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Colloids
and
Surfaces
A:
Physicochemical
and
Engineering
Aspects
j ou rn a l h o m e pag e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f a
Preparation
and
characterization
of
mixed
monolayers
and
Langmuir
−Blodgett
films
of
merocyanine
540/octadecylamine
mixture
Bahri
Gür, Kadem
Meral
∗DepartmentofChemistry,FacultyofScience,AtatürkUniversity,25240,Erzurum,Turkey
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Langmuir–Blodgett films of mero-cyanine 540/octadecylamine mix-turewerefabricated.
Photophysical properties of mero-cyanine540werefollowedas spec-troscopic.
H-aggregateformationof merocya-nine540atsolidandliquidphases wascharacterized.
Surfacemorphologyofthinfilmwas examined by using atomic force microscopy.
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Langmuir–Blodgettfilms(LB)ofmerocyanine540(MC540)mixedwithamphiphilicoctadecylamine (ODA)onglasssubstratearefabricatedbyco-spreadingmethod.TheformationofstableLBfilmofMC540 mixedwithODAonwatersubphaseischeckedbysurfacepressure–area(–A)isotherm.
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Articlehistory:Received6June2012
Receivedinrevisedform31July2012 Accepted22August2012
Available online 10 September 2012 Keywords:
Langmuir–Blodgettfilms
Surfacepressure–area(–A)isotherms Merocyanine540(MC540)
Octadecylamine(ODA) Atomicforcemicroscopy(AFM)
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Mixedmonolayerofmerocyanine540(MC540)dyeandoctadecylamine(ODA)attheair/water inter-facehasbeenpreparedusingtheco-spreadingmethod.Thepressure–area(–A) isotherm studies revealedthatthemixturesofMC540/ODAatadifferentratioformedastablemonolayerattheair/water interfaceandthesefloatinglayerswereeasilytransferredontohydrophilicsubstratesastheY-type Langmuir–Blodgett(LB)film.Thespecificareapermoleculeinthe–Aisothermofthemixedmonolayer ofMC540/ODAwaslargerthanthatofthepristineODA.Theareacanalsogetincreasedupto40mN/m surfacepressurebytheincreaseofthedyeconcentrationinthemixtures.TheLBfilmsofMC540/ODA mixturewereformedat30mN/msurfacepressurebytransferringthemixedmonolayerattheair/water interfaceonhydrophilicglasssubstrateviaverticaldip-coatingasmono-andmultilayerfilms.The photo-physicalpropertiesofMC540inchloroformandLBfilmshavebeeninvestigatedusingtheabsorption, steady-stateandtime-resolvedfluorescencespectroscopytechniques.H-aggregateformationofMC540 bothinchloroformandLBfilmwereconcludedfromthespectroscopicresults.Themorphologyofthe one-layermixedLBfilmofMC540/ODAontheglasssubstratehasbeencharacterizedbyAFM(atomic forcemicroscopy).Thenon-contactmodeAFMimageoftheone-layermixedLBfilmshowedthatthe filmsurfaceconsistedofMC540/ODAnanoclusters.Inconclusion,ourresultscontributeto understand-ingthestronginteractionbetweenMC540andODAattheair/waterinterface,andshowtheeffectsof someparametersonthemixedLBfilmsofMC540.
© 2012 Elsevier B.V. All rights reserved.
∗ Correspondingauthor.Tel.:+902314410;fax:+904422360948. E-mailaddress:kademm@atauni.edu.tr(K.Meral).
1. Introduction
Thinfilmoffunctionaldyemoleculeshasasignificantrolein thedesignofultrafast,miniaturized,optoelectronicandphotonic devices [1]. Thereare several techniquesfor thefabrication of thin filmsoffunctional dyemolecules,suchasspin-coating[2], 0927-7757/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.
Fig.1.Chemical structures ofmerocyanine540 (MC540)and octadecylamine (ODA).
