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Biomaterials based on photosynthetic membranes as potential sensors for herbicides

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Biomaterials based on photosynthetic membranes as potential sensors for herbicides
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  BiosensorsandBioelectronics 26 (2011) 4747–4752 ContentslistsavailableatScienceDirect Biosensors   and   Bioelectronics  j   ournal   home   page:www.elsevier.com/locate/bios Biomaterials   based   on   photosynthetic   membranes   aspotential   sensors   forherbicides Andrea   Ventrella a ,Lucia   Catucci a , b , ∗ ,Tiziana   Placido a , b ,Francesco   Longobardi a ,   Angela   Agostiano a , b , c a Dipartimentodi   Chimica,UniversitàdegliStudidiBari“AldoMoro”,ViaOrabona4,   70126Bari,Italy b IPCF-CNR,sez.Bari,ViaOrabona4,   70126Bari,Italy c INSTM,ViaG.Giusti9,50121Firenze,Italy a   r   t   i   c   le   i   nf   o  Articlehistory: Received1April2011Receivedinrevisedform20May   2011Accepted25May2011 Available online 1 June 2011 Keywords: PhotosyntheticmaterialLayerbyLayerBiosensorHerbicideOpticalassay a   b   s   t   ra   ct In   this   study,   ultrathin   filmmultilayersof    PhotosystemII-enriched   photosynthetic   membranes   (BBY)were   prepared   and   immobilized   on   quartz   substratesby   meansof    aLayerby   Layerprocedureexploitingelectrostatic   interactions   with   poly(ethylenimine)as   polyelectrolyte.Thebiomaterials   thus   obtainedwerecharacterized   by   meansof    opticaltechniques   and   AtomicForce   Microscopy,   highlightingthe   factthattheLayerby   Layerapproachallowedthe   BBYs   tobe   immobilized   with   satisfactory   results.   Theactivityof    these   hybrid   materials   wasevaluated   by   means   of    opticalassaysbased   onthe   HillReaction,   indicatingthat   the   biosamples,which   preservedabout   65%   of    their   srcinal   activityeventen   weeksafter   preparation,wereboth   stable   and   active.   Furthermore,   an   investigationof    the   biochips’   sensitivity   to   the   herbicideterbutryn,   as   a   modelanalyte,gaveinteresting   results:   inhibition   of    photosyntheticactivitywas   observedatterbutrynconcentrations   higher   than10 − 7 M,thus   evidencing   the   potentialof    such   biomaterialsintheenvironmental   biosensor   field. © 2011 Elsevier B.V. All rights reserved. 1.Introduction Theimportanceof    photosynthesisiscommonknowledge,buttodaynewvistasareopeningin   whichphotosyntheticorganismscouldalsobeusefulforbiotechnologicalapplications(Baldinietal.,2003;Croisetiereetal.,2001;GiardiandPace,2006;Koblìzeketal.,2002;Piletskayaetal.,1999).Photosyntheticorganismsincludenotonlyhigherplantsandalgae,butalsophotosyntheticbacte-ria;anyoftheseorganismscouldpotentiallybeusedtoobtainphotosyntheticmaterials.Suchbiomaterialsareofgreatinteresttoresearchersworkingonbiosensingandenvironmentalapplica-tions,duetotheirhighlevelof    sensitivitytoseveralclassesof    toxicspecies,rangingfromherbicidestoheavymetals.Thereasonsforthehighsensitivityof    photosyntheticmaterialstowardsherbicideslie   in   theactionmechanismstypicalofmanysuchtoxiccompounds.Twoclassesdeserveto   