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Electrochimica
Acta
j o ur na l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / e l e ct a c t a
Laccase
biosensor
based
on
screen-printed
electrode
modified
with
thionine–carbon
black
nanocomposite,
for
Bisphenol
A
detection
M.
Portaccio
a,b,
D.
Di
Tuoro
a,b,
F.
Arduini
c,b,
D.
Moscone
c,b,
M.
Cammarota
a,
D.G.
Mita
a,b,d,∗,
M.
Lepore
a,baDipartimentodiMedicinaSperimentale,SecondaUniversitàdiNapoli,Napoli,Italy bConsorzioInteruniversitarioINBB,Roma,Italy
cDipartimentodiScienzeeTecnologieChimiche,UniversitàdiRomaTorVergata,Rome,Italy dInstituteofGeneticsandBiophysicsofCNR,Napoli,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received15May2013
Receivedinrevisedform12July2013 Accepted15July2013 Available online xxx Keywords: BisphenolA Laccase Carbonblack Thionine Screen-printedelectrode
a
b
s
t
r
a
c
t
TherelevanceofBisphenolA(BPA)inhumanhealthiswell-known.Forthisreasonwedesignedand developedabiosensorbasedonabionanocomposite(laccase–thionine–carbonblack)-modified screen-printedelectrode.Thionine,acommerciallyavailabledye,wasusedaselectrochemicalmediatorcoupled withananostructuredcarbonblack.Bymeansofcyclicvoltammetry,theinteractionofthionineadsorbed onmodifiedscreenprintedelectrodewithlaccase/BPAreactionproductshasbeenstudied.Inaddition, theimmobilizationoflaccasebyphysicaladsorptiononthesurfaceofthionine–carbonblackmodified screenprintedelectrodeswasinvestigated.Theresponseofthebiosensorhasbeenoptimizedintermsof enzymeloading,pHandappliedpotentialreachingalinearconcentrationrangeof0.5–50M,a sensitiv-ityof5.0±0.1nA/Mandalimit-of-detection(LOD)of0.2M.Thedevelopedbiosensorhasbeenalso challengedintomatojuicesamplescontainedinmetalliccanswherereleaseofBPAduetotheepoxyresin coatingcanbeassumed.Asatisfactoryrecoveryvaluecomprisedbetween92%and120%wasobtained.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
BisphenolA[BPA, 2,2-(4,4-dihydroxydiphenyl)propane; CAS RegistryNo.80-05-7]isproducedbycombiningacetoneand phe-nolandisanimportantorganicchemicalowingtoitswideuseas intermediateinthemanufactureofpolycarbonateplastics,epoxy resins,andflameretardants[1].Itsmainuseisascoatingofmetallic cans,powderpaints,dentalfillings,andantioxidantinplastics.It hasalsobeenusedasaninertingredientinpesticides,antioxidants andpolyvinylchloridestabilizer[2].Recently,BPAhasreceived considerableattentionduetoitsendocrinedisruptingactivityand possibletoxicimpactonenvironment[3–5].BPAlevelsintheg/L rangehavebeenfoundinbiological,foodandwatersamples[6,7]. ThepresenceofBPAisstilla“hottopic”:theEuropeanFoodSafety Authority(EFSA)isstillinvestigatingonnewriskassessmentof BPAand,veryrecently,initsnewsletter,invited“...MemberStates, researchinstitutions,academia,foodbusinessoperators,packaging businessoperatorsandotherstakeholderstosubmitdataonBPA,in particularitsoccurrenceinfoodandbeverages,migrationfromfood contactmaterials, occurrenceinfood contactsmaterials. Deadline:
∗ Correspondingauthorat:DipartimentodiMedicinaSperimentale,Seconda Uni-versitàdiNapoli,Napoli,Italy.Tel.:+390816132608;fax:+390816132608.
E-mailaddress:mita@igb.cnr.it(D.G.Mita).
31July2012”.Theabovefindingssuggestthatitis necessaryto determinetheBPApresencealsointraceamounts.