layer-by-layer [3], Langmuir–Blodgett (LB)techniques [4,5] etc. Amongthese,theLBtechniqueisauniquemethodthatprovides flexibilityincontrollingthespatialdistributionandtheorientation ofthedyemoleculesinthefilmmatrix[4–6].IntheLBtechnique, themoleculararchitecture and thicknesscan beprecisely con-trolledbymonitoringcertainparameterssuchasthepHofthe subphase,barrierspeed,dippingspeed,molarcomposition, tem-perature, and thesurface pressure of lifting of the LBfilm [7]. Therefore,theLBmethodhasbeenwidelyusedtofabricatethin filmsofalargevarietyoforganicmolecules.Amphiphilicorganic moleculesand numerous polyaromatichydrocarbon derivatives havebeenextensivelyusedforLBfilmapplicationsbecausethey formasuperb monolayerattheair/waterinterface[8].In con-trast,nonamphiphilicandwater-solubleorganicmoleculeshave presentedlimiteduseinLBfilmstudiesbecausenonamphiphilic moleculestendtoformmicrocrystalsattheair/waterinterfaceand water-solublemoleculesmoveintothewatersubphaseduringthe evaporationofthevolatilesolvent[9].Therefore,nonamphiphilic andwater-solublemoleculesaredifficulttotransferontosolid sub-stratesforthepurposeofthegenerationofhigh-qualityLBfilms. Thisproblemcanbeovercomebydopingnonamphiphilicor water-solublemoleculeswithamphiphilicmoleculessuchasfattyacids, octadecylamin and some phospholipids, which form excellent monolayersattheair/waterinterface[9–14].Themixture contain-ingwater-solublemolecules(ornonamphiphilicmolecules)and amphiphilicmoleculesusuallyresultsintheformationofa sta-blefloatinglayerthatcouldpotentiallybeeasilytransferredontoa solidsurface[15].IntheLBmatrix,thewater-solublemoleculescan beembeddedwithinthedopedamphiphilicmoleculesorformed awater-insolublecomplexwiththem.Incontrast,nonamphiphilic moleculestendtoformasandwich-typestructureasaresultof squeezingbetweenheadgroupsoftheamphiphilicmoleculesand watersubphase[13–15].
Dyemoleculeshaveattractedagreat dealofattentioninthe areaofelectronicsandoptoelectronicsduetotheirpotentiallylow costandeaseofdesigningatthemolecularlevel[16].Themost importantrequirement for theuseof functional dyemolecules inthetechnologicalapplicationsisthepreparationoftheirthin films [17]. The thin film of dyes with the desired optical and morphologicalpropertiescanbefabricatedbytheLBfilm tech-nique,easily[4,5].Oneofthesedyemoleculesismerocyanine540 (MC540),whosemolecularsystemshowsanextendedconjugation thatisresponsibleforitsabsorptionatthelongerwavelengthin thevisiblespectrum(Fig.1)[18].MC540isananioniclipophilic
polymethinedyethatisusedasthefluorescentprobeforstudying biologicalmembranesandsensitizersforphotodynamictherapy [9–21].Thephotophysicalpropertiesofthedyestronglydepend onchangesinenvironmentalfactorssuchaspolarity,viscosityand temperature[18].Forexample,thefluorescencequantumyieldof MC540inanaqueoussolutionislowduetoitsabilitytoform non-fluorescentaggregates[21].MC540moleculesalsohaveatendency tobindmicelles,liposomesorvesicles[22].Therefore,their non-fluorescentaggregatesdissociateintofluorescentmonomersinthe presenceofamediumcontainingmicellesorvesicles[21].Although therearenumerousreportsontheLBfilmstructuresofamphiphilic merocyaninedyes[23],thereiscurrentlynostudyregardingthe LBandthemixedLBfilmsofMC540dopedwithODA.Therefore, itwouldbeinterestingtounderstandphotophysicalpropertiesof MC540inLBmatrixandtheroleofinteractionbetweenthedye andODAforbiomedicalapplication.
Herein we report the preparation of the mixed monolayer of MC540withamphiphilicODAat theair/water interfaceand thephotophysicalpropertiesofthedyeinanLBfilm.The spec-troscopic properties of MC540in chloroform and LB filmwere determinedbyusingabsorptionandfluorescence(steady-stateand time-resolved)spectroscopytechniques.Thesurfacemorphology oftheone-layermixedLBfilmshasbeencharacterizedusingthe non-contactmodeAFM(atomicforcemicroscopy).Ourresults con-tribute totheunderstanding ofthe stronginteraction between MC540and ODA at the air/water interface,where the concen-trationsof MC540canmodifythefilm. Additionally,this study supports noteworthyfindings related to thenanostructure and opticalpropertiesofMC540dyeatsolidsurface.
2. Experimental 2.1. Materials
MC540,octadecylamine(ODA),andchloroformwerepurchased fromSigma–Aldrich.
2.2. Methods
AcommerciallyavailableLBtrough(KSV,Minithroughsystem) wasusedforthedepositionofmono-andmulti-layerLBfilms.In LBfilmexperiments,puredeionizedwaterusedforthesubphase wasobtainedfromaKrosClinic(model:KRS-R-75).ThepHofthe subphasewas6.3andthetemperaturewas22◦C.Thepreparation ofLBfilmsofMC540/ODAmixturewasoutlinedintheSupporting Information.
