Application of Localized Surface Plasmon Resonance Spectroscopy to Investigate a Nano − Bio Interface ̌ - Kastner et al. - 2021 - Unknow

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pubs.acs.org/LangmuirArticleApplicationofLocalizedSurfacePlasmonResonanceSpectroscopytoInvestigateaNano−BioInterfaceRineaBarbir,BarbaraPem,NikolinaKalcec,StephanKastner,KatiaPodlesnaia,AndreaCsǎki,́WolfgangFritzsche,andIvanaVinkovićVrceǩ*CiteThis:Langmuir2021,37,1991−2000ReadOnlineACCESSMetrics&MoreArticleRecommendationsABSTRACT:Theaccuratedeterminationofeventsattheinterfacebetweenabiologicalsystemandnanomaterialsisnecessaryforefficacyandsafetyevaluationofnovelnano-enabledmedicalproducts.Investigatingtheinteractionofproteinswithnanoparticles(NPs)andtheformationofproteincoronaonnanosurfacesisparticularlychallengingfromthemethodologicalpointofviewduetothemultiparametriccomplexityofsuchinteractions.Thisstudydemonstratedtheapplicationoflocalizedsurfaceplasmonresonance(LSPR)spectroscopyasalow-costandrapidbiosensingtechniquethatcanbeusedinparallelwithothersophisticatedmethodstomonitornano−biointerplay.Interactionofcitrate-coatedgoldNPs(AuNPs)withhumanplasmaproteinswasselectedasacasestudytoevaluatetheapplicabilityandvalueofscientificdataacquiredbyLSPRascomparedtofluorescencespectroscopy,whichisoneofthemostusedtechniquestostudyNPinteractionwithbiomolecules.LSPRresultsobtainedforinteractionofAuNPswithbovineserumalbumin,glycosylatedhumantransferrin,andnon-glycosylatedrecombinanthumantransferrincorrelatednicelywiththeadsorptionconstantsobtainedbyfluorescencespectroscopy.Thisability,complementedbyitsfastoperationandreliability,makestheLSPRmethodologyanattractiveoptionfortheinvestigationofanano−biointerface.14−16■INTRODUCTIONNMs.Oneofthecommonlyusedmethodsfortheexplorationofnano−biointeractionsisfluorescencequench-Nanomaterials(NMs)havealreadybeenimplementedining,whichreferstoanyprocessthatleadstoadecreaseinbiomedicineatahighratebymeansofdiagnosticandfluorescenceintensityofasampleunderstudyandisusuallyDownloadedviaUNIVOFNEWMEXICOonMay16,2021at16:58:14(UTC).therapeuticmedicaltoolswithimprovedpharmacologicaltheresultofcloseproximity(<10nm)oftheinvolvedprofiles,effectiveness,andsafety.ForanytypeofNMmoleculefluorophoreandquencher.Itcanbeastaticoraapplicationinbiomedicine,carefulevaluationofquality,Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.efficacy,andsafetyattributesshouldbeperformed.Eachofdynamicprocess.Staticquenchingisaresultoftheformationofanon-fluorescentgroundstatecomplexbetweenthetheseattributeslargelydependsonNMbiologicalactionand1−6fluorophoreandthequencher,whereasdynamicquenchingtheirinteractionwithcomponentsofbiologicalsystems.occurswhenaquenchercollideswiththefluorophoreduringOneofthemoststudiedtypesofnano−biointeractionsrelates17−20thelifetimeoftheexcitedstate.MostproteinsaretotheadsorptionofproteinsonthenanosurfaceimmediatelycharacterizedbyintrinsicfluorescenceoriginatingfromtheafterNMexposuretobiologicalmedia.Alayerofadsorbed17,21aromaticaminoacidresidues.Whenboundtonano-proteinsonananosurfaceisknownasproteincorona,whichsurfaces,thefluorescenceintensityoftheseresiduesischangestheNMpropertiesandstabilityandmaymodulatethe7−9quenchedandtheemissionpeakmaximumisshiftedduetoresponseofabiologicalsystemduringsuchinteractions.thechangeinthechemicalenvironmentaroundtheProteincoronaformationimpactsnotonlyNMsbutalsoproteinsthemselvessincetheirfunctionalityorstabilitymaybeimpaired.10EfficacyandsafetyprofilingofNMsdesignedforReceived:December17,2020biomedicalapplicationsdemandsthereforecloseinsightintoRevised:January14,2021themechanismofproteinbindingandinteractionswiththesePublished:January26,2021NMsbyusingreliable,simple,andcost-effectivemethodo-11−13logicalapproaches.Spectroscopictechniquesrepresentagoodoptionduetoopticalpropertiesofbothproteinsand©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c035691991Langmuir2021,37,1991−2000

1Langmuirpubs.acs.org/LangmuirArticle11with10%(v/v)HNO(MerckSuprapur,Darmstadt,Germany)andfluorophores.Thefluorescencequenchingcanbedescribed3bytheStern−Volmerequationthatprovidesinformationonrinsedwithultrapurewater(UPW).Stocksolutionsoftheproteinsbindingconstantsandanumberofbindingsites.11,21,22Thewerepreparedin150mMphosphatebuffersolution(PBS,pH=7.4).methodiswidelyusedbecauseitissimple,accessible,andSynthesisandCharacterizationofCitrateGoldNano-particles.Citrate-cappedAuNPsweresynthesizedusingapreviouslysensitiveandtheprinciplesoftheStern−Volmerrelationship36,37publishedmethoddevelopedbyTurkevichetal.andFrens.20arewellknownandestablished.Briefly,0.5mLof100mMHAuCl4wasaddedto48mLofUPWandMorerecently,techniquesbasedonasurfaceplasmonthesolutionwaslefttoboilunderconstantstirring.Subsequently,1.5resonance(SPR)propertyofmetalshavebeenusedtostudymLofsodiumcitrate(1%w/v)wasaddedandthesolutionwaslefttointeractionsbetweenmetallicnanoparticles(NPs)andcooldownatroomtemperature.PurificationofpreparedAuNPswasbiomolecules.SPRoriginatesfromtheoscillationoffreeperformedbycentrifugationat11,000gfor30min.Theprecipitateelectronsintheconductionbandofametal.UV/VISlightthatwasresuspendedinUPWandstoredinthedarkat4°C.matchesthefrequencyofoscillationwillbeabsorbedbytheInAuNPcolloidalsuspensions,totalgold(Au)concentrationwasdeterminedusinganAgilentTechnologies7500cxinductivelycoupledmetallicsurface,resultingintheenhancementofalocalplasmamassspectrometer(ICP-MS)(Agilent,Waldbronn,Ger-electromagneticfield.SinceNPsaresmallerthanthemany).CalibrationwascarriedoutwithAustandardsolutions(1000wavelengthofincidentlight,thechargedensityisredistributedmg/Lin5%HNO)purchasedfromMerck(Darmstadt,Germany),3andoscillateslocallyaroundtheNP,whichisknownaswhichweredilutedinamixtureof5%HNO3and1%HCl.23−26localizedSPR(LSPR).ThispropertyiswidelyutilizedinHydrodynamicdiameter(dH),sizedistribution,andzeta(ζ)potentialsensors,opticaldevices,lenses,antennas,datastorage,offreshlysynthesizedAuNPswereobtainedbydynamic(DLS)and25electrophoreticlightscattering(ELS)methodsonaZetasizerNanowaveguides,microscopy,andmanyotherfields.AstheabsorbanceintensityandwavelengthofSPRpeakmaximumZS(Malvern,UK)equippedwith523nmgreenlaserata173°angle.(λmax)aredependentonNPcharacteristicsandtherefractiveDLSresultsarereportedfromthesize−volumedistributionfunction,indexofthesurroundingmedium,theabsorbancespectrumasanaverageof10measurements.TheζpotentialvalueswereobtainedfromtheELSmeasurementusingtheHenryequationwithwillthereforerespondtochangesinNPcharacteristics(e.g.,theSmoluchowskiapproximation.Theresultsaregivenasanaverageaggregation)orchangesoftheimmediateenvironment(duetoofthreemeasurements.DatawereprocessedbytheZetasizersoftware26−29ligandadsorption).Trackingthespectralchanges6.32(MalvernInstruments).followingtheexposureofNPs(eitherimmobilizedonachipTheshapeandprimarysizeofAuNPsweredeterminedbyorfreeinsolution)totheanalyteofinterestisthebasisofthetransmissionelectronmicroscopy(TEM,902A;CarlZeissMeditecLSPRmethodinbiosensing.ThetechniquecanbeusedtoAG,Jena,Germany).ThesamplesweredepositedonaFormvar-monitorparametersofthenano−biointeractionsuchascoatedcoppergrid(SPISupplies,WestChester,PA,USA)andair-proteinbindingkineticsonnanosurfaces.30−33TheLSPRdriedatroomtemperature.Themicroscopeoperatedinbright-fieldtechniqueisasensitive,label-free,simple,andcheaptechniquemodeatanaccelerationvoltageof80kV,andtheimageswerecapturedwithaCanonPowerShotS50camera.TheaverageAuNPthatoffersdetection,quantification,and/orbindingcharacter-primarydiameter(d)wasmeasuredfromthemicrographsusingtheizationofdifferentanalyteswithouttheneedforsample25,34,35ImageJprocessingsoftware.Primaryparticlesweredistinguishedfrompurification.Nevertheless,theuseofLSPRforAuNPaggregatesbytracingmanually.Altogether,atleast100determiningNPs−proteinbindingaffinityandkineticshasparticlesperparticletypeweremeasured.stillnotbeenwidelyapplied.TheUV/VISspectraofNPsinwaterwererecordedonaCARYThisstudyaimstoevaluateapplicability,accuracy,and300spectrophotometer(VarianInc.,Australia),withthetemperaturesimplicityofLSPRspectroscopyformonitoringtheNPs−maintainedat25°C.Themeasurementswereperformedinaquartzproteininteractionbycomparingitwiththewell-establishedcuvettewithanopticalpathof1cminawavelengthrangeof400−fluorescencespectroscopy.Duetoahugeamountofscientific700nm.FluorescenceSpectroscopyMeasurements.FluorescencedataongoldNPs(AuNPs)thatisavailableforpropermeasurementswerecarriedoutusinga10mmpathlengthquartzcomparison,citrate-cappedAuNPswereusedasastandardcuvetteonanAgilentCaryEclipsefluorimeter(SantaClara,CA,NMtypewithexcellentopticalproperties.TheirinteractionUSA)at25°C.Fluorescencespectroscopywasusedfortwodifferentwithbovineserumalbumin(BSA),glycosylatedhumanexperimentaldesigns.transferrin(hTRF),andnon-glycosylatedrecombinantInthefirstapproach,fluorescencequenchingoftheintrinsichumantransferrin(recTRF)werestudied,whiletheselectionproteinfluorescencewasobservedbyincubatingdifferentconcen-ofproteinswasbasedontheirabundanceinplasma,theirtrationsofAuNPsintherangefrom0.75to15.05pMwithaconstantfrequentappearanceinproteincoronasaroundNPs,1andconcentrationofproteins(0.2μM).Thereasonforusingdiluteddiversityoftheirstructureandglycosylationpatterns.ObtainedAuNPdispersionswastoavoidtheintrinsicabsorptionofAuNPsand38resultsclearlydemonstratedtheapplicabilityofLSPRconsequentlytheinnerfiltereffect.Throughoutthemeasurements,excitationandemissionslitwidthsweresetat5.0and10.0nm,spectroscopyandfostertheirwideracceptanceinthefieldofrespectively.Awavelengthof280nmwasusedfortheexcitationofnanomedicineandnanosafety.proteins,andtheemissionfluorescencespectraweremeasuredintherangefrom300to450nm.Priortomeasurements,areactionmixture■MATERIALSANDMETHODSconsistingofAuNPsandproteinswasincubatedfor10minat25°C.ReagentsandChemicals.Tetrachloroauric(III)acid(HAuCl4),Obtainedresultswereusedtocalculatethebindingconstantsbysodiumcitrate,aceticacid(CH3COOH,99.5%),hydrochloricacidStern−Volmerequation(eq1):(HCl,37%),andhydrogenperoxidesolution(H2O2,30%)werepurchasedfromSigma-AldrichChemieGmbH(Munich,Germany).F0=+11kKq0τ=+[SVAuNPs]Rotisoland3-triethoxysilylpropylamine(APTES)werepurchasedF(1)fromCarlRothGmbH&Co.,KG(Karlruhe,Germany).BSA(productnumberA-7906;Sigma-AldrichChemieGmbH,Steinheim,whereF0andFrepresentthemaximumofproteinfluorescenceintheGermany)andhTRFandrecTRF(productnumbersPRO-315andabsenceandinthepresenceofAuNPs,respectively;kqisthePRO-747;ProSpecBio,Rehovot,Israel)wereusedasreceivedquenchingconstant,whichindicatestheefficiencyofquenching;withoutfurtherpurification.Priortouse,allglasswarewascleaned[AuNPs]isthemolarconcentrationofAuNPs;KSVistheStern−1992https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

2Langmuirpubs.acs.org/LangmuirArticleVolmerconstant;andτ0isthelifetimeofproteinfluorescence(TrpImmobilizationofNPsonthesurfaceishighlydependentontheresidue)intheabsenceofAuNPs.particletypeandconcentration.Therefore,20μLoffreshlyThemolarAuNPconcentrationwascalculatedusingeq2:synthesizedsphericalAuNPsolution(1.15×1010particles/mL)waspipettedinthecenterofthechipfollowedby30minincubationmolgijjmolyzz[]Au()L×Ar()molatroomtemperatureanddryingundergentlenitrogenflow.A[AuNPs]=jzgcustom-designedLSPRinstrumentconsistedofamicrofluidickL{m()(2)chamberconnectedtoaperistalticpump(IsmatecReglo-ICC,molCole-ParmerGmbH,Wertheim,Germany),halogenlightsourceHLwhere[Au]isthemolarconcentrationofAu,Aristheatomicmassof−2000−FHSA(OceanOptics,USA),andaUV/VISlinearAu(196.97g/mol),andmisthemass(m)of1molofAuNPsphotodiodearrayspectrometerUSB2000+(OceanOptics,USA),ascalculatedaccordingtoeq3:showninFigure1.m(g)=×ρijjgyzzV(nm3)j3zknm{(3)Ineq3,ρisthedensityofgold(1.049×10−20g/nm3)andVisthevolumeofoneAuNP.ForsphericalAuNPs,volumeiscalculatedbasedoneq4:334××rπV(nm)=3(4)whererrepresentstheradiusofAuNPscalculatedasahalfofAuNPdiameterobtainedfromTEMimages(dTEM).Inthesecondapproach,AuNPconcentrationwaskeptconstantat60μM,whileproteinconcentrationswereincreased.Awavelengthof280nmwasusedfortheexcitationoftheproteins,andtheemissionfluorescencespectraweremeasuredintherangefrom300to450nm.Priortomeasurement,eachreactionmixtureconsistingofAuNPsandFigure1.(a)Custom-designedLSPRinstrumentconsistedofaproteinswasincubatedfor10minat25°C.Throughoutthemicrofluidicchamberconnectedtoasyringepump,halogenlightmeasurements,excitationandemissionslitwidthsweresetat5.0andsource,andUV/VISlinearphotodiodearrayspectrometer.(b)10.0nm,respectively.ConcentrationrangesforBSA,hTRF,andMicrofluidicchamberwithdimensions25×16mmwiththechiprecTRFwereasfollows:0.15−0.50,0.3−0.65,and0.4−0.75μM.inside.Arrowsrepresenttheright(red)andleft(blue)fluidicflowUponAuNPs−proteininteraction,initialfluorescenceintensitiesinsideofthechamber.(fluorescenceintensitiesofloneprotein)aresubtractedfromfluorescenceintensitiesofproteinaftertheadditionofNPsinordertocalculateΔFvalues.ObtainedresultswereusedtocalculatePriortoeverymeasurement,theassembledchamberwasflushedconstantsofproteinadsorptiononthenanosurfaceusingthemodifiedwithPBSfor10minandthespectraofthelampwererecorded.ALangmuirequation(eq5):custom-builtPythonprogramwasusedforcontrollingthepumpandΔFK×[protein]thustheflowinthechamber.Measurementswerecarriedoutby=flushingthedifferentconcentrationsofproteinsolutionsinPBSoverΔFmax1p+×[Krotein](5)thechipinthechamberusingthefollowingprocedure.First,PBSwasConstantsofbindingwerecalculatedusingamodifiedlinearformofflushedthroughthechamberfor6minandthentheproteinsolutionLangmuirequation(eq6):for20mininordertoachievecompletebindingoftheproteinsfortheimmobilizedcitAuNPs.Poorlyboundproteinsweredissociatedby111=+flushingwithPBSfor12min.InordertoensurethequalityoftheΔFKF×Δmax×[protein]ΔFmax(6)measurement,foreveryperformedexperiment,anewchipwasusedsinceacompletesurfaceregenerationcouldnotbeachievedevenwithAdditionally,linearplotspresentingtheLangmuircurveweremadehighconcentrationsofHCl.TheheightofthesensorsignalinLSPRfromreciprocalvaluesofconcentrationandΔF.Fromtheinterceptofmeasurementwasdeterminedaftercalibrationwithrefractiveindexthecurve,aΔFmaxvaluewasobtainedandusedtocalculatethestandards.Thus,themeasurementsoftheLSPRshiftduringbindingconstant(K)fromtheslopeofthecurveusingthepreviousequationofdifferentproteinswerecomparableandquantitative.Additionally,(eq6).thesignalintensitywascalculatedwiththreetimesthesignal-to-noiseForbothexperimentaldesigns,thelimitsofdetection(LODs)forratioasathresholdvalue.BSA,hTRF,andrecTRFontheAgilentCaryEclipsefluorimeterwereThedetectionlimitoftheLSPRsensorwas1ng/mLanalyteindeterminedtobe0.01,0.03,and0.05μM,respectively,usingthe39dependencyoftheusedsensorparticleandtheanalytetype.Thelinearregressionmethod.proteinLODswerecalculatedwiththreetimesthesignal-to-noiseEnsembleLSPRMeasurements.ChipsforLSPRsensingwere40ratioasathresholdvalue.WiththeLSPRsensors,therealbindingmadeusingamodifiedprotocolpublishedpreviously.Priortouse,kineticsaredisplayed,withoutbulkeffect,sothevaluecanbeallborosilicateglasschipswererinsedthoroughlywithanalkalinedetermineddirectly.removerandfurtherwithacetone,Rotisol,ethanol,andwaterinanObtainedresultswereanalyzedusingthemodifiedLangmuirultrasonicbathfor10min.Subsequently,theactivationofchipswas41adsorptionmodeldescribedbyequationeq7:conductedbyoxygenplasmaetchingfor1hat380Wusinga200GPlasmaSystem(TePlaGmbH,Wettenberg,Germany).ImmediatelyΔλKa[]Pafteretching,silanizationofthesurfacewasconductedbyincubating=Δλmax1P+[]Ka(7)thechipsin1%APTESin1mMaceticacidfor10minfollowedbywashingwithUPWinanultrasonicbathfor5min.ThisstepiswhereΔλistheshiftintheLSPRpeak,ΔλmaxisthemaximumLSPRrequiredinordertofunctionalizethechipswithaminesurfacegroups,shift,KistheLangmuiradsorptionconstant(M−1),and[P]isthealeadingtoanincreaseintheaffinityofAuNPsforthesurface.proteinconcentration.PreparedchipscanbestoredafterdryingwithnitrogenflowunderanCalculationsofAuNPSurfaceCoverage.Toestimatetheargonatmosphere.PriortoAuNPdeposition,chipsneedtobenumberofproteinmoleculesthatcouldfitontothesurfaceofAuNPsreactivatedbywashingwithUPWinanultrasonicbathfor10min.insolution,twoparametersareneeded:thesurfaceareaofone1993https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

3Langmuirpubs.acs.org/LangmuirArticlenanoparticle(AAuNP)andthesurfaceareaoccupiedbyoneproteinTable1.SizeDistributionandSurfaceChargeofCitrate-molecule(AP).TocalculateAAuNP,theparticleswereassumedtobeCoatedAuNPs,BovineSerumAlbumin(BSA),HumanperfectspheresandtheformulaforthesurfaceofaspherewasusedTransferrin(hTRF),andNon-glycosylatedRecombinant(eq8):aTransferrin(recTRF)inUltrapureWater2AAuNP=4rAuNPπ(8)speciesd/nm(%meanvolume)ζ/mVhwhererAuNPistheradiusoftheAuNPobtainedasahalfoftheparticleAuNPs65.70±0.90(100)−41.60±1.60diameter(dTEM),whichwasmeasuredexperimentallybyTEM.BSA7.33±0.28(100)−30.25±2.08TocalculateAP,thethree-dimensionalsizeofeachproteinwashTRF10.21±2.00(100)−22.32±0.90projectedontoatwo-dimensionalsurfaceasacirclewiththediameterrecTRF7.11±0.37(100)−17.65±2.92correspondingtotheproteindHvalues,measuredexperimentallybyatheDLStechnique.RadiusrPwasthencalculatedasahalfofthedH,Sizedistributionwasobtainedbymeansofhydrodynamicdiameterandtheformulaforthesurfaceareaofacirclewasused(eq9):(dH,nm)fromthesize−volumeweighteddistributionusingthedynamiclightscatteringtechnique.Surfacechargewasdeterminedas2APP=πr(9)zeta(ζ)potentialvalues,inmV,bytheelectrophoreticlightscatteringmethod.Allexperimentsweredoneat25°Cin0.1mMNaCl(pHFinally,thetheoreticalmaximumnumberofproteinmoleculeson5.6)andatAuNPandproteinconcentrationsof15and1μM,thesurfaceofonenanoparticleinsolution(NS)wasobtainedbyrespectively.dividingAAuNPwithAP.InthecaseofAuNPsimmobilizedontheLSPRchip,theavailablesurfaceareaforadsorptionofproteinswasreducedasAuNPswereattachedtothechipononeside.Theamountofsurfacerenderedconductivity(UPW).InordertocalculatetheNSandNLSPRunavailablecanbeapproximatedbyprojectingthethree-dimensionalvalues,dHvaluesweremeasuredforproteins(Table1).ResultsAuNPstoatwo-dimensionalcirclewiththesameparticleradius.TheshowedtheaveragedHforBSAaround7nminaccordance44surfaceofthesaidcirclewassubtractedfromtheAuNPsurfacetowiththepreviouslypublishedresults,whilerecTRFandobtaintheparticlesurfaceavailableforproteinbinding(ANP‑available)hTRFhadhydrodynamicdiametersof7.11and10.21nm,usingeq10:respectively.TheELSmeasurementsrevealednegativeζ22potentialvaluesforallproteinsintherangebetween−17ANPavailable−=−=AAAuNPcovered4rrAuNPππ−AuNPand−30mV.TheSPRpeakofAuNPswasidentifiedata2=3rAuNPπ(10)wavelengthof528nm.ComparisonofSteady-StateFluorescenceQuench-ThetheoreticalmaximumnumberofproteinmoleculesonthesurfaceofoneAuNPimmobilizedontheLSPRchip(NLSPR)wasingandLSPR.AdsorptionkineticsandbindingaffinitiesobtainedbydividingANP−availablewithAP.betweenproteinsandNPsaremostlystudiedusingthefluorescencequenchingtechnique.Proteinsarecharacterized■RESULTSANDDISCUSSIONbyintrinsicfluorescenceoriginatingfromthreetypesofaminoPhysico-ChemicalCharacteristicsofAuNPs.Prepara-acidresidues:tryptophan(Trp),tyrosine(Tyr),andphenyl-tionofmonodispersedAuNPsthatweresphericalinshapealanine(Phe).Trpresidueshavethestrongestfluorescencewithaprimarysizeof60.0±7.4nmwasevidencedbyTEMquantumyield,beingthemostaccountableforintrinsicprotein17,21evaluation(Figure2),whileDLSmeasurementconfirmedfluorescence.Thesteady-stateemissionspectraofproteinsmonomodalsizedistribution,showinganaveragedHof65.7±selectedforthisstudyintheabsenceandpresenceofAuNPs0.9nm(Table1).areshowninFigure3.Steady-statefluorescencequenchingmeasurementswereusedforevaluatingproteinbindingaffinities(Kaff)bytheStern−Volmermethodandproteinbindingconstants(KAd)byLangmuiradsorptionisotherms.ToobtainKaffvalues,afixedconcentrationofproteinwasincubatedwithincreasingconcentrationsofAuNPs(Figure3),andthedecreaseintheintensityoffluorescencewasobservedforallproteinsupontheadditionofAuNPs,clearlyindicatingnano−biointeraction.Duringtheinteractions,aslightblueshiftintheemissionspectrawasnoticed.ItiswellknownthattheemissionfluorescenceisdependentonthepolarityoftheenvironmentaroundTrpresidues.TheblueshiftsofemissionpeaksoccurduetothetransferoftheTrpresiduesintoamorehydrophobicenvironment,whiletheredshiftsarecon-19,45sequencesofmoreexposuretothesolvent.SincetheFigure2.Transmissionelectronmicrograph(TEM)ofcitrate-coatedsurfaceoftheAuNPsisalesspolarenvironmentthantheAuNPs.Thescalebaris100nm.surroundingsolution,theobservedblueshiftconfirmedtheproteinbindingontotheAuNPsurfaceandwasinagreementAsexpected,thedHvalueswerehighercomparedtoprimarywithpreviouslypublishedresultsoninteractionsbetweenBSA4246diameterduetothehydrodynamiclayeraroundAuNPs.TheandAuNPsofdifferentdiameters.TheKSVvaluesforthesenegativevalueofζpotentialindicatedstrongelectrostaticinteractionswereobtainedfromtheslopeoftheStern−Volmerstabilizationbycitrateanions,whichenabledthelong-termplot(Figure4)usingeq1.43colloidalstabilityandgooddispersibilityofAuNPs.ThecalculatedKSVvaluesforBSA,hTRF,andrecTRFwereAllDLSandELSmeasurementswereperformedin0.1mM3.70×1010,4.67×1010,and4.33×1010M−1,respectively.NaClinordertoavoidpolarizationeffectsinsolventofpoorForthecalculationofk,aτvalueof5×10−9swasused,q01994https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

4Langmuirpubs.acs.org/LangmuirArticleFigure3.Fluorescenceemissionspectraof0.2μMaqueoussolutionof(a)bovineserumalbumin(BSA),(b)glycosylatedhumantransferrin(hTRF),and(c)non-glycosylatedrecombinanthumantransferrin(recTRF)intheabsenceandpresenceofAuNPsaddedinaconcentrationrangeof0.75−15.05pM.Theexcitationwavelengthwassetat280nm.Figure4.Stern−Volmerplotsofthefluorescencequenchingof0.2μM(a)bovineserumalbumin(BSA),(b)glycosylatedhumantransferrin(hTRF),and(c)non-glycosylatedrecombinanthumantransferrin(recTRF)byAuNPsaddedinaconcentrationrangeof0.75−15.05pM.Theexcitationwavelengthwassetat280nm.Errorbarsrepresentthestandarddeviationofthemeanofthreereplicatetitrations.Figure5.Logarithmicplotsofthefluorescencequenchingof0.2μM(a)bovineserumalbumin(BSA),(b)glycosylatedhumantransferrin(hTRF),and(c)non-glycosylatedrecombinanthumantransferrin(recTRF)byAuNPsaddedinaconcentrationrangeof0.75−15.05pM.Theexcitationwavelengthwassetat280nm.Errorbarsrepresentstandarddeviationofthemeanvaluesofthreereplicatetitrations.Table2.ResultsObtainedfortheInteractionsbetweenAuNPswithBovineSerumAlbumin(BSA),HumanTransferrin(hTRF),andRecombinantTransferrin(recTRF)ObtainedfromtheFluorescenceQuenchingMeasurements(AffinityConstants(K,M−1),HillCoefficients(n),andProteinBindingConstants(K))andtheLSPRExperiments(ProteinaffAdaBindingConstants(KAdLSPR))inPBSat25°CfluorescencequenchingdataLSPRdataproteinK(109M−1)nNK(105M−1)K(105M−1)NaffAdAdLSPRBSA432,000±121,0001.342105.52±1.675.77±0.95158hTRF3.57±0.850.871081.21±0.121.81±0.3681recTRF4.18±0.970.882241.27±0.131.34±0.24168aTheoreticalmaximalnumber(N)ofproteinmoleculesontheAuNPsurfacewascalculatedfromhydrodynamicdiametermeasurements.whichrepresentsthefluorescencelifeofTrpresidues.ThebetweenproteinsandAuNPswereformedintheground49calculatedkqvaluesforBSA,hTRF,andrecTRFwere7.40×statebeforeexcitationoccurred.1018,9.33×1018,and8.67×1018M−1s−1,respectively.AskSincealltestedsystemscorrespondedtoastaticfluorescenceqvaluesforallproteinsweremuchhigherthanthemaximumquenching,bindingaffinityconstants(Kaff)andHillvalueofthediffusion-controlledquenchingconstant(approx-coefficients(n)werecalculatedusingeq11imately2.0×1010M−1s−1),weassumedthatAuNPsinducedastaticandnotadynamicquenchingprocessfortestedlogijjFF0−yzz=+[logKnlogAuNP]jzproteins.47,48Therefore,thenon-fluorescentcomplexeskF{(11)1995https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