becited,namelythetriazine-derivativesandthephenylurea-derivatives,whichare   abletoinhibittheelectronfluxthroughthePhotosystemII   (PSII)insidethethylakoidmembrane,bylinkingtotheQ  B  siteandsubstitutingtheelectroncarrierplastoquinoneQ  B  (KiddandJames,1991).In addition,theseherbicidesareconsideredasdangeroussubstancesforhumanhealth:bothtriazinesandphenylureasareindicatedas ∗ Correspondingauthorat:DipartimentodiChimica,UniversitàdegliStudidiBari,viaOrabona4,70126Bari,   Italy.Tel.:+390805443443;fax:+390805442128. E-mailaddress: catucci@chimica.uniba.it(L.   Catucci). possiblycarcinogeniccompoundsbyEUofficialbodiesandtheEPA(Wackettetal.,2002),whoseregulationscurrentlyfixthelimits forpesticideconcentrationin   drinkingwaterat0.1  g/Lforanyindividualpesticide,and0.5  g/Lfortotalpesticides(GiardiandPace,2006).Clearly,methodsformonitoringthelevelsofsuchtoxicspeciesareveryimportant,especiallyin   wateror   ecosystemsclosetotheareaswherepesticidesare,orhavebeen,used.Conventionally,theanalyticaltechniquesemployedtoassessthelevelofcontamina-tionbypesticideshavebeengasand/orliquidchromatography,withtraditionalormassspectrometrydetectors.Thesetechniquesarepowerfultoolsfordetectingpesticides,eventhoughtheyaredifficultto   designforrapidandin   situanalyses.Tothisend,newunconventionalmethods,suchasbioassaysandbiosensors,havebeendevelopedinrecentyears   (DelCarloandCompagnone,2010).Thereis   aconsiderablebodyof    datain   theliteratureregardingthepossibilityof    exploitingphotosyntheticmaterialstodetectthepresenceoftoxiccompoundsbymeansof    assaysinsolution.Ontheonehand,forexample,ithasbeenreportedthatchloroplastsandthylakoidmembranesextractedfromspinachleavescanbesuspendedinsuitableaqueousbuffersandthenusedassensingtoolsforopticaldetectionof    atrazine,terbutrynanddiuron,aswellasof    copperandmercurybivalentcations(Campàsetal.,2008;Ventrellaetal.,2009,2010).Ontheotherhand,studiesevaluat-ingphotosyntheticefficiencyin   thepresenceof    toxiccompounds,haveemployedClark-typeelectrodesrecordingoxygenevolutionratechanges(Koblìzeketal.,2002),orelectrochemicaltechniques 0956-5663/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.05.043  4748  A.Ventrellaetal./    BiosensorsandBioelectronics  26 (2011) 4747–4752 basedonamperometrydetectingelectronfluxvariationsthroughthethylakoidmembranes(Croisetiereetal.,   2001).Theuseofassaysin   solutionis   oneof    theestablishedapproachesfor   applyingphotosyntheticmaterialsin   theenvironmentalfield,whileanothersuccessfulapproachreliesonthepreparationof biomaterial-modifiedsubstrates,suitableforbiosensingapplica-tions.Recently,biosensorsinvolvingphotosyntheticmaterialshavebeendeveloped,anddifferentresponseshavebeenobtaineddependingon:thetypeofphotosyntheticmaterials(nativeorengineeredbacterial/plantcells,membranesorproteins);thepro-cedureforimmobilizingthebiocomponents(seebelow);theanalyticaldetectiontechniqueadopted(amperometric,optical).Improvementsinevenoneofthethreeabove-mentionedfactors,couldclearlybeofgreatinterestin   thefieldof    herbicidedetection.Inparticular,to   date,variousmethodsforimmobilizingpho-tosyntheticmaterialshavebeenemployed,suchasphysisorptionontodifferentsubstrates(Frenseetal.,   