2.3. Instrumentation
TheabsorptionspectrawererecordedonaPerkinElmer(Model Lambda35)spectrophotometeratroomtemperature.The absorp-tionspectraofthedyesinchloroformweretakeninaquartzcuvette with a dimension of 0.5cm×1.0cm. Steady-state fluorescence spectraweretakenwithaShimadzuRF-5301PC Spectrofluoropho-tometer.Fluorescencedecaysforthelifetimemeasurementsand theemissionspectrawerecarriedoutwithaLaserStrobeModel TM-3lifetimefluorometerfromPhotonTechnologyInternational. Thedetailsofthismethodhavebeengivenelsewhere[15].All mea-surementsrelatingtofluorescencestudieshavebeenrecordedby usinga 0.5cm×1.0cm fluorescencequartz cuvette. AFMimage oftheLBfilmwasperformedinairusingNanomagnetics instru-mentsobtainedfromAnkara,Turkey.Theimagewasacquiredina non-contactmode.
Fig.2.Surfacepressure–area(–A)isothermsofpristineODAandthemixtureof MC540/ODAatdifferentratio.
3. Resultsanddiscussion
3.1. Surfacepressure–area(–A)isothermofthemixturesof MC540/ODA
Theformationofthestableandfloatinglayersofbothpristine ODAandMC540/ODAmixturesattheair/waterinterfacewas con-firmedbyusingthesurfacepressure–area(–A)isotherms.When 35lof pristineODAinchloroform(0.5mg/mlwasusedforall experimentsandkeptconstant)wasspreadandcompressedon thepurewater subphase,theobtained–Aisothermshoweda smallliquid-phaseregionbeforeitreachedtheclose-packedsolid phase[24].ThespecificareapermoleculeforpristineODAwas cal-culatedas∼0.24nm2,whichiscomparablewiththevaluegiven
in literature[11]. Followingthis,the same–Aisothermstudy wasperformedforpristineMC540.In thisinstance,thesurface pressuredidnotrisesufficientlytobeappliedtoahigh-quality LBfilmwhenpristine MC540monolayerat theair/water inter-face wascompressed at a slow rate. Additionally, fluorescence studiesindicatedthata sectionofMC540moleculespenetrated intothesubphaseduringtheevaporationofvolatilesolvents dur-ingthecompressionprocess(Fig.S1).Therefore,pristineMC540 moleculesdonotformaself-supportingmonolayerattheair/water interfacebecausethedyedoesnothavelongenoughalkylchains, which preventsubmergenceof thedyeintosubphase.In order toovercomethisproblem,itwasanticipatedthatdyemolecules mixedwithanyamphiphilicmoleculescouldbeincorporatedin LBfilms via theacknowledged co-spreading method[10].This procedurehasbeen appliedin many studies including nonam-phiphilicandwater-solublemolecules[9–15].Inthisregard,the performed –A isotherm studies verified that the MC540/ODA mixtureformedhighlystableandfloatinglayersattheair/water interface.TheODA-shaped–Aisothermswereobtainedbythe mixturesofMC540/ODAatdifferentratiosin whichtheMC540 concentrationwasalteredfrom5.0×10−5Mto1.0×10−4M.Fig.2 shows the–A isotherms of pristine ODAand the mixtures of MC540/ODAatadifferentratioat22◦C.The–Aisothermsofthe mixturesshowedanexpansionincomparisontothatofpristine ODA,andthedegreeofthisexpansionincreasedbyincreasingthe levelofMC540inthemixture,whichisclearly showninFig.2 [11].Inotherwords,thespecificareapermoleculeobtainedfrom the–Aisothermofthemixturecontaining1.0×10−4MMC540 is greater than those of the others containing a lower level of MC540(Fig.2).TheexpansionindicatesthatMC540moleculesare