5Langmuirpubs.acs.org/LangmuirArticleFigure6.NormalizedLSPRshiftsfor(a)bovineserumalbumin(BSA),(b)glycosylatedhumantransferrin(hTRF),and(c)non-glycosylatedrecombinanthumantransferrin(recTRF),calculatedasΔλ/Δλmaxwithrespecttoproteinconcentration(nM)inPBS,whereΔλ/ΔλmaxreferstoachangeinLSPRpeakpositiondividedbythemaximumchangeuponadsorptionofproteins.ThedataweremodeledwiththeLangmuiradsorptionisotherm.Errorbarsrepresentstandarddeviationofthemeanofthreereplicatetitrations.andobtainedfromtheslopeandinterceptofthelogarithmicimmediatevicinityoftheAuNPs,anyadsorptionwillcauseaplotsoffluorescencequenching(Figure5).shiftinthepeakposition(Δλ).AdsorptionofproteinsusuallyTheHillcoefficient(n)referstothedegreeofcooperativityresultsinapositiveΔλ(redshift)duetothehigherrefractiveinproteinbindingtotheAgNPsurface.Whenn=1,theindexofproteinscomparedtowater(∼1.50versus1.33,bindingofproteinsisindependent,whilen<1orn>133,60respectively).TheincreaseinΔλfollowedtheincreaseindenotesdecreasedorincreased,respectively,ligandbindingintheamountofboundproteinuntilsaturation,afterwhichitthepresenceofotherligandsonthesurface.ForBSA,n>1reachedaplateau.TheadsorptionofBSA,hTRF,andrecTRFwascalculated(Table2),pointingtocooperativityofbindingontoAuNPswasmonitoredasLSPRpeakshifts(Figure6).andBSAmultilayersontheAuNPs,asalreadyevidencedbyFromthemeasuredrelativeLSPRshifts,anormalized50,51earlierstudies,althoughDominguez-Medinaetal.foundresponsecurvewasconstructedafterAuNPexposuretoanti-cooperativebindingofBSAonto51nmsizedcitrate-proteinsolutionsofdifferentconcentrationsbetween0.30andcoatedAuNPs.Hillcoefficientswerecalculatedasbeinglower37.6μM,whichwasusedtocalculateproteinadsorptionthan1forbindingofbothtransferrins,indicatingrepulsionconstants(KAdLSPR,giveninTable2)usingtheLangmuirforcesbetweenboundandfreeTRFmoleculesthatincreaseadsorptionmodelasdescribedabove.52withthesurfaceoccupancy.Therefore,monolayeradsorp-ProteinadsorptionontotheAuNPsurfaceresultedinthetionontoAuNPsmaybepredictedforhTRFandrecTRF.formationofadynamicbiomolecularlayerwhosecompositionWemeasuredamuchhigherKaffvalue(Table2)forBSAvariesovertimeduetotheassociationanddissociationof14−1(4.32×10M)thanvaluespublishedbysomeauthors61proteins.Thisphenomenonallowedthecalculationofthe(ranging109−1011M−1),whichmaybeexplainedbythe48,50,53constantforproteinadsorptionontothenanosurfacebythesmallersizeofAuNPsusedinthesestudies.Indeed,35,41,6254Langmuiradsorptionisotherms.TheLangmuiradsorp-Lacerdaetal.showedthatKaffvaluesfortheinteractionsoftionisothermisamodelofnonlinearadsorptionthatassumesdifferentbloodproteinsandAuNPsincreasedwiththeincreasemonolayerformation.Alternatively,theFreundlichadsorptionintheAuNPdiameter.ThecalculatedKaffvaluesinourstudyisothermcanbeusedtodescribemultilayerbinding,butthefollowedtheorderBSA>recTRF>hTRF,indicatingthatfittingofourresultstotheFreundlichmodelyieldednoglycosylationofproteinsmayhindertheirinteractionwiththecorrelation.FromthecalculatedKAdLSPRvalues,itcanbeseennanosurface.thatBSAadsorptiontotheAuNPswasstrongerthantheAnotherreasonforstrongerBSA−AuNPassociationadsorptionoftransferrins,whichisinaccordancewithresultscomparedtotransferrinsmaybethepresenceofafreecysteineresidueinBSA,whichmayinteractwiththeobtainedforKaffconstants(seeTable2).However,thesetwo55parameterscannotbedirectlycomparedsincetheLangmuirnanosurfaceduetothehighaffinityofsulfurformetals.ItconstantassumesthatthesurfaceofanadsorbentisisknownthattheinteractionbetweenproteinsandthesurfaceofNPsoccursmainlyduetoelectrostaticforcesandcovalenthomogeneous,whichmeansthateverysiteforadsorptionisbinding.56,57Theinitialbindingmaybestimulatedbyequivalentinthetermsofadsorptionenergy.Moreover,electrostaticforcesfollowedbyformingcovalentbondsmainlyadsorptionofeveryproteinmoleculeisindependentonthe63betweentheAusurfaceandsulfurofthecysteineresidueinmoleculesalreadypresentonthesurface.Alternatively,theproteins.56Ontheotherhand,hTRFandrecTRFdonotStern−VolmermodelusesaHillscoefficientforcalculatingthecontainfreethiolgroupssinceallcysteineresiduesareinvolvedcooperativityofbinding,andtherefore,bindingofproteinindisulfidebonds.58Nevertheless,thisisthefirststudyclearlymoleculesisdependentonthemoleculesalreadyattachedtoshowinganobviousdifferenceinassociationconstantsthesurface.betweenalbuminandtransferrin,whiledisagreementswithSincethesetwomodelsarenotbasedonthesamepreviousstudieslikelystemfromthedifferentNPtypesandprinciples,constantscannotbedirectlycompared.Therefore,59weperformedadditionalsteady-statefluorescenceevaluation,conditionsusedintheexperiments.FortheLSPRevaluationofAuNPs−proteininteractions,wherefixedconcentrationofAuNPswasincubatedwithincreasingconcentrationsofproteinsolutionswereaddedtoincreasingconcentrationofproteins,whichresultedindataAuNPsimmobilizedonthechipsurface.SincetheLSPRpeakusedtocalculateKAdfromthemodifiedlinearformofmaximumisdependentontherefractiveindexintheLangmuirequation(eq7).Asmentionedpreviously,linear1996https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