Traditional methods for BPA detection include chromato-graphic techniques coupled with mass spectrometry, capillary electrophoresisandsolidphasemicroextraction,methodsthatare timeconsuming,cannotbeperformedon-siteandrequire sam-plepre-treatment[8].Electrochemicalsensorscanproviderapid andon-siteBPAdetection.Forthispurposemanyresearchershave developedelectrochemicalsensorsusingtyrosinaseasbiological elementanddifferentimmobilizationprocedures[9–13].Recently, theuseofdifferenttyrosinase-functionalisednanoparticlesystems hasshowninterestingresults[14].
All the developed electrochemical sensors for BPA use the enzymetyrosinase,differentlyfromtheapproachusedforother phenolcompoundsforwhichotherenzymes,suchasperoxidase [15]andlaccase[16],havebeensuccessfullyemployed.In particu-lartheuseoflaccasecombinedwithaproperlychosenredox-active compoundcouldrepresentapotentialgoodcandidatefordesigning andfabricatingnovelBPAsensors.
Laccases(E.C.1.10.3.2)aredimericortetramericglycoproteins, containingfourcopperatomspermonomerdistributedinthree redoxsites: one ineach T1 andT2 sites,and two inT3 site. It isassumedthatthecatalysisfirstlyinvolvesT1Cureductionby thesubstrate,followedbyinternalelectrontransferfromT1Cuto T2andT3Cuand,finally,dioxygenreductionatT2andT3sites
0013-4686/$–seefrontmatter © 2013 Elsevier Ltd. All rights reserved.
[17].Laccasescatalyzetheoxidationofortho-andpara-diphenols, aminophenols,aryldiamines,polyphenols,polyamines,ligninas wellassomeinorganicions,coupledtothereductionof molec-ular dioxygento water [18,19]. In the case of BPA not all the reactionproductshavebeencharacterized.Forinstance,an inter-estingstudywasperformedbyFukudaetal.[20],inwhichthey demonstrated that theBPAwas metabolizedby laccasein two kindsofcompounds:onekindcomposedbyhighmolecularweight compoundsandanotherkindcomposedbylowmolecularweight compounds,oneofwhichwasidentifiedas4-isopropenylphenol bymeansofgaschromatography–massspectrophotometry.The 4-isopropenylphenolasoxidativedegradation producthasbeen identifiedusingchromatographymass–spectrometryalsoinother worksreportedinliterature[21,22].
Ithasbeenalsoreportedthatsomeredox-activecompounds, knownas“mediators”,allowenlargingtherangeofcompounds thatcanbetargetedforoxidationbylaccase[23].Themediator interactswiththeenzymeorwiththereactionproduct.Insome cases,typicallytheenzymefirstlyoxidizesthemediator,which dif-fusesawayfromtheenzymeactivesite,andsequentiallyoxidizes acompoundthatmaybeornottobesubstrateoftheenzyme.The mediatorthenreturnstoitsoriginalformandcansubsequently beusedtoaccomplishtheconversionofmoretargetspecies.The employmentofmediatorsmakestheuseoflaccasemoreattractive becauseitincreasesthenumberofpollutantsthatcanbetargeted [24].Whenmediatorsareinvolvedinlaccase-assistedprocesses, theelectrontransferfromthemediatortotheenzymeisfollowed byelectrondonationfromthetargetmoleculetooxidized media-tor,whichgivesrisetotheregenerationofthemediator[25–27].In othercases,instead,themediatorreactsdirectlywiththereaction products,asitoccursfortyrosinaseinteractingwithphenols[10]. Severalorganicandinorganiccompoundshavebeenreportedas effectivemediatorsfortheabovepurposes.Thelaccasereduction ofdioxygentowaterinthepresenceofdifferentredoxmediators ornanomaterials[28]hasbeenstudiedinotherelectrochemical applications[29–34].
Ithasbeenalsodemonstratedthattheuseofredoxmediators coupledwithcarbonnanomaterialscanfurtherimprovethe elec-trochemicalperformancesofthedevelopedsensor.Forexample carbonnanotubeswereusedtogetherwithferrocenedicarboxylic acid[35],azuredye[36],thionine[37].Amongthecarbon nanos-tructured materialssuchas carbon nanotubesand graphene, it wasrecentlydemonstratedtheusefuluseofcarbon black(CB). CBisanindustriallymanufacturedcolloidalmaterialthatconsists ofapproximatelysphericalcarbonprimaryparticleswith diam-etercomprisedfrom15to100nm,whichtypically formsfused aggregateswithsizesbelow1000nm.CBwasdemonstrated:(i)to haveelectrocatalyticpropertiestowardsmanycompoundssuchas ascorbicacid,dopamine,NADH,benzoquinone,epinephrine, cys-teine,thiocholine,hydrogenperoxide[38–43],and(ii)thatitcan beusedasthebasisforconstructionofatyrosinasebiosensorfor catecholdetection[44].Inaddition,ashighlightedinourpapers [41–43],this materialallows theproduction ofa stable disper-sionusing a cheapcarbon nanostructured material, asrecently confirmedbyCompton’sgroup[45].
InthispaperwedescribeadisposableBPAbiosensorbasedon laccase.Toourknowledge,thisisthefirstreportinliterature con-cerningbiosensorforBPAdetectionbasedonlaccasecoupledwith disposablesensor.Bymeansofcyclicvoltammetry(CV),theeffect ofthionineasamediatorhasbeeninvestigatedwhenthe immobi-lizedlaccaseinteractswithBPA.Inordertoincreasetheanalytical performanceandtheeasinessoftheproposedbiosensor,ascreen printedelectrode(SPE)modifiedbyusingananocompositeformed byCBandthioninehasbeenused.Alsointhiscase,toour knowl-edgethisisthefirsttimethattheCBwasusedcoupledwitharedox mediatorindevelopinganelectrochemicalsensor.
Fig.1.ChemicalstructureofBPA(a)andofthionine(b).
The biosensor response was examinedin terms of immobi-lizedenzymeunits,appliedelectricalpotentialandpH.Finallythe optimizedbiosensorhasbeenusedfortheamperometric determi-nationofBPAinaqueousbuffersolutionsandinpeeledtomatoes storedinmetalliccans.
2. Materialsandmethods 2.1. Materials
Laccase(20Units/mg)fromTrametesVersicolor,BisphenolA(see chemicalstructureinFig.1a),Thionineacetate(seechemical struc-tureinFig.1b),Nafion®andalltheotherchemicalswerepurchased fromSigma(Sigma–Aldrich, Milan,Italy)and usedwithout fur-therpurification.CarbonBlackN220(CB)wasobtainedfromCabot Corporation(Ravenna,Italy).Screen-PrintedElectrodesG-Sensor (SPEs) was obtainedfrom Ecobioservice and Research (Firenze, Italy).
Theworkingelectrodewasmadeofgraphiteanditsdiameter was0.3cm.
2.2. Methods
Theenzymaticworkingelectrodewaspreparedbymodifying thesurfaceofascreenprintedelectrodewith6Lofadispersion ofCB1mg/mLinacetonitrile[41].Aftersolventevaporation,5Lof thionine0.4mMaqueoussolutionwereaddedandthenthe elec-trodewasputinanovenat60◦Cforabout15h[13].Afterthis treatment,theelectrodewasfurthermodifiedbyadding5Lof laccasesolutionatdifferentconcentrations,inordertoobtain enzy-maticunitvaluesrangingfrom0.59Uto6.52U.Afterdrying,the electrodeswerecoveredwith3Lofaneutralizedaqueous solu-tionofNafion®2.2%(v/v)andlefttodryatroomtemperaturefor 30mininordertoavoidrapidenzymeleaking.Thewholescheme isreportedinFig.2a.
The BPA/laccasereaction productswere separately obtained bythefollowingprocedure:500Lof0.05McitratebufferatpH 4.5 were added to100L of 1mM BPA and 100L of laccase (40mg/mL)bothinthesamebufferandallowedtoreactat37◦C for1htoobtaintotalconversionofBPAinproduct.