retainedamongODAmoleculesattheair/waterinterface,where ODAmoleculescanactasasupportingmatrix.Theshapeof–A isothermsofpristineODAandMC540/ODAmixturesatlowdye loading(5.0×10−6Mand1.0×10−5M)arevery similartoeach other, withtheexception of the specificarea permolecule. At thehigherdyeloadings(5.0×10−5Mand1.0×10−4M),the–A isothermsshowedaplateauregionstartingatsurfacepressuresof 40mN/m.Theflatplateaubecomesmoreapparentbytheincrease ofthedyeconcentrationinthemixture.Suchaplateauinthe–A isothermshasbeenobservedin somemoleculesand frequently interpretedinconnectionwithphasetransition,whichis gener-atedbyeffectivechangesinorientationandthearrangementsof moleculesatmonolayerandmolecularaggregation[25].Sincethe plateauformationinthe–Aisothermisonlyobservedathigh dyeloadings,theMC540concentrationinthemixtureis consid-eredforthemoleculararrangementintheLBmatrixasadriving force.ThearrangementofMC540dyemoleculesatahigher sur-facepressurebring abouttheplateauformationdue tothefact that dye molecules at highloadings form aggregate structures thatarearrangedinaside-by-sideandtail-to-tailconformation ofdyemolecules.Additionally,thisplateaucorrespondstoa two-dimensionaltothree-dimensional(2D-to-3D)phasetransitionof MC540 molecules in ODA matrix. The absorptionproperties of MC540in LB filmrevealed that thecompression of mixtureat theair/waterinterfaceforthehighersurfacepressuresinduced theplateauformationduetomolecularaggregationofMC540in ODAmatrix,whichisilluminatedbytheUV–visabsorptionstudy (Fig.S2).Asaresultofspectroscopicstudy,themolecular aggre-gation ofMC540thattookplace atthehigher surfacepressure wasdeterminedbytheincreaseintheabsorbanceoftheMC540 aggregateband.Theexpansionsorchangesinthe–Aisotherm werefollowedbythemolecularareaatseveralsurfacepressures whenODAwasmixedwithMC540atdifferentratios.Themolecular packingofamixedmonolayercouldbedrawnfromtheplotofthe areapermolecule(nm2)versustheconcentrationsofMC540(Fig.
S3).Thesurfaceareawasincreasedbytheincreaseintheratioof MC540/ODAunder40mN/msurfacepressures(Fig.S3).Incontrast, thesurfaceareaathigherpressureswasincreasedup1.0×10−5M andthendecreasedwiththeincreaseoftheMC540concentration. Inthiscase,itisalsopossiblethatthedyemoleculesremain under-neaththeheadgroupsof theODAmonolayer,compressingthe barriersorsomeofthedyesthataresubmergedintothewater subphase,aswellaspreviousexplanationsrelatedtothealteration ofthe–Aisotherm.ThepossibilityofaparticleofMC540 pene-tratingintothesubphasewasfollowedbyafluorescencestudy.The resultsoffluorescencestudyprovedthatMC540moleculesatthe air/waterinterfacedidnotpassintothewatersubphase. Addition-ally,thesurfaceareaatthelowerdyeconcentrationwassharply increased,whilethechangeofthesurfaceareaatthehigherdye concentrationlevelwasgradual(Fig.S3).Thisobservationimplies thatthemolecularaggregationofMC540moleculesintheODA matrixtakeplaceatthehigherconcentrationlevels.Consequently, thestrongelectrostaticinteractionbetweencationicaminogroups ofODAandanionicMC540moleculespreventtheescapeofthedye moleculesintothewatersubphase.Thestronginteractionenables MC540moleculestostandattheair/waterinterface.Thisresult givesaremarkablecontributiontothedyemoleculesbindingto themodelmembranesystems.
3.2. PhotophysicalpropertiesofMC540inchloroformandLBfilm 3.2.1. Absorptionspectroscopy
Theabsorptionpropertiesofdyeinchloroformwereinitially investigated in a wide range of concentration (1.0×10−6 M-1.0× 10−4M) for the determination of the molecular behavior
Fig.3.NormalizedabsorptionspectraofMC540inchloroform.