6Langmuirpubs.acs.org/LangmuirArticleFigure7.LinearplotsobtainedfrommodifiedLangmuirequationataconstantAuNPconcentrationof60μMwithincreasingconcentrationsof(a)bovineserumalbumin(BSA),(b)glycosylatedhumantransferrin(hTRF),and(c)non-glycosylatedrecombinanthumantransferrin(recTRF)inaconcentrationrangeof0.15−0.75μM.Theexcitationwavelengthwassetat280nm.Errorbarsrepresentstandarddeviationofthemeanvaluesofthreereplicatetitrations.plotsweremadefromreciprocalvaluesofproteinconcen-Table3.MainDifferencesandSimilaritiesbetweentrationandΔFvalues(Figure7).FluorescenceSpectroscopyandLocalizedSurfacePlasmonFromtheslopesofthecurves,KAdvalueshavebeenResonanceMethodscalculated(Table2).TheseresultswereingoodagreementfluorescencespectroscopylocalizedsurfaceplasmonresonancewithresultsobtainedfromLSPRmeasurementsastheyshowedthattheadsorptionofBSAtotheAuNPsurfacewas2mLminimum0.25mLhighercomparedtothatoftransferrins.bindingaffinitybetweenmoleculesbindingaffinityofadsorbentfortheadsorptionsitesAdditionally,thetheoreticalmaximumnumberofproteinfluorescencequenchingofproteintheLSPRpeakshiftofNPsbymolecules(N)forbothfreeandimmobilizedAuNPswasuponbindingtotheNPsurfaceadsorptionofproteincalculatedfromtheexperimentaldHvaluesofbothAuNPsandinterferencesduetotheinnerfilterdependsonthechangeintherefractiveproteins.Theestimatedsurfacesoccupiedbyaproteineffectindexofsolutionmoleculewerecalculatedtobe53.7,104.2,and50.6nm2forrequiresfluorescentligandslabel-freemethodBSA,hTRF,andrecTRF,respectively.Thisisingoodhighsamplevolumeandanalytelowsamplevolumeandlowconcentrationconcentrationofanalytesagreementwithliteraturereportsthatestimatedthesizeof264,65NPsdispersedinasolutionthatNPsimmobilizedonachipthatrestrictsTRFfromthecrystallographicstructuretoaround42nm.allowsproteinstoattachtoallthenumberofbindingsitesontheHowever,theseexperimentalstudiesyieldedthesurfaceareabindingsitesnanosurfaceoccupiedbyTRFtobesignificantlyhigherthancalculated,conformationalandrotationalconformationalandrotationalentropyentropyofproteinsnotaffectedofproteinsaffectedlikelybecausetheglycanstructurewasnottakeninto64consideration.Indeed,ourdatashowthatglycosylatedAdsorptionconstantsobtainedbytwocomplementaryhTRFoccupiestwiceasmuchspaceasthenon-glycosylatedapproachesyieldedsimilarresults,withslightlylowerstandardrecTRF.TheestimatedsurfaceareaofBSAisalsoin266deviationsfortheLSPRmethod.Apreviouslypublishedworkagreementwiththereportedvalueof60nm.The32ofBoehmleretal.ontheinteractionsbetweenBSAandcalculationsthusyieldthemaximumcoveragereportedinAgNPsusingtheLSPRmethodshowedthatadsorptionTable2.NforAuNPsimmobilizedonaLSPRchipissmallerconstantsincreasedwithNPdiameter.Contrarily,DennisonetthanNforthefreeAuNPssinceaquarterofthesurfaceis41al.obtainedahigheradsorptionconstantforBSAontotheconsideredinaccessibleforbindingduetotheattachmenttoAuNPsurfacecomparedtoourresults,eventhoughtheyusedthechip.TheN(BSA)isroughlythesameasN(recTRF)sincesmallerAuNPs.ThereasonbehindmaybefoundindifferenttheirdHvaluesweremeasuredtobeverysimilar,whilethesurfacecoatingsastheirAuNPswerestabilizedwithpoly-N(hTRF)issignificantlylower.TheliteraturereportsonTRF(allylaminehydrochloride),whichcanaffecttheresults.41andhumanserumalbuminbindingto5nmFePtNPsshowVangalaetal.69claimedthattheadsorptionconstantofBSAtosimilarrelationofN(TRF)andN(albumin)(22vs20citrate-coatedAuNPsshouldbebetween106to5×107M−165,67moleculesperNP).Also,astudycomparingthebindingbasedontheirLSPRmeasurements,buttheyalsousedsmallerofBSAandhTRFonpoly(lactic-co-glycolicacid)NPsyieldedAuNPscomparedtous.However,noneofthesestudiesN(BSA)tobealmostdoublethanN(hTRF),whichconfirmsreportedvaluesthatcanbedirectlycompared,aspresentedin68ourmodel.InrelationtotheobtainedHillcoefficientsourstudy(KAdandKAdLSPRinTable2).However,ourresults(Table2),itcanbeconsideredthatthemaximumsurfacecorrespondquitewelltothoseobtainedbyTsaietal.,70whocoveragewithBSAwouldbeestablishedmoreeasilysincetheemployedamulti-methodapproachtostudyBSAadsorptionBSAbindingiscooperative(n>1).Likewise,sincen<1foronAuNPs.bothtransferrins,theirbindingisun-cooperative,andmoreMeasuredvaluesforKAdandKAdLSPRwerequitesimilarforenergywouldbeneededtoachievesaturation(i.e.,toreachN).allthreeproteins(Table3),implyingnosignificantdifferenceAdvantagesoftheLSPRMethodforNano−BiobetweentheadsorptionofalltheseproteinstotheAuNPStudies.FluorescencespectroscopyandLSPRmethodsdiffersurface.Thiswasnotthecaseforthebindingaffinities(Tableinexperimentalapproachesandcalculationmethods.Thekey2).ThereasonmaybefoundinthefactthattheadsorptiondifferencesandsimilaritiesbetweenbothmethodsareexperimentsmeasuredbyLSPRprovideonlytheinformationsummarizedinTable3.ontheproteintransportfromthesolutiontotheAuNPsurface1997https://dx.doi.org/10.1021/acs.langmuir.0c03569Langmuir2021,37,1991−2000

