Acetylcholinesterase biosensor based on self-assembled monolayer-modified gold-screen printed electrodes for organophosphorus insecticide detection

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ContentslistsavailableatSciVerseScienceDirect

Sensors

and

Actuators

B:

Chemical

j o u r n al hom e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b

Acetylcholinesterase

biosensor

based

on

self-assembled

monolayer-modiﬁed

gold-screen

printed

electrodes

for

organophosphorus

insecticide

detection

Fabiana

Arduini

a,c,∗

_,

_Simone

_Guidone

a

_,

_Aziz

_Amine

b

_,

_Giuseppe

_Palleschi

a,c

_,

_Danila

_Moscone

a,c

a_Dipartimento_di_Scienze_e_Tecnologie_Chimiche,_Università_di_Roma_Tor_Vergata,_Via_Della_Ricerca_{Scientiﬁca,}₀₀₁₃₃_Rome,_Italy b_Faculté_de_Sciences_et_Techniques_de_Mohammadia,_B.P._146,_Mohammadia,_Morocco

c_Consorzio_{Interuniversitario}_Biostrutture_e_Biosistemi_“INBB”,_Viale_Medaglie_d’Oro,₃₀₅_Roma,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30June2012 Receivedinrevisedform 28September2012 Accepted3October2012 Available online 12 October 2012 Keywords:

Acetylcholinesterase Pesticide

Organophosphate Self-assembledmonolayer Goldscreen-printedelectrode

a

b

s

t

r

a

c

t

Amono-enzymaticacetylcholinesterase(AChE)amperometricbiosensorfororganophosphatedetection wasdevelopedimmobilizingtheAChEenzymeviaglutaraldehydeonapreformedcysteamine self-assembledmonolayer(SAM)ongold-screenprintedelectrodes(Au-SPEs).Theenzymaticactivitywas monitoredmeasuringtheenzymaticproduct,thiocholine,atanappliedpotentialof+400mVvs.Ag/AgCl usingferricyanideinsolutionaselectrochemicalmediator.Theelectrocatalyticactivityofferricyanide towardsthiocholinewasinvestigatedbyusingNicholson–Shainmethodandﬁndingasecondorder homogenousrateconstantksequalto(5.26±0.65)×104M−1s−1.Inordertodevelopasensitive biosen-sor,theeffectofcysteamineconcentration,durationofSAMdepositionandAChEconcentrationwere optimized.Usingparaoxonasmodelcompound,thebiosensorshowedalinearrangeupto40ppbwith adetectionlimitof2ppb(10%ofinhibition).Thebiosensorwassuccessfullychallengedwithdrinking watersampledemonstratingtobeausefulanalyticaltoolfororganophosphorusinsecticidedetection.

1. Introduction

Pesticidesareamongthemostimportantenvironmental pol-lutantsbecauseoftheirsigniﬁcantpresenceintheenvironment [1].Theorganophosphorusinsecticidesareoneofthemostused insecticidesduetheirhightoxicitybutlowpersistenceinthe envi-ronmentwhencomparedwiththeorganochlorinepesticides.Their toxicityisbasedontheabilitytoirreversiblyinhibitAChEwhichis akeyenzymeofnervoustransmission.AChErapidlyconvertsthe neurotransmitteracetylcholinetocholineandaceticacidafterthe transmissionofanerveimpulse.Aspartofnormalcholinergic neu-rotransmission,aproperlyfunctioningofAChEisacriticalstep;in fact,theinhibitionofthisenzymeleadstocholinergicdysfunction anddeath[2,3].Thedetectionoforganophosphorusinsecticides isgenerallycarriedoutusinggaschromatographyorhigh perfor-manceliquidchromatographythatrequireskilledpersonneland laboratoryset-up[4,5].Simpleandsensitivestrategiesfordetecting organophosphoruscompoundsarethereforecriticallyimportant inordertoperformthemeasurement“insitu”usingminiaturized, cost-effectiveandeasytouseanalyticalsystems.Inthiscontext, theuseof cholinesteraseenzymes hasshown great promiseto assemble enzyme sensorsfor environmentalscreening analysis [6–9].BymeasuringtheAChEactivitybeforeandafterexposure

∗ Correspondingauthor.Tel.:+390672594404;fax:+390672594328. E-mailaddress:fabiana.arduini@uniroma2.it(F.Arduini).

ofthebiosensortoenvironmentalsamples,itispossibleto quan-tifytheamountoforganophosphorusinsecticidespresentinthe sample.

ThedevelopmentofanAChEbiosensorrequiresthe immobi-lizationofAChEonthetransducerandtheimmobilizationisakey pointtoobtainasensitivebiosensor.Infact,theenzymeshould retainitsquaternarystructure,shouldbeclosetothetransducer andthefilmofthemembraneshouldbethininordertoavoida limiteddiffusionoftheenzymaticsubstrate.Inliteratureseveral techniquesforimmobilizingenzymesontransducersarereported, suchasadsorption[10],entrapmentbymeansofsol–gels[11,12] andcross-linking[13,14].Unfortunately,usuallyusingthesetypes ofimmobilizationnocontrolovertheorientationoftheenzymeis achieved[15].Inordertoavoidthisdrawback,theimmobilization byusingSAMhasbeenreportedasa successfullyalternativeto fabricatebiosensors[16].SAMswereusuallypreparedusingthe affinity of thiols such as alkanethiols for some metal surfaces, particularly gold. In this case, the main advantage consists in theimmobilizationoftheenzymeclosetotheelectrodesurface withahighdegreeofcontroloverthemoleculararchitectureof therecognitioninterface[17,18].Fewpapersinliteraturereport the assemblingof a AChE-SAMbiosensor. Somerset et al. have developedagoldelectrodemodifiedwithmercaptobenzothiazole andeitherpoly(o-methoxyaniline)orpoly(2,5-dimethoxyaniline) [19,20].AnAChEbasedamperometricbiosensorwasdeveloped by immobilizing the enzyme onto a self assembled modified goldelectrodeusing3-mercaptopropionicacid,glutaraldehydeor

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(N-cyclohexy-N -(2-morpholinoethyl)carbodiimide)methyl-p-toluenesulphonatebyPedrosa etal.[21,22].AnAChE biosensor wasalso constructed by meansof gold nanoparticlesand cys-teamineassembledonglassycarbonpaste[23]orbysinglewalled carbon nanotubes wrapped by thiol terminated single strand oligonucleotide(ssDNA)ongold[24].