absorptionspectraofMC540withrespecttothemonomer max-imum in chloroform atdifferent concentrations. MC540dye in chloroformhastwoabsorptionbandsatlowconcentrationlevels (Fig.3).Theintensebandat570nmbelongstoMC540monomersin chloroform.Thisbandmaximuminchloroformwasreportedtobe 568nmand572nm[18,26].Theotherbandat∼528nmrelatedto H-dimerformationofMC540.Theequilibriumbetweenthe maxi-mumabsorbancevaluesoftwobandsinchloroformwaschanged byincreasingthedyeconcentrationlevels.Theabsorbanceofthe monomerbanddecreasedandthatoftheH-dimerincreasedwith theshiftofthebandmaximumwhenthedyeconcentrationwas increasedto1.0×10−4M.Inthemeantime,theH-dimerband max-imumofMC540inchloroformshiftedtotheblueregion,which appearedat512nmfor1.0×10−5Mandat509nmfor1.0×10−4M. Additionally,theH-dimerbandwasbroadercomparedtothatin thelowerdyeconcentrations.The absorptionbandobserved at theblueregionrevealsthedifferentmolecularbehaviorofMC540 moleculesintheconcentratedsolution.Therearetwoabsorption bandsinthedimerspectrumofMC540inchloroformasreadily seenin Fig.3.Thisis illuminated bythesecond derivativeand deconvolution ofthe absorptionspectrum ofMC540 in chloro-form[27].Thesemethodsareimportantforthedeterminationof theabsolutemaximaofabsorptionbands,whichareparticularly usedforthecharacterizationofoverlappingabsorptionbandsdue totheirintenseaggregation[28].Thesecondderivativespectraof MC540inchloroformdependingonthedyeconcentrations(Fig.S4) demonstratedthatthereweretwobandsobservedat571nmand 528nmat1.0×10−6Mdyeconcentration,whichwereattributedto monomerandH-dimer,respectively.Anewbandappearedinthe blueregionwithincreasingdyeconcentrationcomparedtothat inthediluteddyeconcentration.Themaximumofthisbandwas observedat504nmwhentheotherbandmaximawereconstant. TheabsorptionbandmaximaofMC540inchloroformwere con-firmedbythedeconvolutionspectrumofMC540at1.0×10−4M dyeconcentration(Fig.S5).Thedeconvolutionspectrumcomposed ofthreeabsorptionbandswhicharelocatedat503nm,530nmand 570nm.Thebandpositioninginthedeconvolutionspectrumare compatiblewiththoseinthesecondderivativesanalysisofMC540 absorptionspectrainchloroform.Accordingtothespectral anal-ysisofMC540inchloroform,thebandat∼528nmand∼504nm attributedtoH-dimerandhigheraggregates(H-aggregates)while theabsorptionbandat571nmbelongingtoMC540monomers. H-aggregateformationofMC540iswell-knowninthepresenceofa cationicsurfactantinnonpolarsolvents[29].Additionally,the for-mationofthebandobservedat504nminchloroformcontributes
Fig.4.AbsorptionspectraofmixedLBfilmsofMC540/ODA.
colorchangeofthedyesolution(Fig.S6).Therefore,the spectro-scopiccharacterizationoftheH-aggregateformationofMC540in asolutionisimportantforthebiomedicalapplications.
AbsorptioncharacteristicsofMC540intheLBfilmwere inves-tigated by the preparation of mono- and multilayer films. For this purpose, the mixed LB films of MC540/ODA at a certain (1.0× 10−4M)dyeconcentrationwerefabricatedat30mN/m
sur-facepressure.Fig.4shows theabsorptionspectraof monoand multilayerLBfilmsofMC540/ODAmixture.Itcanbeseenfrom Fig. 4 that there are two intense absorption bands aroused at 530nmand571nmintheabsorptionspectrumoftheone-layer mixed LB film of MC540/ODA. The absorption bandat 530nm revealedtheH-dimerstructureof MC540,while theabsorption bandat571nmattributedtomonomericformofthedyeinLB film.Thesmallred-shiftobservedattheabsorptionbandmaxima of theH-dimerand monomer in the case of themixed mono-layer,withrespecttothoseinchloroformstemsfromtheorganized aggregationofMC540inLBfilm.ThenumberofMC540/ODA lay-erstransferredonto glasssubstratewasincreasedto11.It was concludeduponthecarefulexaminationofFig.4thatabsorption characteristicsofMC540inLBfilmweredrasticallyaffectedbythe mixedlayersbeingtransferredontoglasssurfaces.Thisobservation fortheabsorptionpropertyofMC540impliesthattheinteraction betweenthetransferredlayerstakesplaceandthisalsotriggers themoleculararrangementofthedye.It canalsobeconcluded fromFig.4 that theincrease in thenumber of transferred lay-ersdecreasedtheintensityofthemonomerbandat571whenit increasedH-dimerbandobservedat530nminLBfilms. Addition-ally,themultilayerLBfilmofMC540/ODAresultedintheformation of a newabsorptionshoulder of approximatelyat 470nm.The newabsorptionbandimpliestheformationofhigheraggregates of MC540 in LB film knownas H-aggregate. The clear absorp-tionmaximumofthisbandwasconfirmedbythedeconvolution ofthetotalabsorptionspectrum.Fig.5demonstratedthe decon-volutionabsorptionspectrumofthenine-layermixedLBfilmof MC540/ODA.The analysisresult exposed thepresence of three absorptionbandsintheabsorptionspectrumofnine-layerLBfilm, whichwaslocatedat476nm,532nmand575nm.Inthemultilayer LBfilms,theincreaseinthedyeaggregationisduetothe inter-actionofdyemoleculesinonemonolayerwithODAandMC540 moleculesofanothermonolayer.Asresultsofthesecond deriva-tiveanddeconvolutionspectraofMC540indifferentmedia,the increaseinthedegreeofaggregationcausedablue-shiftin aggre-gatebandmaximumofthedye.Spectroscopicdifferencesandband splittingobservedinaggregatedsystemareexplainedaccording
Fig.5. Deconvolutionofabsorptionspectraofnine-layerMC540/ODALBfilm. totheexcitontheorybasedonmonomerdipole–dipole interac-tionintheaggregates[30,31].Strongelectroniccouplingbetween thedyemoleculesinaggregateunitscausestheformationof H-aggregates,J-aggregatesanddimers.InthecaseoftheH-aggregate, theabsorptionbandmaximumarisesattheblueregioncompared tothemonomerbandandtheydecreasefluorescencepropertiesof dyemolecules.IncontrasttotheH-aggregate,J-aggregateshavea red-shiftandnarrowabsorptionbandwithrespecttothemonomer andtheyenhancethefluorescenceintensityofthedyes.The infor-mationrelatedtospectroscopicandphotophysicalpropertiesof aggregatestructuresisavailableinliterature[15,30,31].