Toobtainathionine–CB-modifiedSPE,twostepswerecarried out:(1)6LofadispersionofCB1mg/mLinacetonitrilewere placedonSPEasdescribedinourpreviouswork[41];(2)thionine wasmadetoadsorbonthemodifiedSPEaspreviouslydescribed.
AllthemeasurementswerecarriedoutusingSPEsconnected to a PalmSensinstrument (Palm Instruments,the Netherlands) coupled to a PC and were performed at room temperature (25.0±0.5◦C). The potentials were referred to the internal Ag
Fig.2. (a)Fabricationprocessoflaccasebiosensorbasedonscreen-printedelectrodemodifiedwiththionine–carbonblackandNafion.(b)Schemeofbiosensorelectrocatalytic mechanism.
pseudo-referenceelectrode of the SPE. The cyclic voltammetry experimentswerecarriedoutin0.1Mphosphatebufferplus0.1M KCl,pH=6.5andatscanrateof100mV/s.
Foramperometricexperiments,a magneticstirreranda stir-ringbarprovidedtheconvectivetransportintheelectrolyticcell filledwithavolumeof10mL.Thesemeasurementswerecarried outintheabsenceofKClinthereactionvolume,becausesalinity affectsthesolubilityofthereactionproducts,leadingto inactiva-tionorchangesintheenzymaticprocessesasaresultofinteractions withtheproduct.Inparticular,chlorinatedcompounds substan-tiallyinhibittheconversionofBPAand thenchlorineionisthe maincauseoflowconversion[24].
Eachexperimentalpointinthefiguresistheaverageoffour experimentscarriedoutunderthesameconditions.The experi-mentalerrorneverexceeded5%.
MicrographsofCB-SPE,thioninemodifiedCB-SPEandlaccase biosensorbasedonthioninemodified CB-SPEwereobtainedby scanning electron microscopy (SEM) using a Carl Zeiss model SUPRA40microscope.
3. Resultsanddiscussion
3.1. Cyclicvoltammetrymeasurementsusingthionineinsolution Preliminaryexperimentswereperformedinordertostudythe electrocatalyticpropertiesofthioninetowardsBPA/laccase reac-tionproducts.Thionineis anartificialorganicdyederivativeof phenothiazine,anditisusedasamediatorsinceitsformalpotential fallsbetween0.08and−0.25V,neartheredoxpotentialofmany biomolecules[13,37].
Initially100Lof 0.4mM thionine werepoured ontheSPE without any modification and the typical thionine voltammo-gramobtainedisshownincurve1ofFig.3a,withEpa=−330mV, Epc=−360mV,Ipa=1.47AeIpc=1.33AandaIpa/Ipcratioequal to1.10.
Afterwards,20LofBPA/laccasereactionproduct,obtainedas reportedinSection2.2,wereaddedtothe100Lof0.4mM thion-ineandpouredonthescreenprintedelectrode.TheCVinpresence ofBPA/laccasereactionproductandthionineisreportedincurve2 ofFig.3a.Itiseasytonoticearelevantchangeinthevoltammogram shapeaftertheadditionofBPA/laccasereactionproduct.The cur-rentsignalduetothioninereductionprocessincreasesinrespectto theoneinabsenceofBPA/laccasereactionproduct.Conversely,the
anodicpeakofcurrentsignalduetothethionineoxidationprocess decreases,asexpectedformediatedreactions.Theenzymatic reac-tioncausedanincreaseoftheamountofthionineoxontheworking electrodesurface,withtheconsequentincreaseofcathodic cur-rentpeak.Onthecontrary,theanodiccurrentpeakisproportional to theamount of thioninered and this quantity decreasesafter theenzyme/BPAreaction.Theseresultsconfirmthatthioninehas anelectrocatalyticactivitytowardsthereduction ofBPA/laccase oxidationproductsasshownbytheelectrocatalyticmechanism schemereportedinFig.2b.ThentheSPEsareanusefultoolfor mon-itoringtheelectrocatalyticeffectwiththeadvantage,inrespect totheconventionalelectrodessuchasglassycarbonelectrode,of usingafewmicrolitersofsolution.