1998;Naessensetal.,2000),inclusioninanaturalorsyntheticgelmatrix(Koblìzeketal.,2002)orchemicalcovalentbinding/cross-linking,althoughthemateri-alsproducedbythesemethodshavesofartendedto   becomelessactiveoveraperiodof    time(Rouillonetal.,2006).Averypromisingtechniqueforthedepositionof    biologicalmaterialsontosubstratesistheLayerbyLayer(LbL)method(Crespilhoetal.,   2006;Decheretal.,1992).It   allowstheadsorptionofalternateanionicandcationiclayersofpolyelectrolytesand/orproteins(whicharegenerallynegativelycharged)ontosolidsup-portsbyexploitingelectrostaticinteractions.Thistechniquehasgreatpotential,sinceitcanbeappliedtodifferentkindsof    materi-als(Oliveiraetal.,2002;Raposoetal.,   1997).Theversatilityof    thisapproachcouldallowthecreationof    severalhetero-structureswithspecializedfunctions.Inaddition,theadsorptionprocessesdonotdependonthesubstratesizeandtopology,andincreasingthenum-berofdepositedlayersresultsinalinearincreasein   filmthickness.Severalexamplesarereportedintheliteratureaccountingfortheimmobilizationof    proteinsbythisprocedure,althoughonlyfewexamplesdealwithphotosyntheticmaterialsandtheseexamplesfocusonthephotosyntheticbacteriaReactionCenter(Kongetal.,1999;Mallardietal.,2007).Inthisstudy,forthefirsttime,quartz(Qz)substrateswerefunc-tionalizedbyPSII-enrichedthylakoidmembranefractions(BBY)obtainedfromspinachleavesemployinga   LbLmethodandusingpoly(ethylenimine)(PEI)aspolyelectrolytetomodifythesubstrate.Thepreparedhybridinorganic-biomaterialwascharacterizedbymeansofAFMandopticaltechniquesinorderto   obtaininforma-tiononthesurfacedistributionandtoevaluatethebiocomponent’sactivity.Inparticular,thebioactivityof    thehybridmaterialswasevaluatedbyadaptingtheHill   Reactionopticalassaytosolidsub-strates.TheHillassayis   widelyemployedto   establishtheintegrityoftheelectrontransferchainefficiencyforphotosyntheticsam-plesinsolution(Hill,1937),buttotheauthors’knowledgethisis thefirsttimeithasbeenappliedto   asolidsubstrate.Thestabilityof thehybridmaterialswasevaluatedintermsofageingeffects,andtheresponseofthepreparedbiochipsin   thepresenceof    theherbicideterbutryn,atdifferentconcentrations,wasinvestigatedinviewofpossiblebiosensoristicapplications. 2.Materialsandmethods  2.1.Chemicals TritonX-100,[2-N-morpholine]ethane-sulphonicacid(MES),NaCl,CaCl 2 ,   NaHCO 3 ,   2,6-dichlorophenolindophenol(DCIP),ter-butryn,isopropanol,methanol,hydrogenperoxide(30%),sulphuricacid(96%)andPoly(ethylenimine)(PEI,50%(w/v)inH 2 O,MW ∼ 750000Da)werepurchasedfromSigma–Aldrich(St.Louis,MO);acetone(99.8%)waspurchasedfromCarloErbaReagents(Milano,Italy).Qz100 × 100 × 1(mm)slides(fusedsilica)werepurchasedfromEOT(Polcenigo-Pordenone,Italy)andsmallslideswerecut   withdimensionsofabout30 × 10(mm)fortheexperi-mentsreportedinthispaper.  2.2.IsolationofPSIIenrichedmembranefractions BBYmembraneswereisolatedfrommarketspinachleavesfol-lowingHankamer’sprocedure(Bertholdetal.,   1981;Hankameretal.,1997).TheconcentrationoftheBBYsampleswas   quantifiedaschlorophyllconcentration,inmgmL  − 1 (Arnon,1949).Thepho- tosyntheticmaterialswerestoredat − 80 ◦ Cafterfreezinginliquidnitrogen.  