3.2.2. Fluorescencespectroscopy
In order to determine fluorescence properties of MC540 in chloroformandLBfilm,steady-statefluorescencespectraofthe sampleswerestudiedatthe536nmexcitationwavelength.The presenceof intensenon-fluorescentH-aggregation ofMC540in chloroformdrasticallyinfluencesthefluorescentpropertiesofthe dye.Therefore,thecharacterizationoffluorescencepropertiesof dyeinchloroformandLBfilmareessentialfortechnological appli-cations.ItwasreportedthatthefluorescencemaximumofMC540 in chloroform was formed at 589±1nm [18,26]. The fluores-cencepropertiesof MC540ina wide concentrationrangefrom 1.0×10−6Mto1.0×10−4Mwereexamined.Fig.6showsthe fluo-rescencespectraofMC540inchloroform.Oneintensefluorescence
Fig.6. FluorescencespectraofMC540atdifferentconcentrationinchloroform.
Fig.7.NormalizedfluorescencespectraofmixedLBfilmsofMC540/ODA.
bandat583nmwasobservedatdilutedyeconcentrationwhichis attributedtothemonomericformofMC540moleculesin chlo-roform. The fluorescence maximum and intensityof MC540in chloroformwasalteredwithanincreaseinthedyeconcentration. Forexample,thefluorescencemaximawereobservedat588nm for5.0×10−5Mand593nmfor1.0×10−4Mandthefluorescence intensitywasstronglyquenched atconcentrations greaterthan 1.0×10−5M.Thefluorescencequenchingandred-shiftinthe fluo-rescencespectrumofMC540dependingonthedyeconcentrationis duetotheintenseaggregationandchangingpolarity[21,28,29,32]. Additionally,thefluorescencequenchingisattributedtothe reab-sorptioneffect,which canbeobserved intheconcentrated dye solution[33].AlthoughtheH-aggregateofMC540isobservedat concentrationsgreaterthan5.0×10−6M(Fig.3),theincreasein thefluorescenceintensityofMC540canbeexplainedbyincreasing theamountoffluorescentMC540monomers(Fig.S7).Incontrast, thestrongquenchingobservedat1.0×10−4MofMC540in partic-ularindicatesthepresenceofthereabsorptionprocessaswellas intenseaggregation.
The normalized fluorescence spectra of mixed LB films of MC540/ODAwerepreviouslypresentedinFig.7.AsshowninFig.7, thefluorescencebandmaximumofone-layermixedLBfilmwas observedat576nm.Thefluorescencebandascribedtomonomeric MC540intheLBfilm.ThefluorescencebandmaximumofMC540 intheLBfilmisblue-shiftedincomparisontothatinchloroform. Theblue-shiftcanberelatedtothevibrationalenergylevelsinthe groundstateoftheMC540moleculesbyvaryingthelocal envi-ronmentaroundthedyemolecules[28].Theretentionofthedye moleculesintheLBmatrixsupportsthisphenomenon.The rela-tivelyhigherrigidityofthedyemoleculesprovided bythelocal environmentaroundMC540moleculesconstrictsthefreedomof rotation.Thesignificantdifferencesinthefluorescencespectrum ofMC540intheLBfilmwerenotobservedbyanincreaseinthe numberoflayersonthesubstrateandthereweresmalldifferences intheintensitiesofthefluorescencespectra.Additionally,the flu-orescencebandmaximumwasred-shiftedfrom576nmto578nm withincreaseinthenumberoflayer.Thesmallred-shiftinthe fluo-rescencebandmaximumcanbeascribedtomolecularaggregation becauseincreasingthenumberoflayersenhancesaggregationof MC540intheLBfilm(Fig.4).
