetheresidualenzymaticactivitywasmeasured inanewsubstratephosphatebuffersolution,inabsenceofreal sample.Inaddition,theenzymaticinterferencesduetoreversible inhibitors are avoided because after the incubation step, the biosensor iswashed with distilled waterand,in this way, only theinhibitorcovalentlylinkedtotheenzyme(organophosphorus andcarbammiccompounds)ismeasured.

Conclusions

Inthispaperwehavereportedtheresultsofthedeterminationof the concentrationofParaoxon and Dichlorvosby meansof an AChEcarbonpastebiosensors.Themethodologywasbasedonthe inhibitionoftheactivityofAChEentrappedinamixture(5:4:1by weight)ofcarbonpaste,oil and AChE,respectively. Theoxime TMB-4 resulted more effective than 2-PAM in reactivating the inhibitedAChE.

Otherpropertiesofour biosensorare:(a) adetectionlimitof 0.86ppbforParaoxonandof4.2ppbforDichlorvos;(b)an intra-electrodeerrorneverexceedingthe3%,whiletheinter-electrode reproducibilitywasaround7%;and(c)anoperationalstabilityof about40days.

Tocomparethecharacteristicsofourbiosensorswiththeones ofsimilarbiosensors,wemustremind,forexample,thataccording to the literature, the detection limit of differently developed biosensorsdepends onthe severalparameters suchaspH, tem-perature, the immobilization matrix and the time of reaction betweentheenzymeand theinhibitor.Thus,directcomparison ofthesensitivitybetweendifferentbiosensorsisnoteasyandthe citedparametersshouldbetakenintoconsideration.Furthermore, thedefinitionsofLODandlinearrangearenotunique.Therefore, todothis comparison,the LODand linearrangevaluesof bio-sensorswithdesigncharacteristicssimilartoourandtakenfrom

20 18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 60 70 80 90 100 Reactivation (%) [Pesticide] (μM) FIGURE6

Reactivationpercentagesasafunctionofpesticideconcentration.Symbols: (&)Paraoxon;(&)Dichlorvos.

300 250 200 150 100 50 0 0 10 20 30 40 50 60 70 80 90 3 2 1 Current (nA) Time (s) FIGURE5

Currentintensitymeasuredintheabsenceofinhibitor(trace1),inpresence ofinhibition(trace2),afterreactivationwith3.5mMTMB-4(trace3). Experimentalcondition:T=258C,[ATCl]=100mMinphosphatebuffer0.1M pH7.5containing0.1MKCl,appliedpotential:+410mVvs.Ag/AgCl; pesticideincubationtime=30min.

TABLE1

Recoverystudiesofspikedrealwatersamples Samples Paraoxon concentration added(ppb) Paroxon concentration found(ppb) Recovery (%) Site1 5 6 120 15 13 87 Site2 5 7 140 15 14 93 Samples Dichlorvos concentration added(ppb) Dichlorvos concentration found(ppb) Recovery (%) Site1 10 12 120 30 32 107 Site2 10 9 90 30 25 83

Experimentalconditions:ATCl(100mM),appliedpotential:+410mVvs.Ag/AgCl, phosphatebuffer0.1M+KCl0.1M,pH7.5.Incubationtime=30min.Allthevaluesare meanoftriplicatemeasurements.

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literatureandreportedinTable2,havebeenintegratedwiththe definitionsused toobtainthem. ExaminationofTable 2shows that our results, even under the indicated limitations, can be competitive withthe other reported in the tablewhen a more attentivecomparisonisattempted.Infact,insomecasesthebetter LODischaracterizedbyaverylimitedlinearrange(seeforexample ref. [41]) or LODreachesa very lowvalueonly whendifferent definitionsofthisparameteraregiven(seeref.[42],whoseresults werenotreportedinthetableduetomanydifferentLOD defini-tionsgivenalongthepaper).

Althoughmanyothertechnologicalapproachesarenowadays available[44],inthisworkwehavehighlightedthatoptimising theoperativeconditions,itispossibletorealizeasimplebiosensor based on enzyme immobilization in the carbon paste, which allows satisfactoryresultsin terms ofsensitivity and reproduci-bility.Moreover,theelectrochemicalsurfacecanbeeasilyrenewed andthebiosensorcanbereadytouseforsuccessiveanalysis.

InadditionweknowthatthereareothermoresensitiveACHE basedbiosensors,buttheseweredonewithengineeredenzymes

[45]andthentheyaremoreexpensive.

Inconclusionswheneconomyandeaseofusearemandatory, our biosensor can show characteristics betteror comparable in respecttothoseobtainedbyotherauthors.

Acknowledgements

ThisworkwashasbeensupportedbytheItalianConsortiumINBB withafellowshiptoDanielaDiTuoro,bytheItalianMinistryof HealthundertheNationalStrategicProjects‘Salutedelladonna’ (ISS/ISPESL)and‘EndocrineDisruptors’(ISPESL),andalsobythe ItalianMinistryofHealth/ISZM(Portici-Italy).

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TABLE2

LODsandlinearrangesforamperometricAChEbiosensors

Paraoxon Dichlorvos Ref.

LOD (ppb) Linearrange (ppb) LOD (ppb) Linearrange (ppb) 0.86 0.86–22.93 4.2 4.2–33.16 Presentpaper 51.6a 51.6–154.8e N.A. N.A. [35]

3b _{N.A. N.A. N.A. [31]}

2.86c _{N.A. 2.21}c _{N.A. [36]}

N.A. N.A. 0.69a N.A. [37]

N.A. N.A. 0.0025c _0.01–10e _[38]

0.29d

N.A. N.A. N.A. [39]

N.A. N.A. 132a _{N.A. [40]}

0.3a _{N.A. N.A. N.A. [41]}

10d

14–173e N.A. N.A. [28]

N.A. 300–1800f _{N.A. N.A. [26]}

94d

N.A. N.A. N.A. [29]

N.A. N.A. 0.02c N.A. [42]

0.025a _{0.025–0.175 N.A. N.A. [43]}

N.A.standsfornotavailable.

a

LODvaluecorrespondstotheconcentrationoftheinhibitorforwhichthereis10%of inhibition.

b

LODvaluecorrespondstotheconcentrationoftheinhibitorforwhichthereis5%of inhibition.

c

NoLODdefinition.

d

LODdefinedinasthispaper.

e

Linearrangedefinedinfunctionoflog(pesticideconcentration).

f

Linearrangedefinedinfunctionofpesticideconcentrationasinourpaper.

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