2.3.PreparationofPEI–BBYmultilayers TheimmobilizationoftheBBYsontoQzslideswasachievedexploitingtheLbLprocedure.TheQzslidewaspreviouslytreatedwithcleaningsolventsfor10min,respectivelymethanol,iso-propanolandacetone.AfterwardstheQzsubstratewas   negativelychargedbydippingitfor4min   insidea   “piranha”solution,i.e.oleumH 2 SO 4 /30%H 2 O 2  intheratio3:1,thenrinsedwithMilli-Q wateranddriedbynitrogenstream.Inthenextstep,a   layerof    posi-tivelychargedPEIwas   adsorbedby10min   incubationoftheQzslideinastirredsolutionof    1.6mg/ml   PEIinMilli-Qwater.AnegativelychargedBBYlayerwasthenadsorbedontothePEI-modifiedslide,bya10min   incubationina   suspensionof0.015mg/ml(chlorophyllconcentration)BBYinanaqueousbuffer(MNCB)containingMES(25mM),   NaCl(10mM),   CaCl 2  (5mM)   andNaHCO 3  (10mM)   at   pH6.5,understirringconditionsat4 ◦ C.TheslidewassubsequentlyrinsedwithMilli-Qwateranddriedwithnitrogenstream.Insuchaway,thefirstBBYlayer,called“layer1”,wasimmobilizedandfur-therlayerswereobtainedbyrepeatingthestepsreportedabove,untilsuitablePEI–BBYmultilayershadbeenproduced.ThedriedPEI–BBYmultilayersonQzwerestoredat4 ◦ C.  2.4.Absorptionmeasurements UV–visabsorptionspectrawererecordedusinga   VarianCARY 5000spectrophotometer(Varian,PaloAlto,USA).Foropticalchar-acterizationof    thebiosamplesin   aqueoussuspension,1cmpathlengthquartzcellswereused,whileformeasurementsonthebiomodifiedQzchips,asuitablesupportwasemployed.  2.5.Photochemicalassays Inthisstudy,theelectrontransferefficiencywasdeterminedbymeansoftheHillReactionopticalassay,i.e.DCIPphotoreduc-tionbyanelectrondonor(inthecaseofBBYs,water)catalyzedbythebiomaterialsstudied(Hill,1937;Ventrellaetal.,2009).Absorbancemeasurementswerecarriedoutat600nm.   Asetof four13Wfluorescenttubes(Osram,Munich,Germany),eachpro-ducingbasiccoolwhitelight,wasusedtoinduceDCIPreductionin   saturatingconditions;thephotonfluxdensityof    thewhitelightwas880  mol   m − 2 s − 1 .Thisassaywas   employednot   onlyfortheanalysisof    theBBYsuspensions,butalsoforthatof    theBBYmodi-fiedQzsupports;inbothcasesDCIPwas60  MandMNCBbufferwasused.  2.6.AtomicForceMicroscopy(AFM)characterization HeightmodeAFMinvestigationswereperformedonthebiomodifiedsubstratesin   air,atroomtemperature,bymeansof    A.Ventrellaetal./    BiosensorsandBioelectronics  26 (2011) 4747–4752 4749 Fig.1. CharacterizationofPEI–BBYmultilayersby   UV–visabsorptionspectroscopy.Panel“a”:Spectraof    BBYsimmobilizedontoQzatincreasingnumbersof    layers.Panel“b”:absorptionpeaksat   680nmfortheBBYsolutionusedforthe   immobilization,asafunctionof    thenumberof    layers.Inset:absorptionpeaksat680nmof    immobilizedBBYs   asafunctionof    the   numberoflayers.Reporteddatarepresentmeanvalues ± standarddeviationsobtainedfromthreereplicates. aPSIAXE-100SPMsystemin   AFMmodeandcantileverswithsili-connitridetipswereused,operatingin   non-contactmodeata1Hzscanrate.AsiliconSPMsensorfornon-contactAFM(PSIA),havingaforceconstantof42Nm − 1 anda   resonancefrequencyof320KHzwasused.AFMimageswereprocessedbyusinga   XEIProgramto   flattenthetopographicmicrographs,andremovetheslopeandcurvatureartefactsproducedbythescanningprocess. 