3.2.3. Time-resolvedfluorescencespectroscopy
To determinethe fluorescencelifetime ofMC540 in chloro-form and the one-layer LB film, fluorescence decay spectrum of the samples were recorded upon excitation at 536nm. The
Table1
ThespectroscopicdataandfluorescencelifetimevaluesofMC540inchloroformandLBfilm.
[MC540] abs.max.(nm) fluo.max.(nm) 1(ns) 2(ns) 2 Inchloroform 1.0× 10−6M 571a/528b/– 583 1.20 – 1.00 1.0× 10−5M 571a/528b/504c 588 1.40 – 0.95 1.0×10−4M 571a/528b/504c 593 1.60 – 1.10 InLBfilm One-layer 571a/531b/– 576 0.50 2.67 1.10 Nine-layer 571a/531b/476c 578 – – – aMonomer. b H-dimer. c H-aggregate.
fluorescence lifetime values were calculated by using specific fit-softwareofPTI(PhotonTechnologyInternational).The fluores-cencedecayspectraofMC540withexponentialfitinchloroform were presented in Fig. 8. The exponential analysesof the flu-orescence decays of MC540 in chloroform were fitted to the single-exponentialdecayswiththeacceptablestatistical2value.
The obtained single-exponential decay indicates homogeneous environmentaroundthedyemolecules.Asaresultofdecay analy-sis,thelifetimevalueofdilutedMC540(1.0×10−6M)inchloroform was1.20ns.ThelifetimeofMC540inchloroformwasdependenton theincreaseindyeconcentrationandthelifetimesat1.0×10−5M and1.0×10−4Mdyeconcentrationwerefoundtobe1.40nsand 1.60ns, respectively. The value of thelifetime for 1.0×10−4M MC540 in chloroform was markedly greater than the lifetime observedfor MC540ata dilutedconcentration.Theincrease in the lifetime of dye molecules is explained by the presence of thereabsorptionprocesseswhilenon-fluorescentH-aggregatewas stronglyobserved[34].Iftherewasnoreabsorptionprocessinthe system,it would beobservedasa decreasein thefluorescence lifetimeofconcentratedMC540comparedtothatofthediluted oneduetothefactthatH-aggregatesandH-dimerquench fluo-rescenceintensityanddecreasefluorescencelifetimeduetotheir fastinternalconversionprocess,inwhichtheradiativetransition isforbidden[28].
IntheLBfilm,thefluorescencedecayofMC540wasfoundtobe bi-exponential(Fig.9).Thebi-exponentialdecaywasinterpreted bytheinhomogeneousdistributionofthedyemoleculesandthe factthattheprobeencountersdifferentenvironmentsdueto dif-fusionwithinitslifetime.Accordingtothebi-exponentialanalysis ofthefluorescencedecays,fluorescencelifetimevaluesofMC540 intheLBfilmwerecalculatedas1=0.50nsand2=2.67ns.The
longlifetimecomponentwasassignedtofreeMC540monomers
Fig.8.FluorescencedecayspectraofMC540withexponentialfitsinchloroform.
intheLBfilmwhenshortlifetimesresultedfromdifferent orien-tationofthedyemonomers.Additionally,theshortlifetimemight beduetotheexcitationenergytransferredtothenon-fluorescent aggregatesresultinginadecreaseinfluorescencelifetime[14,28]. ComparisonoffluorescencelifetimesofMC540moleculesin chlo-roformandLBfilmrevealsthatthefluorescencelifetimeintheLBis increasedduetoamorerigidenvironmentforthedyemoleculesin theLBmatrix.Thespectroscopicresultsandfluorescencelifetimes ofMC540indifferentmediaweresummarizedinTable1. 3.3. AFMobservationsofone-layerLBfilmofMC540/ODA
AFMisausefultechniqueforgaining informationonsurface morphologyofthethinfilms,especiallyfortheflatLBfilms[34]. One-layerofthemixedLBfilmofMC540/ODAwastransferredonto hydrophilicglasssurfacetotakeitsAFMimages.Fig.10showed theAFMimageofY-typemixedLBfilmofMC540/ODAwithphase imageand3Dimages.AFMimagedepictedthattheMC540/ODA nanoclusters were formed in LB film. These nanoclusters are nearlythesamesizesandhaveuniformdistributiononthe sur-face.Thedimension(width×length×height)ofthenanoclusters weredeterminedasanaveragevalueof300nm×500nm×10nm for MC540/ODA mixture by examination of AFM images (Fig. S4). Additionally, the phase and 3D AFM images (Fig. 10 and Fig.S8) reveal thatthe stablenanoclusters are actuallyformed byagglomerationofseveralsmallernanoclusterswhose dimen-sion is 300nm×150nm×10nm. Since the morphology of LB filmdependsonthefilmmaterials,thenonamphiphilicdopant moleculesaffectthemonolayershape ofamphiphilicmolecules suchasODA,fattyacidand phospholipidswhich mayformbig
Fig.9.Fluorescencedecayspectrumofone-layermixedLBfilmofMC540/ODAwith exponentialfits.