In all these cases, however, keeping in mind that the organophosphorus insecticides are able to irreversibly inhibit the AChE, after each measurement the biosensor need to be re-preparedorreactivated.TheAChEbiosensor,infact,canbe reac-tivatedputtingincontactthebiosensor,afterthemeasurement, forseveralminuteswith2-PAM(pyridine-2-aldoxime methylio-dide)solution[25]or,forexample,obidoximesolution[26].The reactivation should be performed immediately after the mea-surement, in order to avoid the enzyme phenomenon called “ageing”thatrenderstheinhibitedenzymemoreresistanttothe reactivationbecomingpermanentlyinhibited[27].Inordertoavoid thesestepswithdrawbacksintermoftimeofanalysis,theuseof screenprintedelectrodes(SPEs)canbeasuccessfullyalternative.

ThegoldSPEshavetheadvantagestobeminiaturized,mass pro-duced,andcost-effective,thussuitableforasinglemeasurement, propertiesveryusefulinthecaseofirreversibleinhibitionbased biosensors.Inthepresentworkwereportthedevelopmentofa novelamperometricmono-enzymaticAChEbiosensorinwhichthe enzymeisimmobilizedviaglutaraldehydeonapreformedSAM ofcysteamineontoAu-SPE. Inordertohaveamono-enzymatic biosensor,theacetylthiocholinewasusedassubstrate.The enzy-maticproductthiocholinewasdetectedbyusingferricyanidein solutionaselectrochemicalmediator.

2. Materialsandmethods

2.1. Apparatus

Amperometric measurements were carried out using a VA 641amperometricdetector(Metrohm,Herisau,Switzerland), con-nectedtoanX-trecorder(L250E,Linseis,Selb,Germany).

Cyclic voltammetry (CV) was performed using an Autolab electrochemicalsystem(EcoChemie, Utrecht, TheNetherlands) equippedwithPGSTAT-12andGPESsoftware(EcoChemie,Utrecht, TheNetherlands).Electrochemicalimpedancespectroscopy(EIS) measurements were carried out in the same cell with a PC-controlledAutolab. A sinusoidal voltage perturbation of 10mV amplitudewasapplied over thefrequency range of 100kHz to 0.1Hzwith10measurementpointsperfrequencydecade.Forthe ﬁttingofthedataobtainedbyEIS,Z-viewssoftware(Scribner Asso-ciates,Inc.)wasused.

2.2. Electrodes

Au-SPEswereboughtfromEcobioservice(Florence,Italy).The diameteroftheworkingelectrodewas0.3cmresultinginan appar-entgeometricareaof 0.07cm2_._Before_thiol_{measurements,}_the referenceelectrodewaschlorinatedbyapplyingapotentialof0.6V betweenthesilverandanexternalAg/AgClelectrodefor20sina phosphatebuffersolutioninthepresenceof0.1MKCl[28].

2.3. Reagents

Allchemicalsfromcommercialsourceswereofanalyticalgrade. Potassium ferricyanide from Carlo Erba (Milano, Italy), acetyl-cholinesterase(AChE)fromelectriceel,acetylthiocholinechloride, cysteamine,glutaraldehyde andparaoxonwere purchasedfrom SigmaChemicalCompany(St.Louis,USA).

2.4. Thiocholinemeasurements

Thiocholine was produced enzymatically by AChE using acetylthiocholineassubstrate(becausethiocholineisnot commer-ciallyavailable). For thispurpose, 1mLof1Macetylthiocholine solution was prepared in phosphate buffer 0.1M (pH=8), and 100 units of AChE were added to this solution. After 1h, the concentration of thiocholine produced by AChE was estimated spectrophotometrically by Ellman’s method. For this purpose, 900␮L of phosphate buffer solution (0.1M, pH=8), 100␮L of 0.1MDTNB,and5␮Lthiocholinesolution(diluted1:100inwater) wereputinaspectrophotometriccells.Theabsorbancewas mea-sured, and the real concentration was evaluated by using the Lambert–Beerlawwith theknownmolar extinctioncoefﬁcient ofTNB(ε=13,600M−1cm−1)[29].After1h,theacetylthiocholine hydrolysisiscompleted,and1mLsolutionof1Mthiocholineis obtained.Thesolutionisstablefor1dayat4◦C.

Thiocholinemeasurementswerecarriedoutusing amperomet-ricbatchanalysisatAu-SPEinastirred0.05Mphosphatebuffer solution+0.1MKCl,pH7.4(10mL)containing1mMofferricyanide ionsatanappliedpotentialof+400mVvs.Ag/AgCl.Afteraround 7min,timerequiredforbaselinestabilization,thethiocholinewas addedandtheresponsewasrecorded.

2.5. AChEbiosensor

The Au-SPE, as receivedby Ecobioservice, wasimmersed in 100mM cysteamineaqueoussolutionfor 15hatroom temper-ature in darkness to formcysteamine monolayer. Then, it was thoroughlywashedwithdoubledistilledwatertoremovethe phys-icallyadsorbedcysteamine.Afterthat,thesensorwascoveredwith anaqueoussolutionofglutaraldehyde5%(v/v)for30min,then, itwasthoroughlywashedwithdoubledistilledwatertoremove theunreactedglutaraldehyde.Finallytheresultingelectrodewas incubatedwithAChEsolution(10U/mL)for 15h;afterthat,the biosensorwaswashedandmaintainedinphosphatebufferat4◦C (Scheme1).

2.6. Acetylthiocholinemeasurement

TheacetylthiocholinewasmeasuredusingAChEbiosensorat anappliedpotentialof+400mVvs.Ag/AgClinstirred0.05M phos-phatebuffersolution+0.1MKCl,pH7.4(10mL)containing1mM offerricyanideions.Whenastablebaselinecurrentwasreached, theacetylthiocholinewasaddedandtheanalyteresponseatthe steadystatewasrecorded.

2.7. InhibitionmeasurementusingAChEbiosensors

Paraoxoninaqueoussolutionswasusedasstandardinsecticide fortheAChEinhibitionmeasurements.Theenzymaticactivityof thebiosensorwasmeasuredbefore(i0)andafter(ii)itsexposure toparaoxon for a certain time (incubationtime) at 3mM sub-strate(acetylthiocholine)concentration.Aftertheincubationtime, thebiosensorwasrinsedthreetimeswithdistilledwaterandthe responsetowardsthesubstratewasmeasuredasdescribedabove. Thedegreeofinhibitionwascalculatedasarelativedecayofthe biosensorresponse.