3.Resultsanddiscussion  3.1.PreparationandcharacterizationofPEI–BBYmultilayers Asreportedin   Section2,   followingtheLbLprocedure,PEI–BBY multilayerswereassembledontoQzsubstrates;thesupportmate-rialwaschoseninfunctionof    theanalytictechniqueusedtomonitortheimmobilizationprocessandtheactivityof    thebioma-terialsdeposited.Indeed,in   thiswork,thehybridmaterialswereanalysedstepbystep,bymeansofUV–visabsorptioninordertoverifytheeffectiveloadingof    thebiocomponent,richin   chlorophyllpigments.IntheUV–visabsorptionspectra(Fig.1a),thetypicalabsorp- tionbandsofBBYatabout680and438nm(correspondingtochlorophyll a )andtheshouldersintherangeof    450–550nm(duetocarotenoidsandluteine)areclearlyvisible;itshouldbenotedthatPEIistransparentin   theexaminedwavelengthrange.It   canbeseenfromFig.1that   increasingthenumberoflayersincreasestheabsorptionsignalintensity,suggestingahigherquantitativeloadingofthebiocomponent.Nevertheless,anexcessivelayernumber(higherthan6–7),wasfoundtoproducenofurtherincrementintheabsorptionbands,asismoreeasilyobservableintheinsetof Fig.1b,wheretheabsorp- tionpeakvaluesofPEI/BBYmultilayersat680nmarereportedasafunctionoftheBBYlayernumber.Theseobservationswerecon-firmedbytheUV–visspectraoftheBBYsuspensionemployedfortransferringthebiosamplesontothesubstrate(Fig.1b).Therefore 6–7-layerscanbeconsideredastheoptimalnumbertoobtaintheimmobilizationofthegreatestamountof    BBYs.Thiswas   foundtobe   sufficienttoproduceintenseopticalsignals.Furtherconfirmationoftheimmobilizationof    theBBYmem-branefragmentswasachievedbyAttenuatedTotalReflectance-FourierTransformIRSpectroscopy(ATR-FTIR).TheATR-FTIR spectraofthePEI–BBYmultilayers(seeSupportinginformation)showedtheAmideIandAmideII   absorptionbandsat1653cm − 1 and1541cm − 1 respectively,thataretypicalof    proteinstructures,aswellasabandat1738cm − 1 ,   indicatingthepresenceof    lipidstructures.Thetopographyof    BBYsonthebiomodifiedsupportswas   inves-tigatedbynon-contactmodeAFM.Animagetakenofa   PEI–BBY 7-layermodifiedslide,obtainedon5  marea,is   showninFig.2a, togetherwiththecorrespondingheightprofiles(Fig.2b).The patchyanddensedistributionof    structures,observedevenat40  m   (imagesnotshown),canbesafelyattributedtoBBYsam-plessincenocomparablesignalswereobservedforQzsupportsmodifiedwithonlyPEI(seeSupportinginformation).Asreportedinthelineprofile(Fig.2b),the  z  -axisdimensionsof    thesestructuresareapproximately20nm,   40nm,   evenreaching80nm,   asidentifiedbytheincreasingbrightnessinFig.2a.Such heightvaluesareingoodagreementwiththesizeof    thestackedgranafragmentsof    our   biosamples.Eventhoughitis   notpossibleto   evaluateaccuratelythelateraldimensions,wecanassumethelatterarewithinafewhundrednanometres,in   accordancewiththeaveragediameterof    granadisks,reportedto   beintherangeof 350–600nm(ArvidssonandSundby,1999;Kirchhoffetal.,   2008;StaehelinandVanderStaay,1996).Interestingly,a   3D   magnificationofa   2  marea(Fig.2c)shows steepincreasesin   heighttodifferentplateaus.Accordingtothelit-erature,a   valueof    about10–11nmcouldbeexpectedforasinglegranafragment(i.e.asinglebilayerfragment),andmostBBYmem-branesconsistofa   stackof    two   granadisks;therefore,theheightlevelsobservedherecouldrepresentastackoftwo,fourandeightgranafragments,respectively(Nieldetal.,   2002;Kirchhoffetal.,2008).Thevariabilityinheightobservediscertainlydueto   thedif-ferentdegreeof    fragmentationof    granathylakoidsoccurringduringtheBBYpreparation.  