3.2. Cyclicvoltammetrymeasurementsusingthionineadsorbed ontheworkingelectrodesurface
In order todevelop a ready-to-usebiosensor modified with thionine,this mediator wasadsorbedontheworkingelectrode surface,accordinglytoourpreviouslyoptimizedprocedureinthe caseofthioninemodifiedcarbonpasteelectrode[13].The thionine-modifiedSPEwasthuscheckedbycyclicvoltammetryinorderto testitsworkingstability.AsshowninFig.3b,theredoxpeaksof thioninecanbeobservedatEpa=−320mVandatEpc=−360mV withalargeincreaseinthepeakcurrents.However,after10scans, adecreaseofabout70%inanodicandcathodicintensitycurrentof thionineisobserved,demonstratingthatthesimplemodification byadsorptiongivesanunsatisfactoryworkingstability.
Inorder toavoidthis drawback,weconstructeda biosensor modifiedwithCB–thioninenanocomposite.Theapproachusedin thisworkwasthebilayerone,inwhichfirstly,alayerofCBwas depositedontheworkingelectrodesurfaceby“drop”coating, fol-lowedbyanotherlayerofthionine.Inthisway,athionine–CB–SPE was obtained. This latter was tested by cyclic voltammetry in ordertoinvestigateitsworkingstability.AsshowninFig.3c,the potentialofanodic andcathodicpeakswereEpa=−220mVand Epc=−260mV,respectively,andstablecyclicvoltammogramswere obtained.ThedifferenceoftheexperimentalEpfromtheideal surfaceredoxprocess(withEp=0)isduetolimitations associ-atedwithchargepropagationinthefilm,thechemicalinteraction betweenthe ionsand the modified film, and thepolarizability ofthecationinfluencingitspenetrationin oroutoffilm.Using thionine–CB–SPE,after10 scansonly asmall decreaseofabout
-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 -6000 -5000 -4000 -3000 -2000 -1000 0 1000
a)
2 1 C ur rent (n A) potential (V) -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4b)
time C ur rent ( A) potential (V) -0.4 -0.3 -0.2 -0.1 0.0 -20 -15 -10 -5 0 5 10 15 timec)
Cu rrent ( A) potential (V) -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 -10 -8 -6 -4 -2 0 2 4d)
2 1 Cu rr en t ( A) potential (V)Fig.3. (a)CyclicvoltammetryforSPEwiththioninesolutionintheabsence(curve1)andinthepresences(curve2)ofthelaccase/BPAreactionproducts.(b)Cyclicvoltammetry foranadsorbedthioninemodifiedSPE.(c)CyclicvoltammetryforanadsorbedthionineandCB-SPE.(d)CyclicvoltammetryforanadsorbedthionineandCB-SPEintheabsence (curve1)andinthepresences(curve2)ofthelaccase/BPAreactionproducts.Forallthemeasurementsvscan=100mV/s.
5% in themeasuredanodic and cathodic intensitycurrent was observed.Moreover,thepeakcurrents(Ipa=11AandIpc=16A) weremuch higherthan theonespreviousfoundinthecase of thionine–SPE(Ipa=0.39AandIpc=0.29A),demonstratingthe improvementduetothepresenceofcarbonblack,becausethe pres-enceofthenanomaterialCBN220,characterizedbyahighsurface area(120m2/g),allowstheadsorptioninastablewayofahigher amountofthionineontheworkingelectrodesurfaceinastable way.
InordertotesttheelectroactivitytowardstheBPA/laccase reac-tionproduct,cyclicvoltammetrywascarriedoutinabsenceand inpresenceofBPA/laccasereactionproducts.Fig.3dshowsthat thecurrentduetothethioninereductionincreaseswithrespect tothatobtainedintheabsenceofBPA/laccasereactionproducts, whileinthereversescan thecurrent oftheanodicpeakdueto thethionineoxidationdecreases,asexpectedformediated oxida-tivereactions.Theseresultsconfirmtheelectrocatalyticactivityof thionineadsorbedonCB-SPEtowardsthereductionofBPA/laccase reactionproducts.Thethionine–CB–SPEwasthuschosenfor fur-therstudies.