I%= i0−ii i0 ×

100 (1)

wherei0andiirepresentthebiosensorresponsebeforeandafter theincubationprocedure,respectively.

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Scheme1.TheschemeoftheassemblingoftheAChEbiosensor.

2.8. Samplecollection

ThedrinkingwatersamplewascollectedatTorVergata Univer-sityLaboratory,theSaccoriverwatersamplewascollectednear Ceccano;realsampleswerediluted1:2with0.1Mphosphatebuffer solution+0.2MKCl,pH7.4anddirectlyanalysed.

3. Resultsanddiscussion

3.1. AmperometricthiocholinedetectionatAu-SPEbymeansof ferricyanide

Themono-enzymaticAChEbiosensorusestheacetylthiocholine assubstrate,thustheﬁrstgoalwasthedevelopmentofahighly sensitiveenzymaticproductthiocholine(RSH)sensor.Themajor drawbackrelativetotheelectrochemicaldetectionofthiocholine isthehighoverpotentialrequiredatmostconventionalelectrode surfaces(gold,platinumandcarbonpaste)andthefoulingofthe workingelectrodesurface.Toovercometheseproblems,the elec-trochemical detection of thiocholine could beperformed using nanostructuredmaterialsorredoxmediators[30–35].Inthelast case,theelectrochemicalmediatorcanbeadsorbedonthe work-ingelectrodesurfacesuchasinthecaseofPrussianBlue[33],cobalt hexacyanoferrate[34]oritcanbedirectlymixedtotheinkusedto printtheworkingelectrode,suchasinthecaseofcobalt phthalo-cyanine(CoPc)[35].In thecase ofAu-SPEsmodiﬁedwithSAM, ourchoicewastousetheelectrochemicalmediatorferricyanide insolutionbecauseitisabletoelectrocatalysetheoxidation of thiocholine[36].

The electrochemicalbehaviour of ferricyanidetowards thio-cholineoxidationwasﬁrstlyinvestigatedusingaCVtechniqueand aAu-SPE. Beforethethiocholinemeasurement,theAu-SPEwas conditionedbymeansof30CVs usingferricyanide10mM pre-paredin0.05Mphosphatebufferplus0.1MKCl,pH7.4atscanrate of50mV/s.Thisconditionstepwasnecessaryinordertodecrease thepeaktopeakseparationfrom314mVto93mVafter30scansas wecanseeinFig.1,thisbehaviourcanbeascribedtotheenhanced kineticrateafterelectrochemicalpretreatmentasreportedin lit-erature using carbon SPE [37]. Next, we have investigated the responseofthesoconditionedAu-SPEtowardsferricyanidebyCV overascanrangeof0.010–0.200V/s.Thecurrentofbothanodicand cathodicpeaksincreaseslinearlywiththesquarerootofthescan rate(datanotshown),indicatingasemi-inﬁnitelinear diffusion-controlledcurrent.Afterthat,theAu-SPEwasstudiedtoevaluate theelectrocatalyticeffectofferricyanidetowardsthethiocholine oxidation.

Fig.1. CVsofAu-SPEin10mMferricyanidein0.05Mphosphatebuffer+0.1MKCl, pH=7.4,scanrate50mV/s(continuousline=ﬁrstscan,longdashedline=10thscan, shortdashedline=20thscananddottedline=30thscan).

Fig. 2 shows a typical response of ik (kinetically controlled current)obtainedinpresenceofthiocholineandid(diffusion con-trolled current)obtainedinabsenceof thiocholine.Thegeneric reactioncanbedescribedbythefollowingequations:

ferricyanide+RSH→ferrocyanide+RSSR (2)

Fig.2.CVsofferricyanide4mMin0.05Mphosphatebuffer+0.1MKCl,pH=7.4, scanrate20mV/sinabsence(continuousline)andinpresenceofthiocholine10mM (dashedline)or20mM(dottedline).

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ferrocyanideelectrode←→ ferricyanide (3) According to the above equations, the addition of thiocholine causesanincreaseinconcentrationoftheferrocyanide,resultingin anincreaseoftheanodicpeakcurrent.Onthecontrary,thecathodic peakcurrent isproportional totheamountofferricyanide that decreasesaftertheadditionofRSH(thiocholine).Moreover,the increaseofthiocholineconcentrationleadstoafurtherincrease oftheanodicpeakcurrentand adecreaseofthecathodicpeak, conﬁrmingthepropertyofferricyanideaselectrocatalystfor thio-cholineoxidation.

Inordertocalculatethesecond-orderhomogeneousrate con-stantksforthereactionbetweenferricyanideandthiocholine,the theoryofstationaryelectrodevoltammetrydevelopedby Nichol-sonandShain[38]wasused.Calculationofkshasbeenperformed byvaryingscanratesintherangeof5–100mV/sandthiocholine concentrationintherangeof10–20mM,ﬁxingtheconcentration offerricyanideat4mM.Thereactionschemecanbedescribedas:

ferrocyanide−e−→ferricyanide (4)

thiocholine+ferricyanide←→ferrocyanidekf (5)

wherekfisthepseudo-ﬁrstorderrateconstant.Usingthe experi-mentalvaluesofik/idavalueof(kfRT/nFv)canbeobtainedfrom theworkingcurvereportedin Nicholsonand Shainpaper [38]. Plotting(kfRT/nFv)vs.1/

v

ateachconcentrationofthiocholine,it ispossibletocalculatekf.Then, aplotofkf vs.thiocholine con-centration wascarried out obtaining a linear behaviour whose slopewasequaltothesecond-orderhomogenousrateconstant ksforreactionbetweenferricyanideandthiocholine.Itwasfound equalto(5.26_±0.65)_×104_M−1_s−1_,_{demonstrating}_the_good elec-trontransferbetweenthiocholineasthiolandferricyanide[39]. Thisvalueis,forexample,higherthantheonefoundforthe electro-catalyticreductionofnitriteusingferricyanide(2.75×103_M−1_s−1₎

[40].