3.2.FunctionalityandstabilityoftheimmobilizedBBYs OncethePEI–BBYmultilayershadbeenprepared,measure-mentswerecarriedoutto   testwhetherBBYactivitywaspreservedbyobservingtheelectrontransferefficiencythrougha   Hill   Reactionbasedopticalassay(seeSection2).Thismethod,asmentionedabove,isusuallyemployedinsolution,andhasbeenadaptedhere,forthefirsttimeto   solidbiofunctionalizedsubstrates.Thedecreaseinabsorbanceintensityat600nmfora   solutioncontainingoxidizedDCIPandafreshlyprepared7-layerPEI–BBY biochipshowedthatthebiochipwasactiveafterpreparation,sincetheimmobilizedBBYlayerswerefoundtobecapableof performingDCIPreduction.Thedependencyofthe“specificelec-trontransferefficiency”forPEI/BBYmultilayersonthenumberof depositedlayerswasverified;theparameterindicatedas“specificelectrontransferefficiency”was   calculatedastheratiobetweentheabsorbancechangeat600nm(fromtheHill   assay)andtheabsorbanceof    thebiochipat680nm,   inordertoexcludeanyeffect  4750  A.Ventrellaetal./    BiosensorsandBioelectronics  26 (2011) 4747–4752 Fig.2. Morphologicalcharacterization.Panel“a”:AFMimageof    theBBYmultilayers(7-layers)ataresolutionof    5  m.Panel“b”:   heightprofilecorrespondingto   thesectionindicatedbythelineinpanel“a”.Panel“c”:3Dmagnificationof    a2  marea. onthechipresponsesdueto   possiblesmallvariationsof    absorbance(i.e.   ofamountofimmobilizedbiomaterial)in   thedifferentbiochipsusedforthisexperiment.As   result,noremarkableactivitywasfounduntillayer4wasdeposited,whilefora   highernumberoflay-ers,theactivitywasfoundtoincreaseasa   functionoftheamountof immobilizedBBYs(Fig.3).Itcanbededucedthat   theoptimalnum-berfordepositedlayersis7,   sinceforhighernumbersoflayersnoincrementintheabsorptionsignals(i.e.amountofimmobilizedbiomaterial)wasobserved.Inordertoevaluatethelossof    activityproducedbytheBBY multilayerpreparationwithrespecttoBBYsolutionsbeforeimmo-bilization,HillReactionopticalassayswereperformedona   BBY suspensioncharacterizedbythesameabsorbanceastheimmo-bilizedsamples.Theresultsshowedthatafterimmobilizationtheactivitywaspreservedat85–90%.ThestabilityoftheBBYmultilayerpreparationwas   theneval-uatedbycomparingtheactivitiesofchipsstoredfor1,4and10weekswithfreshlypreparedchips.Theresults,reportedasper-centagesoftheresidueelectrontransferefficiency,areshowninFig.4.Itcanbeseenthatthebiochipsshowedahighpercentageof activityduringthefirstweek,andinterestinglyaconsiderableactivity(about65%)wasobservedeven10weeksafterprepara- Fig.3. EffectofthenumberoflayersonPEI–BBYmultilayeractivity.Dependencyof the“specificelectrontransferefficiency”ofaPEI–BBYmultilayerslideon   thenum-ber   ofimmobilizedlayers,after30min   illuminationassay.Reporteddatarepresentmean   values ± standarddeviationsobtainedfromthreereplicates. tion.It   isworthnotingthatthisstabilityvalueis   considerablyhigherthanthoseobtainedimmobilizingphotosyntheticmateri-alsbymeansof    othertechniques(Rouillonetal.,   2006).Moreover,itshouldbehighlightedthatnot   onlydid   thegeneralaspectofthePEI–BBYmultilayerabsorbancespectrumnot   changethroughouttheperiodof    observation,butadecreaseinthemaximuminten-sityvalueswasobservedonlyduringthefirstweek.Afterwards,nosignificantchangein   theabsorbancevalueswas   noticed,evi-dencethattheimmobilizationprocedureherereportedallowsthepreparationof    relativelystablesampleswithoutanexcessivelossofbiomaterialduringstorageperiod.  