3.3. Electrochemicalcharacterizationofthionine–CB–SPE
Thenthethionine–CB–SPEwaselectrochemicallycharacterized. Thecurrent oftheanodic () andcathodic()peaksincreases
linearlywithincreasingscanrateupto200mV/s(Fig.4), demon-stratingthatthereisasurfacecontrolledelectrochemicalreaction. Infact,forthinfilmsandlowscanrates(iffinitediffusionoccurs withinthefilm),thin-layerbehaviourpredominates,andthepeak
0,0 0,2 0,4 0,6 0,8 1,0 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 curre nt ( A) scan rate (V/s)
4.0 4.5 5.0 5.5 6.0 6.5 7.0 -0.28 -0.26 -0.24 -0.22 -0.20 -0.18 -0.16 -0.14 -0.12 -0.10 -0.08 E 0 (V ) pH
Fig.5.FormalredoxpotentialasafunctionofpH.
current(Ip)isproportionaltothescanrate
v
accordingtothe fol-lowingequation: Ip= n2F2 4RT Awherenisthenumberoftheelectronsinvolvedintheredox reac-tion,FistheFaradayconstant,Risthegasconstant,Tistheabsolute temperature,Aisthesurfaceoftheworkingelectrodeassuming thatitssurfacecorrespondstothegeometricalareaand is sur-facecoverage.Athigh-scanratesitcanbeobservedthattheanodic andcathodicpeakcurrentsdeviatefromalineartrend.
Theeffectof pHontheredoxreactionof thionineadsorbed on the CB-SPEs was also evaluated as reported in Fig. 5. The thionineresulted tobeelectroactive whenit is workingin the pH range from 4.0 to 7.0 (Britton–Robinson buffer). The
rela-tionship between E◦ (V) and pH is described by the equation E◦(V)=(−0.057±0.003)pH+(0.136±0.016)(R=0.997).Theslope valueisequalto(−57±3)mV/pHanditisingoodagreementwith thetheoreticalvalueof−59mV/pH(at20◦C)forareversible elec-trontransfer processcoupledwithidenticalnumberof protons andelectrons[46].Inordertoevaluatethenumberofelectrons involved,thepeakwidthathalfheightofthecathodicpeak(Fig.3c) wasmeasuredandresultedequaltoof92mV[47],thusmeansthat theredoxreactionofthionineinvolvesatransferofoneelectron coupledtothetransferofoneproton.
3.4. SEMmicrographs
Inordertounderstandthemorphologicalchangesdueto differ-entstepsinelectrodepreparation(seeFig.2a),SEMimagesofbare SPE(Fig.6a),CB-SPE(Fig.6b),thioninemodifiedCB-SPE(Fig.6c) andlaccasebiosensorbasedonthioninemodifiedCB-SPE(Fig.6d) arereported.ThecomparisonbetweenFig.6aandbshowsthat thepresenceofCBiswellevidenced;thenanostructurematerial conferstoelectrodesurfacearoughtopography,whilethe pres-enceofthethioninelayerontheCB-SPEcausesonlyadifferent contrastinrespecttotheSPE-CBSEMimage.Arelevantsurface changewasobservedinFig.6dwhenNafionmembraneispresent ontheenzymaticelectrode.
3.5. Laccase-CB-SPEbiosensor
InordertodevelopalaccasebasedCB-SPEbiosensor charac-terizedbysatisfactorysensitivity,severalparametersaffectingthe analytical performancesofthebiosensor wereinvestigated and optimized.