3.2. AmperometricthiocholinedetectionatAu-SPEmodiﬁedby meansofferricyanide

3.2.1. Choiceofappliedpotential

Aplotofcurrent/appliedpotentialusingathiocholineand fer-ricyanideconcentrationof3×10−4Mand1mMrespectively[36], wasconstructedintherangeoftheappliedpotential0–800mVvs. Ag/AgCl(datanotshown).Asexpected,theamperometricresponse followedthebehaviouroftheoxidationcurrentintheCV.The oxi-dationcurrentincreasedfrom0to+100mVvs.Ag/AgClreachinga plateauataround+200mVvs.Ag/AgCl.However,weobservedthe highestratiosignal/noiseat+400mVvs.Ag/AgCl,thusthisapplied potentialwasselectedfortherestofwork.

3.2.2. Analyticalfeaturesofthiocholinemeasurement

Thegoodelectroanalyticalperformancesofthedeveloped sys-tem were then conﬁrmed by performing amperometric batch measurements.Infact,ourpurposewasthedevelopmentofa sen-sorforthiocholinedetectionasaplatformfor anamperometric biosensorforinsecticidedetection.

Using the previous selected applied potential (+400mV vs. Ag/AgCl) and a ferricyanide concentration equal to 1mM [36], calibrationcurves were carried out obtaininga detection limit (S/N=3) of 3×10−6M together with a linear range up to 1×10−3M (R2₌_0.9964). _The _sensor _showed _also _a _sensitivity equalto113mAM−1cm−2,which ishigherthantheoneshown bytheSPEmodiﬁedwithCoPc(24mAM−1cm−2)and compara-blewiththatobtainedinthecaseofSPEmodiﬁedwithPrussian Blue(143mAM−1cm−2)[13].Moreover,thereproducibilitywas

Fig.3. CVsofferricyanide4mMin0.05Mphosphatebuffer+KCl0.1M,pH=7.4, scanrate20mV/susingabaregoldSPE(continuousline)andacysteaminemodiﬁed goldSPE(dashedline).

evaluated by studying the response of 5_×10−5M thiocholine, foundingaRSD%equalto5%(n=4).

3.3. FerricyanidebehaviouratAu-SPEmodiﬁedwithaSAMof cysteamine

Ourgoalwasthedevelopmentofbiosensorforinsecticide detec-tionimmobilizingtheAChEbySAM.Inthiscase,cysteaminewas selectedasalkanethioltakinginconsiderationthatglutaraldehyde willbe used tolink theAChE tocysteamine. Firstly, theeffect ofcysteaminecoverageofAu-SPE onferricyanideresponse was investigated,becausetheelectrontransferforredoxreactionsof differentmoleculesatSAMcanbecontrolledbyelectrostaticand hydrophobiceffects[41].

ACVstudywasperformedusingferricyanideatbareAu-SPEand Au-SPEmodiﬁedwithaSAMofcysteamine,preparedbydipping theAu-SPEina100mMcysteaminesolutionovernight (Cyst–Au-SPE).WeobservedthatinthecaseofbareAu-SPE,thepeaktopeak separationwas314mV;inthecaseofcysteaminemodiﬁed Au-SPE,instead,itwas89mV(Fig.3);itseemsthatthepresenceof cysteamineonthesurfaceoftheworkingelectrodecanimproveits electrochemicalperformance.Thisbehaviourshouldbeascribedto theelectrostaticinteractionbetweencysteamineandferricyanide. ThepKaofthecysteamineimmobilizedonthegoldelectrodewas estimatedbyShervedanietal.tobeequalto7.6,thusinthepH regionlowerthan7.6,theAusurfaceispositivelycharged[42].

Inourcase,weworkedatpH=7.4,thusthesurfaceofgold elec-trodemodiﬁedbySAMofcysteamineshouldbecharacterizedby positivecharge,reason forthatprobablytheCVofferricyanide, whichis negativelycharged,ischaracterizedbya peaktopeak separationlowerthanatthebareAu-SPE.

Inconclusion,inourcasethecysteaminewillbeusedto immo-bilizetheAChEbycross-linkingwithglutaraldehyde,butwehave alsodemonstratedthataSAMofcysteaminecanfacilitatethe elec-trontransferofferricyanideatworkingelectrodesurfaceofAu-SPE.

3.4. AChEbiosensor

3.4.1. Optimizationofbioactivelayer

InordertodevelopasensitiveAChEbiosensor,theAChEandthe cysteamineconcentrationsandthedepositiontimeofcysteamine onthegoldelectrodesurfacewereinvestigatedandoptimized.

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3.4.2. EffectofAChEconcentration

Theeffect of AChE concentrationon thebiosensor response wasinvestigated.TheAChEconcentrationwasvariedinarange comprisedbetween0.1and10U/mLusingaSAMpreparedwith cysteamine0.1mMandadepositiontimeof15h,anda glutaralde-hydesolutionat5%(v/v).Theresponseofthebiosensorswastested usingacetylthiocholine3× 10−4_M._We_found_that_in_the_case_of AChE0.1U/mL,1U/mLand10U/mL,acurrentequalto0,116and 209nAwasobserved, respectively.Theresultsshowedthatthe responseofthebiosensorincreasedwiththeincreaseoftheenzyme amountasexpected,butalsotheworkingstability(successively measured) increasedwith theincrease of the enzyme amount. Keepinginmindthat:(i)theAChEinhibitionbyorganophosphorus insecticidesisairreversible,thelowerpossibleamountofenzyme shouldbeused[6]and(ii)anenzymeloadinglowerthan10U/mL doesnotallowasatisfactoryworkingstability, thusanenzyme amountof10U/mLwasﬁnallyselectedforfurtherwork.