Fig.10. AFMimage(5.0m×5.0m)ofone-layermixedLBfilmofMC540/ODA(a)and3D-AFMimages(b).
clustersatair/waterinterface.Inthisregard,itisreportedthatthe differentdomainmorphologieshavebeenobservedinthemixedLB filmofmethyleneblue(MB)mixedwithdimyristoyl-phosphatidic acid(DMPA)asafunctionofMBsurfacedensity[12].AFMstudyhas demonstratedthecrystallinedomainstructuresofN,N-bis (2,6-dimethylphenyl)-3,4,9,10-perylenetetracarboxylicdiimide(DMPI) mixedwithstearicacid(SA)in themixedLBfilm[35].In addi-tion,wehaverecentlyshowedthemixedmonolayerofpyronin dyes/SAmixtureinLBfilmhasasurfacemorphologiesconsisting ofnanoclusters[15].Itisconcludedthatthemorphologicfindings ofMC540/ODA-LBfilmarecompatible withtheexplanationsin thesurfacepressure–area(–A)isothermsstudiesandthe spec-troscopicresults.Theformationofthenanoclustersisattributed toself-assembleddyemoleculesinODAmatrixandthecomplex formationofdyemoleculesinteractedwithODA.
4. Conclusions
Thisstudyshows that mixed monolayerof MC540/ODAcan bereadilypreparedonpurewatersubphasewhileMC540cannot merelyform.TheformationofthemixedmonolayerofMC540/ODA attheair/waterinterfacewasconfirmedby–Aisothermstudies. ThestronginteractionbetweenMC540and ODAforma water-insolublecomplexandproducethefloatinglayersattheair/water interface. The specific area per molecule obtained from –A isothermsrevealedthattheMC540dyemoleculesretainedamong
thearrangedODAmoleculesattheair/waterinterface. Addition-ally,theplateauformationathighsurfacepressurewasobserved due to molecularaggregationin the higher dyeconcentrations atthemixedmonolayer.OpticalpropertiesofMC540in chloro-formand LBfilmwerestudiedspectroscopicallyand theprobe environmentaffectedthephotophysicalpropertiesofMC540. H-aggregatesofMC540inchloroformwereobservedbyincreasing thedyeconcentration,whilethemonomericdyeindiluteddye concentrationwaspredominant.TheintenseH-typeaggregation andreabsorption processesobservedinchloroform inducedthe strongquenchinginthefluorescenceintensityofMC540aswellas thestrongred-shiftingoffluorescencemaximumofthedye.Inthe LBfilm,theincreaseinthenumberoflayersbroughtabout form-ingthemolecularaggregationstructureofMC540.Theabsorption spectraofMC540/ODAdemonstratedthatthemonomerand H-dimerformationofMC540wereavailableinone-layermixedLB filmwhenH-aggregatesofthedyewereformedinthemultilayer mixedLBfilm.OurresultsconcludedthattheaggregationofMC540 iseasilycontrolledbythenumberoflayers.Themolecular organi-zationbasedonthenumberoftransferredlayerscausedtothesmall changingintheintensityandmaximumoffluorescencespectrum ofMC540intheLBfilm.Accordingtotime-resolvedstudies,the flu-orescencedecayspectraofLBfilmwerebi-exponentialwhenthe decaysinchloroformindicatedcompliancewithmono-exponential kinetic.Additionally,MC540moleculesembeddedinODAmatrix increasedthefluorescencelifetime.TheAFMimageofone-layer
mixedLBfilmshowedasurfacecoveredwithnanoclusterswhich areclosetoequalsizeandhaveuniformdistribution.Consequently, thisstudyisanexcellentexamplerelatedtothequalityofmixed monolayerapplicationsofthefunctionaldyemolecules,whichare incapableofformingamonolayerbythemselvesattheair/water interface.Ourresultscontributetotheunderstandingofthestrong interactionbetweenMC540andODAat theair/waterinterface, wheretheamountsofMC540canmodifythefilmpropertiesand revealhowtobuildthevariousopticalpropertiesofMC540thin films.
Acknowledgment
TheauthorsthanktheResearchFundofAtatürkUniversityfor thefinancialsupportofthiswork.
AppendixA. Supplementarydata
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfa.2012.08.067.
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