7Langmuirpubs.acs.org/LangmuirArticleandnotontheactualattachmentkineticsthatwasobtained■ACKNOWLEDGMENTSfromthefluorescencequenchingstudy.Moreover,LSPRThisstudywasfinanciallysupportedbythe“Researchresultsmayexplainchangesinadsorption-inducedreorienta-Cooperability”ProgramoftheCroatianScienceFoundationtion,clustering,andaggregationoftheproteinonafundedbytheEuropeanUnionfromtheEuropeanSocialFundnanosurfaceasafunctionofproteinconcentrationinsolution,undertheOperationalProgrammeEfficientHumanResourceswhilethedynamicequilibriumadsorptionprocesswas2014−2020(grantHRZZ-PZS-2019-02-4323),bytheCro-obstructedduetotheimmobilizationofAuNPsontotheatianScienceFoundationgrantnumberHRZZ-IP-2016-06-chip.Ontheotherhand,theStern−Volmerapproachallows2436,andbythePPPProgrammesforProject-RelatedtheinvestigationofthekineticsofaphotophysicalPersonalExchangebetweenGermanAcademicExchangeintermoleculardeactivationprocess.ServiceandMinistryofScienceandEducationoftheRepublic■ofCroatia(ID57392451).CONCLUSIONSThisstudydemonstratedacomplementaryandcombined■REFERENCESapproachtoinvestigatethenano−biointerface.Fluorescence(1)Akter,M.;Sikder,M.T.;Rahman,M.M.;Ullah,A.K.M.A.;quenchingtitrationisanestablishedtechniquewithawell-Hossain,K.F.B.;Banik,S.;Hosokawa,T.;Saito,T.;Kurasaki,M.AdevelopedtheoreticalbackgroundthatemploystheStern−SystematicReviewonSilverNanoparticles-InducedCytotoxicity:PhysicochemicalPropertiesandPerspectives.J.Adv.Res.2018,9,1−Volmerequation.However,itrequireshighervolumesand16.concentrationsofthesamplesandcostlierequipmentandis(2)Sukhanova,A.;Bozrova,S.;Sokolov,P.;Berestovoy,M.;pronetoopticalinterferences,comparedtoLSPR.TheLSPR,Karaulov,A.;Nabiev,I.DependenceofNanoparticleToxicityonestablishedinthepastdecades,hasstillbeenlessknownandTheirPhysicalandChemicalProperties.NanoscaleRes.Lett.2018,13,appliedinbindingandkineticstudies.Here,theviabilityof44.LSPRforthecharacterizationofNPs−proteininteractionswas(3)Gatoo,M.A.;Naseem,S.;Arfat,M.Y.;MahmoodDar,A.;demonstrated.ThebindingconstantsofthreeplasmaproteinsQasim,K.;Zubair,S.PhysicochemicalPropertiesofNanomaterials:ImplicationinAssociatedToxicManifestations.BiomedRes.Int.2014,(albumin,glycosylated,andnon-glycosylatedtransferrins)to2014,1−8.thesurfaceofcitrate-coatedAuNPswerecalculated.Theorder(4)Moore,T.L.;Rodriguez-Lorenzo,L.;Hirsch,V.;Balog,S.;ofproteinadsorptionconstantsontotheAuNPsurfacewastheUrban,D.;Jud,C.;Rothen-Rutishauser,B.;Lattuada,M.;Petri-Fink,sameregardlessofthemethodused,whilebindingaffinitiesA.NanoparticleColloidalStabilityinCellCultureMediaandImpactobtainedbythefluorescencequenchingmethodshowedtheonCellularInteractions.Chem.Soc.Rev.2015,44,6287−6305.trendofBSA>recTRF≈hTRF.Thisstudy,therefore,clearly(5)Misra,S.K.;Dybowska,A.;Berhanu,D.;Luoma,S.N.;Valsami-demonstratedtheusefulnessoftheLSPRapproachinJones,E.TheComplexityofNanoparticleDissolutionandItsImportanceinNanotoxicologicalStudies.Sci.TotalEnviron.2012,nanotoxicologicalstudieswhileadvisingtheconsiderationof438,225−232.theirdifferences,limitations,andtransparencyinreporting(6)Docter,D.;Westmeier,D.;Markiewicz,M.;Stolte,S.;Knauer,S.experimentalconditions.K.;Stauber,R.H.TheNanoparticleBiomoleculeCorona:LessonsLearned-ChallengeAccepted?Chem.Soc.Rev.2015,44,6094−6121.■(7)Feliu,N.;Docter,D.;Heine,M.;delPino,P.;Ashraf,S.;AUTHORINFORMATIONKolosnjaj-Tabi,J.;Macchiarini,P.;Nielsen,P.;Alloyeau,D.;Gazeau,CorrespondingAuthorF.;Stauber,R.H.;Parak,W.J.InVivoDegenerationandtheFateofIvanaVinkovićVrceǩ−InstituteforMedicalResearchandInorganicNanoparticles.Chem.Soc.Rev.2016,45,2440−2457.OccupationalHealth,Zagreb10000,Croatia;orcid.org/(8)Cai,R.;Chen,C.TheCrownandtheScepter:Rolesofthe0000-0003-1382-5581;Email:ivinkovic@gmail.comProteinCoronainNanomedicine.Adv.Mater.2019,31,1805740.(9)delPino,P.;Pelaz,B.;Zhang,Q.;Maffre,P.;Nienhaus,G.U.;AuthorsParak,W.J.ProteinCoronaFormationaroundNanoparticles−fromthePasttotheFuture.Mater.Horiz.2014,1,301−313.RineaBarbir−InstituteforMedicalResearchand(10)Bargheer,D.;Nielsen,J.;Gebel,G.;Heine,M.;Salmen,S.C.;́OccupationalHealth,Zagreb10000,CroatiaStauber,R.;Weller,H.;Heeren,J.;Nielsen,P.TheFateofaDesignedBarbaraPem−InstituteforMedicalResearchandProteinCoronaonNanoparticlesinVitroandinVivo.BeilsteinJ.OccupationalHealth,Zagreb10000,CroatiaNanotechnol.2015,6,36−46.NikolinaKalceč−InstituteforMedicalResearchand(11)Li,L.;Mu,Q.;Zhang,B.;Yan,B.AnalyticalStrategiesforOccupationalHealth,Zagreb10000,CroatiaDetectingNanoparticle−ProteinInteractions.Analyst2010,135,StephanKastner−LeibnizInstituteofPhotonicTechnology,1519−1530.Jena07745,Germany(12)Lima,T.;Bernfur,K.;Vilanova,M.;Cedervall,T.Under-KatiaPodlesnaia−LeibnizInstituteofPhotonicTechnology,standingtheLipidandProteinCoronaFormationonDifferentSizedPolymericNanoparticles.Sci.Rep.2020,10,1129.Jena07745,Germany(13)Vilanova,O.;Mittag,J.J.;Kelly,P.M.;Milani,S.;Dawson,K.AndreaCsáki−LeibnizInstituteofPhotonicTechnology,JenaA.;Radler,J.O.;Franzese,G.UnderstandingtheKineticsofProtein-̈07745,GermanyNanoparticleCoronaFormation.ACSNano2016,10,10842−10850.WolfgangFritzsche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