3.4.3. Effectofcysteamineconcentrationanddepositiontimeon goldelectrodesurface

Thedensityof hydrocarbonchainsongoldelectrode surface wasduetotheconcentrationofalkanethiolsusedandtheduration ofSAMdeposition.FortheinvestigationofSAMtimedeposition, Au-SPEwasimmersedfor 1and 15hin cysteaminesolutionat concentrationof0.1mM,usingAChEatconcentrationof10U/mL and glutaraldehyde at 5%(v/v). Theresponse of thebiosensors wastestedusingacetylthiocholine3×10−4M.Wehaveobserveda responsetowardsthesubstrateonlyinthecaseofbiosensor prea-paredwith15hasdepositiontime,thusthistime wasselected for furtherexperiments. In order toevaluate theeffect of cys-teamineconcentration,theAu-SPEwasimmersedincysteamine solutionatdifferentconcentrations:0.1,1,10and100mM,using AChEatconcentrationof10U/mLandglutaraldehydeat5%(v/v). Theresponseofthebiosensorswastestedusingacetylthiocholine 3_×10−4M.Thebiosensorspreparedusingcysteamine100mMhad thehighestresponse(around200nA)andthehighestworking sta-bility.Forelectrodesimmersedincysteamine0.1,1and10mMthe biosensorsallowedresultscharacterizedbylowworkingstability. Lookingattheseresults,weselectedasmostsuitablebiosensorin termsofworkingstability,reproducibilityandsensitivitytowards acetythiocholine,theonedevelopedusingSAMpreparedwith cys-teamine100mM,adepositiontimeof15h,AChEat10U/mLand glutaraldehydeat5%(v/v).Thisbiosensorwascharacterizedbya reproducibility(RSD%)intra-andinter-electrodeof2.3%and16%, respectively.ThehighvalueofRSD%inthecaseofinter-electrode reproducibilitydoesnotaffect theaccuracyof insecticide mea-surementsbecausetheywerecarriedoutmeasuringtheresponse beforeandaftertheexposuretotheorganophosphate,thus mea-suringtherelativedecayofenzymaticactivityofasinglebiosensor. 3.5. Electrochemicalimpedancespectroscopymeasurements

Electrochemical impedance spectroscopy can provide useful information ontheimpedance changesoftheelectrode surface duringthefabricationprocess ofbiosensors,giving information abouthowtheinterfacialregionofthegoldelectrodesurfacein presenceofSAMofcysteaminechangesbeforeandaftertheAChE immobilization,measuringthevalueofelectrontransferresistance (Rct).TheRct,estimatedaccordingtothediameterofthesemicircle presentatthehighfrequencyregion,represents,infact,the diffi-cultyofelectrontransferofferro/ferricyanideredoxprobebetween thesolutionand theelectrode. Fig.4showsthetypicalNyquist plot obtained for cysteamine modified Au-SPE (Cyst–Au-SPE), glutaraldehyde–Cyst–Au-SPEand AChE–glutaraldehyde–Cyst–Au-SPE(biosensor).In thisfiguretheNyquistplotofthebaregold SPE was omitted because characterized by a much higher Rct

Fig.4.ComplexplaneimpedanceplotsatopencircuitpotentialforCyst–Au-SPE(a), glutaraldehyde–Cyst–Au-SPE(b)andAChE-glutaraldehyde–Cyst–Au-SPE (biosen-sor)(c)using5mMferro/ferricyanidein0.1MKCl.Inset:Randlescircuit.

(72,187±315)thantheoneobtainedinthecaseofCyst–Au-SPE (407±9),conﬁrmingthedataobtainedusingtheCVtechnique that showed a better electron transfer of ferro/ferricyanide at Cyst–Au-SPEthantheatbareAu-SPE.

The fabrication process of biosensors was followed mea-suring the Rct values, observing that the Rct increases in the following order: Rct AChE–glutaraldehyde–Cyst–Au-SPE (2431±30)>glutaraldehyde–Cyst–Au-SPE (681±9)>Rct Cyst–Au-SPE (407±9), conﬁrming also the deposition of a glutaraldehydelayerandoftheAChEenzymeontheCyst–Au-SPE.

3.6. AChEbiosensorforinsecticidedetection

The optimized biosensor was challenged with the enzy-matic substrate acetylthiocholine. Fig. 5 shows the calibration curve obtainedfor different substrate concentrations described by MichaelisMenten equation. It waspossible tocalculate the apparent Michaelis Menten constant (KMapp), found equal to 1.1±0.2mM.Asubstrateconcentrationof3.0mMwaschosenfor theinhibitionmeasurements.

Fig.5.CalibrationplotofacetylthiocholinechlorideusingtheAChEbiosensor. Appliedpotential:+400mVvs.Ag/AgCl.0.05Mphosphatebuffer+0.1MKCl,pH 7.4+1mMferricyanide.

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Fig.6. Studyofincubationtime.Appliedpotential:+400mVvs.Ag/AgCl,0.05 phosphatebuffer+0.1MKCl,pH7.4+1mMferricyanide,3mMacetylthiocholine assubstrateconcentration,40ppbofparaoxonasinhibitor.

3.6.1. Studyofincubationtime

Theincubationtime isthereactiontimeoftheenzymewith theinhibitor.ForirreversibleinhibitionsuchastheoneofAChE byorganophosphorusinsecticides,itispossibletoachievelower detectionlimitsusinglongerincubationtimes;infact,thedegree oftheenzymeinhibitionusuallyincreaseswiththeincubationtime untilreachingaplateau[6].Inourcase(Fig.6)arapidincreaseofthe inhibitiondegreeupto15min,usingaconcentrationofparaoxon of40ppb,wasfound,reachingaplateauafter20min.Thus,atime of15minwasselectedasacompromisebetweenasensitive mea-surementandareasonablemeasurementtime.

3.6.2. Paraoxondetectioninstandardsolutionsandinwaterreal samples

We selected paraoxon as organophosphorus compound for insecticidedetectionusingthedevelopedbiosensor.Theparaoxon measurementwasperformedusingtheprocedurecalled“medium exchange”inwhichitispossibletoavoidelectrochemical inter-ferencesfromelectroactivespeciesandfromreversibleinhibitors duringthepesticidemeasurement[6,13].Briefly,theenzymatic activitymeasurementbeforeinhibitioniscarriedoutinbuffer solu-tioninpresenceoftheenzymaticsubstrate.After,thebiosensor isputincontactwiththesamplecontaminatedwithinsecticides for a selectedtime;then, the biosensoris rinsed severaltimes withdistilled water and the enzyme residualactivity is finally measuredin anewbufferaliquot inpresenceof theenzymatic substratebutinabsenceofanyinterferingspecies.Inthisway,it waspossibletoavoidbothelectrochemicalinterferencesandthe possiblepresenceofreversibleAChEinhibitorssuchasfluoride, Cd2+_,_Cu2+_,_Fe3+_,_Mn2+_and_{glycoalkaloids}_[43]_._The_measurements were performed using the selected incubation time of 15min, obtainingacalibrationcurvedescribedbythefollowingequation: y=(1.07±0.03)x+(8.09±0.51),R2₌_0.9868_with_a_linear_range_up to40ppband adetectionlimit(LOD),calculatedastheamount of paraoxon for obtaining a 10% of inhibition, equal to 2ppb. Theanalytical performances obtainedare competitivewiththe onesreportedinliteratureusing,forinstance,cobalt phthalocya-ninemodifiedcarbonepoxycompositewithAChEimmobilizedon nylonnet(LODequalto12ppbforparaoxon)[44],AChEbiosensor basedonapolishable7,7,8,8-tetracyanoquinodimethane-modified graphite-epoxybiocomposite(LODequalto27.5forparaoxon)[45], AChEcapturedinagelatinmembranecoupledwithcarbon screen-printedelectrode(LODequalto2.5ppbforparaoxon)[46],AChE immobilized byglutaraldeyde onto carbon screen-printed elec-trodemodifiedwithPrussianBlue(50%ofdegreeofinhibitionfor