3.5.1. Effectofenzymeloading
Theamountofenzymeimmobilizedontotheelectrodesurface isakey parametertoobtaina highsensitivityofthebiosensor. In detail we investigated the electrochemical response of the
electrode as a function of the amountof immobilized enzyme (0.59U,1.19U,2.36U,4.74Uand6.52Uoflaccaseatapotential of−200mVin0.05Mcitratebuffer,pH4.5).AsshowninFig.7a, theelectricresponse increaseswiththeamountofimmobilized laccase,reachingamaximumvaluewhenusing1.19Uofenzyme. Forgreateramounts,thereisasignificantdecreaseinthecurrent value.Thisbehaviourcouldbeattributedtotheincreaseoffilm thickness,leading toan increaseof interfacial electrontransfer resistance,makingtheelectrontransfermoredifficult[48].Forthis reason,subsequentexperimentswerecarriedoutusing1.19Uof laccaseimmobilizedontheworkingelectrodecoveredbyalayerof Nafiontoavoidtheleakageoftheenzymeduringthemeasurement. 3.5.2. Effectofappliedpotential
Inordertoobtainahighsensitivityintheelectrochemical detec-tionofenzymaticproduct,thebiosensorresponsetowardsBPAhas beenstudiedasafunctionoftheappliedpotentialin0.05M cit-ratebuffer,pH4.5.Weinvestigatedthebiosensorresponsevarying theappliedpotentialintherangebetween−300and−100mVvs internalAgpseudo-referenceelectrode.AsevidencedinFig.7b,the highestcurrentpeakisobtainedatthepotentialof−200mV,sothis workingpotentialhasbeenappliedinthesuccessiveamperometric measurements.
3.6. EffectofpH
ItiswellknowntheinfluenceofpHonthebiosensorresponse, thusthisinfluenceonthebiosensorresponsetoBPAata concen-trationof10MhasbeenstudiedinapHrangebetween3and5.8 in0.05Mcitratebuffer.TheresultsarereportedinFig.7c, show-ingthatcurrentvaluesarepHdependentandthattheoptimum pHoccursat4.5.ForlowerorhigherpHvalues,acurrentdecrease occurs,probablyduetolossorinactivationoftheenzyme activ-ity.TheseresultsareinagreementwiththeoptimumpHrange of 3.5–5reportedfor free laccase[24,49]and indicate thatthe immobilizationproceduredidnotalterthelaccaseactivity.Once establishedthattheoptimumvalueofthepeakcurrentoccursat pH4.5,theexperimentshenceforwardwerecarriedoutunderthese conditions.
3.7. BPAconcentrationdetermination
Onceoptimized the biosensor response in terms of enzyme loading(1.19U),pH(0.05McitratebufferatpH4.5)andapplied potential(−200mV),thesignalcurrentwasstudiedasafunction oftheBPAconcentration(Fig.8).
Intheinset(a)theamperometricsignalobtainedwithBPAata concentrationequalto20Misreportedanditshowstheresultof twosuccessiveadditionsofBPAstandard.
AscanbeseenfromFig.8,theelectricalresponseasafunction ofBPAconcentrationissimilartoaMichaelis–Mentenbehaviour, with a Kapp
m =161±18 M and Imax=1073±57 nA. The Kmapp valueissimilartoKmbiochemicalonereportedin[50]andobtained fromexperimentwithBPAandfreelaccase.
Linearity was obtained in a BPA concentration range of 0.5–50M,while thesensitivitywas5.0±0.1nA/M (seeinset (b)).Thelimit-of-detection(LOD)wasequalto0.2M.TheLODhas beencalculatedasratiosignal/noise(S/N)=3.Theintra-electrode repeatabilitywasaroundthe3%(n=5),whiletheinter-electrode reproducibilitywasaround5%.Concerningthestability,the elec-trodehadanoperationalstabilityofabouttwoweeks.
The LOD obtained is comparable with the one (0.15M) obtainedinourpreviousstudyinwhichatyrosinase–thionine car-bonpasteelectrodewasdeveloped,butbetterthantheone(23M) foundbyDempseyetal.[10]usingtyrosinase-thionineglassy car-bonelectrode. Moreover, in ourcase there is theadvantage of
0 1 2 3 4 5 6 7 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
a)
cu rr ent (n A )laccase unit
-300 -250 -200 -150 -100 35 40 45 50 55
b)
cu rr ent (n A) potential (mV) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 30 40 50 60 70 80 90 100c)
re la tive cu rr e nt (% ) pHFig.7.(a)Effectofenzymeunitsonthebiosensorresponseinpresenceof10M BPAin0.05Mcitratebuffer,pH4.5,appliedpotentialequalto−200mV,T=25◦C.