3.3.Sensingresponsestoamodelanalyte TheBBYmodifiedQzsupportswereusedto   testtheirabilitytosensethepresenceof    herbicidesbelongingto   theclassoftriazine-derivatives;specifically,fortheseteststheherbicideterbutrynwasemployed.Inordertofindtheoptimalconditionsforthetests,threeBBY chipswerefloodedfordifferentincubationperiods,i.e.for10,30and60min,respectively,in   suitablebeakerscontainingterbutrynsolutionsatthesameconcentration;aftertheincubationwithter-butryn,theelectrontransferefficiencyforeachchipwasevaluated Fig.4. StabilityofthePEI–BBY7-layerslides.Residueelectrontransferefficiency,atdifferentsamplingtimesafterpreparation,calculatedasapercentageofthevalueobtainedfromfreshlypreparedPEI–BBY7-layerslides.Reporteddatarepresentmeanvalues ± standarddeviationsobtainedfromthreereplicates.   A.Ventrellaetal./    BiosensorsandBioelectronics  26 (2011) 4747–4752 4751 Fig.5. Effectofincubationtimewithterbutryn.Residueelectrontransferefficiencyof    aPEI–BBY7-layerslideimmersedina   solutioncontaining10 − 4 Mterbutryn,asafunctionofincubationtime,calculatedasa   percentageof    the   valueobtainedfroma   PEI–BBY7-layerslidenot   treatedwithterbutryn.Reporteddatarepresentmeanvalues   ± standarddeviationsobtainedfromthreereplicates. by   meansoftheHillReactionopticalassay,andcomparedwiththatofaBBYchipwhichhadnotbeenincubatedwithterbutryn.Therelevantactivitywasplottedina   graph(see   Fig.5)asafunctionofincubationtimewiththeherbicide:itcanbeclearlyobservedthatanincubationtimeof    10min   didnotproduceanyremarkableinhibitoryeffectontotheBBYchips,while30min   werefoundtobesufficientfora   completeinhibitionoftheelectrontrans-ferprocess.Followingthis,thedependencyoftheBBYchips’responseonterbutrynconcentrationwasevaluated.Thegraphin   Fig.6showsthe“specificelectrontransferefficiency”of    BBYchipsplottedagainstthelogarithmof    theterbutrynconcentration,intherangeof10 − 10 –10 − 4 M.Ascanbeeasilynoted,thehighertheherbicideconcentration,themorePEI–BBYactivitywasinhibited.Thechipsallowedter-butryntobedetectedatconcentrationshigherthan1.58 ×   10 − 7 M.Thisvaluewasobtainedasfollows:let  y   and  x   beconsideredasthedependentandindependentvariablesin   Fig.6;afterrecord-ingtheassayresponsesforfiveblankreplicates,theaverageblankvalue(  y B )wasfoundtobeequalto   2.41(withtheunitsreportedinFig.6).Thenot-detectableintervalwasconsideredas  y B ± 3 S  B ,where S  B  representsthecontributionof    theblankstandarddevia- Fig.6. Effectofterbutrynconcentration.Dependencyof    the“specificelectrontrans-ferefficiency”of    aPEI–BBY7-layerslideon   theterbutrynconcentration,aftera   30min   incubation.Reporteddatarepresentmeanvalues ± standarddeviationsobtainedfromthreereplicates. tion,equalto   0.13fortheblankherein.Insucha   waythedependentvariablevalueofthedetectionlimit(  y lod )wasfoundtobeequalto   