Fig. 7.Storage stability as percentageof residual activity. Appliedpotential: +400mVvs.Ag/AgCl,0.05phosphatebuffer+0.1MKCl,pH7.4+1mMferricyanide, 3mMacetylthiocholineassubstrate.

paraoxon25ppb)[13].Theresultsfoundinthisworkarealso sat-isfactorywhencomparedwithAChEimmobilizedontoaSAMgold electrode(LODequalto9.3ppbforparathion)[22]withthe advan-tagein thecaseof goldscreen-printedelectrodesthattheyare massproducedandcost-effectivethussuitableforasingle mea-surement,propertyveryusefulinthecaseofirreversibleinhibition basedbiosensors.

Inordertoevaluatetheaccuracyofthemethod,thebiosensor waschallengedinspikeddrinkingwatersample.Drinkingwater samplecollectedandtestedinourlaboratorygavenodegreeof inhibition.When,thesamplewasfortiﬁedwith10ppbofparaoxon, arecoveryof103±3%(n=3)wasobtained.Inaddition,asample collectedfromtheSaccoriverwasanalysed,obtaininginthiscase adegreeofinhibitionequalto10.9±0.4%(n=3),corresponding to5.2±0.7ppbofparaoxoninthesample,keepinginmindthat therealsamplewasdiluted1:2withphosphatebuffer(seeSection 2.8).InordertoevaluatetheaccuracyofthebiosensorintheSacco riversample,thesamplewasalsofortiﬁedwith10ppbofparaoxon, obtainingarecoveryequalto97±5%(n=3).

3.6.3. Storagestabilityofbiosensor

Inordertotestthepracticabilityofthedevelopedbiosensor, thestoragestabilitywastested.Whenthebiosensor wasnotin use,itwasstoredat4◦Cinphosphatebuffersolution.Thestability wastestedmeasuringthebiosensorresponseina3mM acetylth-iocholinesolution. We observed a rapid decrease of enzymatic activityduringtheﬁrstweekuptoaround60%ofresidual enzy-maticactivity(Fig.7),afterthatitremainedalmoststableupto1 month.

4. Conclusions

Inthiswork,anAChEbiosensorfororganophosphorus insecti-cidesbasedonenzymeinhibitionwasdeveloped.TheAChEenzyme wasimmobilizedviaglutaraldehydeonapreformedcysteamine SAM on Au-SPEs. As reportedin literature, theimmobilization usingaselfassembled monolayerallowsobtainingahighly ori-entatedenzymeimmobilization,leadingtoalowdetectionlimit. Thestrategyofusingferricyanideinsolutionaselectrochemical mediatorallowedtoobtainasensitivesensorfortheenzymatic productdetection.Moreover,theelectrochemicalandenzymatic interferenceswereavoidedbecausethe“mediumexchange” mea-surement method was used. The biosensor was challenged in

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drinkingwatersampleobtainingsatisfactoryresults.Inaddition, theuseofAu-SPEsallowsasingleinsecticidemeasurementwhichis agreatadvantagesincethesecompoundsinhibittheAChEenzyme inirreversibleway.Multipleinsecticidemeasurementswiththe samebiosensorrequire,infact,areactivationoftheimmobilized enzyme or theuse of a renewable enzymatic membrane, both time-consumingprocedures. The developed biosensor hasthus demonstratedtobeanusefulanalyticaltoolforscreeninganalysis oforganophosphorusinsecticides.

Acknowledgements

This work was supported by National Industria 2015 (MI01 00223)ACQUA-SENSEproject.

References

[1]K.Y.Abdel-Halim,A.K.Salama,E.N.El-khateeb,N.M.Bakry, Organophospho-ruspollutants(OPP)inaquaticenvironmentatDamiettaGovernorate,Egypt: implicationsformonitoringandbiomarkerresponses,Chemosphere63(2006) 1491–1498.

[2]J.E. Storm, K.K. Rozman, J. Doull, Occupational exposure limits for 30 organophosphate pesticidesbasedoninhibitionof redbloodcell acetyl-cholinesterase,Toxicology150(2000)1–29.

[3] K.D.Spradling,J.F.Dillman,Themoleculartoxicologyofchemicalwarfarenerve agents,AdvancesinMolecularToxicology5(2011)111–144.

[4]http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8141b.pdf

(accessed26.06.12).

[5] L.BetowskiDonnelly,T.L.Jones,Theanalysisoforganophosphoruspesticide samplesbyhigh-performanceliquidchromatography/massspectrometryand high-performanceliquidchromatography/massspectrometry/mass spectrom-etry,EnvironmentalScienceandTechnology22(1988)1430–1434. [6] F. Arduini, A. Amine, D. Moscone, G. Palleschi, Biosensors based on

cholinesteraseinhibitionforinsecticides,nerveagentsandaﬂatoxinB1 detec-tion(review),MicrochimicaActa170(2010)193–214.

[7]S.Andreescu,J.L.Marty,Twentyyearsresearchincholinesterasebiosensors: frombasicresearchtopracticalapplications,BiomolecularEngineering23 (2006)1–15.

[8]M.Pohanka,D.Jun,H.Kalasz,K.Kuca,Cholinesterasebiosensorconstruction—a review,ProteinandPeptideLetters15(2009)795–798.

[9] M.DelCarlo,A.Pepe, M.Sergi,M.Mascini,A.Tarentini,D. Compagnone, Detectionofcoumaphosinhoneyusingascreeningmethodbasedonan elec-trochemicalacetylcholinesterasebioassay,Talanta81(2010)76–81. [10]C.Bonnet,S.Andreescu,J.L.Marty,Adsorption:aneasyandefﬁcient

immobili-sationofacetylcholinesteraseonscreen-printedelectrodes,AnalyticaChimica Acta481(2003)209–211.