(b)Dependenceofbiosensorresponseontheappliedpotentialinthepresenceof 10MBPAin0.05Mcitratebuffer,pH4.5,T=25◦Cand1.19Ulaccase.(c)Effect
ofpHonthebiosensorresponseinpresenceof10MBPAin0.05Mcitratebuffer, appliedpotentialequalto−200mV,T=25◦Cand1.19Ulaccase.
0 50 100 150 200 250 300 0 100 200 300 400 500 600 700 cur ren t ( n A) [BPA] ( M)
Fig.8.DependenceofbiosensorresponseontheconcentrationofBPAin0.05M citratebuffer,pH4.5,appliedpotentialof−200mV,1.19Uoflaccase.Inset:(a) amperometricsignalobtainedwithtwosuccessiveadditionofBPAstandard,of 20M;(b)linearrangeforbiosensorresponse.
Table1
Recoverystudiesofspikedtomatojuicesamples.
Sample BPAconcentration added(M) BPAconcentration found(M) Recovery(%) A 10 12 120 A 20 18.5 92 B 10 9 90 B 20 19 95
Experimentalconditions:0.05Mcitratebuffer,pH4.5,appliedpotentialof−200mV, 1.19Uoflaccase.Allthevaluesaremeanoftriplicatemeasurements.
acost-effective biosensor,becausewe haveused(i)disposable,
cost-effectiveandminiaturizedscreen-printedelectrodeinstead
ofcarbonpasteorglassycarbonelectrodes(ii)thecarbonblack,
whichisacost-effectivenanostructuredmaterialand(iii)the
lac-caseenzyme,whichiscost-effectivethanthetyrosinaseenzyme.
3.8. BPAmeasurementsinrealsamples
Tochallengethebiosensorwithrealsamples,thebiosensorwas
thentestedusingtomatojuicecomingfrommetalcanscoatedwith
epoxicresins,whichareknowntocontainandleakBPA.Two
differ-entcommercialbrandswereinvestigated.Thejuiceswereproperly
treated[51]andeachsample(Aand B)wasexaminedwithour
biosensorusingtheprocedurepreviouslydescribed.No apprecia-blecurrentsignalswereobtained,accordingtothecircumstance thatthesamesamplesanalyzedwithHPLCgaveaBPA concentra-tionsmallerthanthevalueofourLOD.Toevaluatetheaccuracy ofourbiosensor,thesampleswerespikedwithknownamountof BPA.TwodifferentconcentrationsofBPAwereusedforeachsample andTable1showstheobtainedresults.Arecoveryvaryingbetween 92%and120%wasobtained.Therelativestandarddeviation(RSD) ofthreesuccessivemeasurementsofthesamesamplewasaround 5%.Theseresultssuggestagoodrecoveryvaluesandalowmatrix effectonthebiosensorsamples.
4. Conclusion
In this paper it hasbeen reported,for the first time toour knowledge,thedesignanddevelopmentofabiosensorbasedon
abionanocompositelaccase–thionine–carbonblackforBPA detec-tion.
Inparticular theinteractionwithlaccase/BPAreaction prod-uctsbythionineadsorbedonscreen-printedelectrodesmodified withnanostructuredcarbonblackwasinvestigated.Thethionine seemstobeaneffectiveelectrochemicalmediatortowardstheBPA detectioninbothtyrosinase[10,13]andlaccasebasedbiosensors. Theresponseofthebiosensorwasoptimizedintermsofenzyme loading,pHand applied potentialreachinga detection limit of micromolarslevel.
The biosensor proposed here shows better results when compared with laccase carbon paste biosensor [52] and tyrosinase–thionineglassycarbonelectrode[10].Thecouplingof thioninewithnanostructuredmaterials(carbonblackinourcase) thusallowsthedevelopmentofastable,miniaturizedandlowcost deviceforBPAdetection.
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