2.02.Thelinearregressionobtainedbyfittingthefourexperi-mentalpointsrelevantto   concentrations10 − 8 ,10 − 7 ,10 − 6 ,10 − 5 M(10 − 10 M   was   notconsideredsinceclearlyoutof    thedynamicrangeofthegraph)wasusedtofind  x lod =   − 6.80,thatisthelogarithmof theminimumconcentrationdetectable,andsotherelevantdetec-tionlimitwas   foundtobe   1.58 × 10 − 7 M.Althoughthisdetectionlimitvalueis   higherthanthoseobtainedbyconventionalmethodsorinnovativemethodsbasedongenet-icallyengineeredphotosyntheticproteins(Giardietal.,   2009),itiscomparabletothoseobtainedbynon-conventionaltechniquesandsensorsinvolvingnativephotosyntheticmaterials(DelCarloandCompagnone,2010). 4.Conclusions In   thisstudy,theLbLprocedurewas   usedfortheimmobilizationofBBYsontoQzslides,obtainingbiomaterialswithdifferentnum-bersof    layers;PEIwas   employedaspolyelectrolyte.ThePEI–BBY multilayerswerefoundtobewellcoveredandatthesametimethebiologicalactivitiesof    theBBYsused,estimatedbymeansofopti-calassaysaselectrontransferefficiencieswerefoundtoremainhighimmediatelyafterimmobilization.Inaddition,thebiomateri-alswerefoundtopreserveremarkablyhighactivityvalues(about65%of    theirsrcinalvalues)eventenweeksafterpreparation.Theseresultsareofconsiderableinterestsincetheyare,toourknowledge,thefirstexamplesofLbLperformedonthylakoidbiomembranes.Theuseofmembranes,insteadofproteins,couldproveveryusefulinnumerousbiosensorapplications,sincethepurificationof    mem-braneproteinsandenzymes,i.e.thesensingpartof    a   biosensor,notonlyrequiresgreatlossof    timeandmoney,butisalsomoredifficulttoperform,comparedwiththeextractionofthemembranescon-tainingsuchproteins.ThePEI–BBYmultilayersweretestedasa   toolforsensingherbicidesbelongingtotheclass   of    triazine-derivatives,andaninhibitionopticalassaywasperformedbyincubatingthebiomaterial,inthepresenceof    terbutryn,atdifferentconcentra-tions.Interestingresponseswerefoundatterbutrynconcentrationshigherthan10 − 7 M,a   valuethatis   stillhigherthantheofficiallegallimitsforthisherbicideindrinkingwater,butin   competitionwiththevaluesdetectablebyothernon-conventionalmethodsbasedonbiosensorsthatexploitnativephotosyntheticmaterials.ThePEI–BBYmultilayerscouldevenbeusefulfor   labora-toriesnot   equippedwithelectrochemicalfacilities,buthavingspectrophotometricinstruments,sincerapidoptical(colorimetric)assayscouldbe   performedwiththeBBYbiochips.Furthermore,thisstudycouldprovideusefulinformationto   researcherswish-ingtoperformimmobilizationof    other,different,“proteinenrichedbiomembranes”bymeansof    theLbLtechnique.Infuture,theauthorsintendtoevaluateparametersthatcouldimprovethequalityof    BBYimmobilizationandthedetectionlimitvalue,inordertoobtainmoreresistant,stableandsensi-tivebiochips,forinstanceusingphotosyntheticmembranesfromgeneticallyengineeredorganisms.  Acknowledgements Thisworkwas   supportedbyPRIN2008“Architettureibridemul-tifunzionalibasatesubiomolecoleperapplicazioninelcampodellasensoristica,dellaconversionedienergiaedelbiomedicale”.  AppendixA.Supplementarydata Supplementarydataassociatedwiththisarticlecanbe   found,intheonlineversion,atdoi:10.1016/j.bios.2011.05.043.
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