[11]S.Andreescu,L.Barthelmebs,J.L.Marty,Immobilizationofacetylcholinesterase on screen-printedelectrodes:comparative studybetween three immobi-lization methods and applicationsto the detection oforganophosphorus insecticides,AnalyticaChimicaActa464(2002)171–180.

[12]K.Anitha,S.VenkataMohan, S.Jayarama Reddy,Developmentof acetyl-cholinesterasesilicasol–gelimmobilizedbiosensor—anapplicationtowards oxydemeton methyl detection, Biosensors and Bioelectronics 20 (2004) 848–856.

[13]F.Arduini,F.Ricci,C.S.Tuta,D.Moscone,A.Amine,G.Palleschi,Detectionof car-bamicandorganophosphoruspesticidesinwatersamplesusingcholinesterase biosensorbasedonPrussianBluemodiﬁedscreenprintedelectrode,Analytica ChimicaActa580(2006)155–162.

[14]F.Arduini,F.Ricci,A.Amine,D.Moscone,G.Palleschi,Fast,sensitiveand cost-effectivedetectionofnerveagentsinthegasphaseusingaportableinstrument andanelectrochemicalbiosensor,AnalyticalandBioanalyticalChemistry388 (2007)1049–1057.

[15]Th.Wink,S.J.vanZuilen,A.Bult,W.P.vanBennekom,Self-assembled mono-layersforbiosensors,Analyst122(1997)43R–50R.

[16]J.J.Gooding,D.B.Hibbert,Theapplicationofalkanethiolself-assembled mono-layerstoenzymeelectrodes,TrendsinAnalyticalChemistry18(1999)525–533. [17]A. Fragoso,N.Laboria, D. Latta, C.K.O’Sullivan, Electron permeable self-assembledmonolayersofdithiolatedaromaticscaffoldsongoldforbiosensor applications,AnalyticalChemistry80(2008)2556–2563.

[18] R.K. Shervedani, A. Hateﬁ-Mehrjardi, M.K. Babadi, Comparative electro-chemical study ofself-assembled monolayersof 2-mercaptobenzoxazole, 2-mercaptobenzothiazole,and2-mercaptobenzimidazoleformedon polycrys-tallinegoldelectrode,ElectrochimicaActa52(2007)7051–7060.

[19]V.S. Somerset, M.J. Klink, M.M.C. Sekota, P.G.L. Baker, E.I. Iwuoha, Polyaniline–mercaptobenzothiazole biosensor for organophosphate and carbamatepesticides,AnalyticalLetters39(2006)1683–1698.

[20] S.Somerset,P.Baker,E.I.Iwuoha,Mercaptobenzothiazoleon-goldorganic phasebiosensorsystems:1.Enhancedorganophosphatepesticide determi-nation,JournalofEnvironmentalScienceandHealthPartB:Pesticides,Food Contaminants,andAgriculturalWastes44(2009)164–178.

[21]V.A.Pedrosa,J.Caetano,S.A.S.Machado,M.Bertotti,Determinationofparathion andcarbarylpesticidesinwaterandfoodsamplesusingaselfassembled monolayer/acetylcholinesteraseelectrochemicalbiosensor,Sensors8(2008) 4600–4610.

[22]V.A.Pedrosa,J.Caetano,A.S.Sergio,S.A.S.Machado,R.S.Freire,M.Bertotti, Acetylcholinesterase immobilization on 3-mercaptopropionic acid self assembled monolayer for determination of pesticides,Electroanalysis 19 (2007)1415–1420.

[23]D.Du,W.Chen,J.Cai,J.Zhang,H.Tu,A.Zhang,Acetylcholinesterase biosen-sorbasedongoldnanoparticlesandcysteamineselfassembledmonolayerfor determinationofmonocrotophos,JournalofNanoscienceandNanotechnology 9(2009)2368–2373.

[24]S.Viswanathan,H.Radecka,J.Radecki,Electrochemicalbiosensorfor pesti-cidesbasedonacetylcholinesteraseimmobilizedonpolyanilinedepositedon verticallyassembledcarbonnanotubeswrappedwithssDNA,Biosensorsand Bioelectronics24(2009)2772–2777.

[25]A.N.Ivanov,R.R.Younusova,G.A.Evtugyn,F.Arduini,D.Moscone,G.Palleschi, Acetylcholinesterasebiosensorbasedonsingle-walledcarbonnanotubes—Co phtalocyaninefororganophosphoruspesticidesdetection,Talanta85(2011) 216–221.

[26] L.G. Zamﬁr, L. Rotariu, C. Bala, A novel, sensitive, reusable and low potentialacetylcholinesterasebiosensorforchlorpyrifosbasedon 1-butyl-3-methylimidazoliumtetraﬂuoroborate/multiwalled carbonnanotubesgel, BiosensorsandBioelectronics26(2011)3692–3695.

[27]R.M.Dawson, Tacrineslows therate ofageing ofsarin-inhibited acetyl-cholinesterase,NeuroscienceLetters100(1989)227–230.

[28]F.Ricci,F.Arduini,C.S.Tuta,U.Sozzo,D.Moscone,A.Amine,G.Palleschi, Glu-tathioneamperometricdetectionbasedonathiol–disulﬁdeexchangereaction, AnalyticaChimicaActa558(2006)164–170.

[29]G.L.Ellman,Tissuesulfhydrylgroups,ArchivesofBiochemistryandBiophysics 82(1959)70–77.

[30] K.A. Joshi,J. Tang,R. Haddon, J.Wang, W.Chen,A.Mulchandani,A dis-posable biosensor for organophosphorus nerve agents based on carbon nanotubes modiﬁed thick ﬁlm strip electrode, Electroanalysis 17(2005) 54–58.

[31] G.Liu, S.L.Riechers,M.C.Mellen,Y. Lin,Sensitive electrochemical detec-tion of enzymatically generated thiocholine at carbon nanotube modi-ﬁed glassy carbon electrode, Electrochemistry Communications7 (2005) 1163–1169.

[32] F.Arduini,C.Majorani,A.Amine,D.Moscone,G.Palleschi,Hg2+_detection_by

measuringthiolgroupswithahighlysensitivescreen-printedelectrode mod-iﬁedwithananostructuredcarbonblackﬁlm,ElectrochimicaActa56(2011) 4209–4215.

[33] F.Ricci,F.Arduini,A.Amine,D.Moscone,G.Palleschi,Characterisationof Prus-sianBluemodiﬁedscreenprintedelectrodesforthioldetection,Journalof ElectroanalyticalChemistry563(2004)229–237.

[34]F.Arduini,A.Cassisi,A.Amine,F.Ricci,D.Moscone,G.Palleschi, Electrocat-alyticoxidationofthiocholineatchemicallymodiﬁedcobalthexacyanoferrate screen-printedelectrodes,JournalofElectroanalyticalChemistry626(2009) 66–74.

[35]J.P. Hart, I.C. Hartley, Voltammetric and amperometric studies of thio-cholineatascreen-printedcarbonelectrodechemicallymodiﬁedwithcobalt phthalocyanine:studies towards a pesticide sensor, Analyst 119 (1994) 259–263.

[36]T. Neufeld, I. Eshkenazi, E. Cohen, J. Rishpon, A micro ﬂow injection electrochemicalbiosensorfororganophosphoruspesticides,Biosensorsand Bioelectronics15(2000)323–329.

[37]W.Y. Su, S.M. Wang, S.H. Cheng, Electrochemically pretreated screen-printed carbon electrodes for the simultaneous determination of aminophenol isomers, Journal of Electroanalytical Chemistry 651 (2011) 166–172.

[38]R.S.Nicholson,I.Shain,Theoryofstationaryelectrodepolarography.Single scanandcyclicmethodsappliedtoreversible,irreversible,andkineticsystems, AnalyticalChemistry36(1964)706–723.

[39]O. Nekrassova,G.D.Allen, N.S. Lawrence, L.Jiang, T.J. Jones,R.G. Comp-ton,The oxidation of cysteineby aqueous ferricyanide: a kinetic study usingborondopeddiamondelectrodevoltammetry,Electroanalysis14(2002) 1464–1469.

[40]R.Ojani,J.B.Raoof,E.Zarei,Electrocatalyticreductionofnitriteusing ferri-cyanide;Applicationforitssimpleandselectivedetermination,Electrochimica Acta52(2006)753–759.

[41]V.A.Pedrosa,T.R.L.C.Paixao,.R.S.Freire,M.Bertotti,Studiesonthe electro-chemicalbehaviorofacystineself-assembledmonolayermodiﬁedelectrode usingferrocyanideasaprobe,JournalofElectroanalyticalChemistry602(2007) 149–155.

[42]R.K.Shervedani,M.Bagherzadeh,S.A.Mozaffari,Determinationofdopamine inthepresenceofhigh concentrationofascorbicacidbyusinggold cys-teamineself-assembledmonolayersasananosensor,SensorsandActuators B115(2006)614–621.

[43]F.Arduini,A.Amine,D.Moscone,G.Palleschi,Reversibleenzyme inhibition-basedbiosensors:applicationsandanalyticalimprovementthroughdiagnostic inhibition(review),AnalyticalLetters42(2009)1258–1293.

[44]P-Skadal,M.Mascini,Senstivesetectionofpesticidesusing amperomet-ricsensorsbasedoncobalt–phthalocyanine-modiﬁedcompositeelectrodes and immobilizedcholinesterases, Biosensorsand Bioelectronics 7 (1992) 335–343.

(8)

[45]D.Martorell,F.Cespedes,E.Martinez-fabregas,S.Alegret,Determinationof organophosphorusabdcarbamatepesticidesusingabiosensorbasedona polishable7,7,8,8,tetracyanoquinodimethane-modiﬁedgraphite-epoxy bio-composite,AnalyticaChimicaActa337(1997)305–313.

[46]M.Pohanka,D.Jun,K.Kuca,Amperometricbiosensorsforrealtimeassaysof organophosphates,Sensors8(2008)5303–5312.

Biographies

FabianaArduiniisaresearcherinanalyticalchemistryattheChemicaland Technol-ogyDepartmentoftheUniversityofRome“TorVergata”.Shegraduated“cumlaude” inchemistry(2004)andobtainedherPh.D.degreeinchemicalscience(2007).Her researchinterestsincludethedevelopmentofelectrochemicalsensorsand biosen-sorsandtheirapplicationintheﬁeldofclinical,foodandenvironmentalanalytical chemistry.ShewasinvolvedinseveralNationalandEuropeanprojects.Sheisauthor ofmorethan30papersininternationalandnationalscientiﬁcjournalsandbooks andofmorethan50posterandoralpresentationsatNationalandInternational Congresses.

SimoneGuidoneisgraduatedinchemistryatUniversityofRome“TorVergata”in 2010withtheThesis:developmentofbiosensorsforpesticidedetection.

AzizAmineisaprofessorofbiochemistryintheFacultyofSciencesandTechniques oftheUniversityofHassanII-Mohammedia(Morocco).HereceivedPh.D.degree

fromtheFreeUniversityofBrusselsin1993.ProfessorAmine’sresearchoverthe last20yearshasfocusedonsensorsandbiosensorsandtheiruseinanalytical chem-istry.Heisauthorofmorethan100papersandscientiﬁccontributionsandhas servedascoordinatorofseveralnationalandinternationalresearchprojects.Heisa reviewerforseveralscientiﬁcinternationaljournals.Hewasco-ordinatorofvarious co-operationprojects.

GiuseppePalleschi,fullprofessorofanalyticalchemistry,hasbeentheHeadofthe DepartmentofChemicalScienceandTechnologyoftheUniversityofRome“Tor Vergata”for1995–2007.In2000heobtainedthe“LaureaHonorisCausa”fromthe UniversityofBucharest.Prof.Palleschi’sresearchoverthelast30yearshasbeen focusedonthedevelopmentofchemicalsensorsaswellasbio-andimmunosensors foruseintheareasofbiomedicine,foodandenvironmentalanalysis.Heistheauthor ofmorethan200papersininternationalscientiﬁcjournalsandaninvitedspeaker inmanyInternationalCongresses.

DanilaMosconeisfull professorinanalytical chemistryattheChemicaland TechnologyDepartmentoftheUniversityofRome“TorVergata”.Shehasbeen involvedinthefieldofbiosensorsforabout30years,andisanexpertin elec-trochemicalbiosensorandimmunosensorsassembling,theiranalyticalevaluation andapplicationforsolvinganalyticalproblems.Sheisactuallyinvolvedin Euro-peanprojectsandisresponsibleforNationalprojects.Herscientificactivityis summarizedinmorethan150papersoninternationalandnationalscientific jour-nalsandbooks,andinmorethan300oralandposterpresentationsatscientific meetings.