HandbookofSurfacePlasmonResonanceToFemkeandNick,MargotandNielsHandbookofSurfacePlasmonResonanceEditedbyR.B.M.SchasfoortandAnnaJ.TudosUniversityofTwente,Enschede,TheNetherlandsISBN:978-0-85404-267-8AcataloguerecordforthisbookisavailablefromtheBritishLibraryr TheRoyalSocietyofChemistry2008AllrightsreservedApartfromfairdealingforthepurposesofresearchfornon-commercial purposesorforprivatestudy,criticismorreview,aspermittedundertheCopyright,DesignsandPatentsAct 1988 and the Copyright and Related Rights Regulations 2003, this publication may notbereproduced, storedor transmitted, inanyformor byanymeans, without thepriorpermission in writing of The Royal Society of Chemistry, or in the case of reproduction inaccordance with the terms of licences issued by the Copyright Licensing Agency in the UK,orinaccordancewiththetermsof thelicencesissuedbytheappropriateReproductionRightsOrganizationoutsidetheUK.Enquiriesconcerningreproductionoutsidethetermsstated here should be sent to The Royal Society of Chemistry at the address printed on thispage.PublishedbyTheRoyalSocietyofChemistry,ThomasGrahamHouse,SciencePark,MiltonRoad,CambridgeCB40WF,UKRegisteredCharityNumber207890Forfurtherinformationseeourwebsiteatwww.rsc.orgForewordMakenobonesabout it, Ilovesurfaceplasmonresonance(SPR)-basedbio-sensortechnology. Afterspendingthreeyearstryingtomeasurebindingcon-stantsusinganalyticalafnitychromatography,IimmediatelysawthebenetsofSPRthersttimeIsatdowninfrontofaBiacorein1991.Eventoday,nocomparable technology exists to characterize molecular interactions in real timewithout labeling in an automated and robust fashion. But as the technology hasexpandedovertheyears, Indthattherearethreegeneral attitudestowardsSPR. Therearethenay-sayerswhohatethetechnology. Therearelong-timeusers who think they are experts. And there are the users who recognize they donotknoweverythingaboutSPRbutareeagertoimprovetheirskills.Ever since the rst commercially viable instrument was unleashed in 1990 bythebiosensorgroupatPharmacia(whichwasspunoutintoaseparatecom-panycalledBiacorein1996,onlytobeacquiredrecentlybyGeneralElectric,which previously bought Amershamwho at one time had merged withPharmacia, soinfact nowthe biosensor grouphas come full circle, eventhough they have always shared the samecafeteria in Uppsala, Sweden), therehave beencritics of SPRtechnology. Somuchsothat in2003I createdacharacter called Dr. Evil Pessimist, who represents a composite of thevariousdetractorsof SPR. Dr. Pessimist rantsandragesaboutproblemshehaswiththetechnology,includingnonspecicbinding,instrumentdrift,masstransportandavidityeffects.HearguesthatsinceSPRusesasurfacetherateconstantswemeasurewill neverreect solution-basedbindingconstants. Infact, muchof hisresentment of thetechnologystemsfromthefact that hisexperimentsfailorhisdatanevertasimplemodel.IthasbeenmyexperiencethattherearetwoprimarycausesofthisSPRa-phobia: poor-qualityreagentsand/orpoorexperimental design. Perhapsthemolecules Dr. Pessimist is studying do not in fact interact or the preparations ofsamples are not active to begin with. Dont shoot the messenger. Dr. Pessimistasserts that his proteins are of high quality because they are a single band onanSDSpagegel. Hefails torealizethat this is not evidenceof anactivepreparationor aconformationallyhomogeneous sample. I thinkbiosensorexperiments are akin to protein crystallography. No structural biologist I knowwouldattempttocrystallizeanimpure,half-denaturedpreparationofproteinvthat hasprecipitatedat thebottomof anEppendortube. Thesadthingisthat garbage intoabiosensor will oftengive complexresponses that usersmisinterpretassomeinterestingbindingevent.Ihavefoundthatwhenexperimentsaredesignedappropriatelywithgood-quality reagents and data are processed and analyzed properly, bindingresponsescanberoutinelyttoasimpleinteractionmodel. However, unlikeDr. Pessimist, I do not expect to obtain perfect binding responses when I set upexperimentsonanewinteraction.Irealizethatobtaininghigh-qualitydataisan iterative process. In my research group, we usually set up a trial experimenttoverifythat thebindingpartners actuallyinteract. Thenwewill oftentrydifferent couplingchemistries, surfacedensities, and/or buer conditions tooptimizesurfaceactivity.And when it comes to the number one complaint about SPR technology (thatthe surface will automatically change the thermodynamics of the system), whatDr. Pessimist fails to realize is that most biosensor experiments do not use a atsurface. Instead, thesurfaceis coatedwithadextranlayer, whichsuspendsthemoleculeinsolution. Weandothershaveshownwithnumeroussystemsthat whenexperimentsareperformedproperly, bindingconstants(includingthermodynamic parameters) measured with SPRdo in fact match thoseobtainedfromsolution-basedmeasurements.However,IagreewithDr.Pessimistinoneregard.Since1991,Ihavereadevery paper that reported using a commercial SPR biosensor and Dr. RebeccaRichandIhavecomposedayearlyreviewoftheliteraturesince1998.Thisisbecomingafairlydauntingtasksince more than1000researchpapers arepublished annually. More, unfortunately, is not always better. We nd that thedata in most biosensor articles are not worth the paper they are printed on. Forexample, abouthalfthetimeauthorsevenfail topresentguresshowingthebindingresponses andyet theyexpect us tobelievetherateconstants theyreportfortheirinteractions.Withoutavisualinspectionofthedata,wehaveno idea if the experimentswere runproperly. Andoftentimes, evenwhen dataarepresented,itisclearthattheinvestigatorsdonotknowhowtoutilizethetechnology properly. Also, while a fundamental dogma of science is to replicateandrandomize samples, less than3%of publishedbiosensor dataincludereplicateinjections evenwithinasingleexperiment. Anoverlayof replicateinjectionsdemonstratesthestabilityofthereagentsandmultipleindependentexperimentsyieldanaverageandstandarddeviationforthereportedbindingconstants, yetthisattentiontodetail inabiosensorexperimentismorerarethan nding a four-leaf clover in the outeld at Fenway Park. In addition, lessthan 5% of the authors who report kinetic constants include an overlay of thebindingresponsewiththettedmodel. Andnally, evenfromabriefglancethroughtheliterature,itisapparentthatthemajorityofinvestigatorsdonotunderstandthattheshapeoftheresponseproleshouldbeanexponentialinboththeassociationanddissociationphases(maybemanyusersdonotevenunderstandwhatanexponentialis).Itisnowonderthatscientistsoutsidethebiosensor use community think SPR technology does not work. I would thinkthesamethingifallIhadtorelyonwasthepublisheddata.vi ForewordYou might ask yourself, how did it get to this point? I often wonder if allscientists are sopoorlyeducatedinbasic scientic technique (whichcouldactually explain why we havent found a cure for the common cold). I place theblame on the kit mentality that was introduced with molecular biology backin the early 1990s, back when we were listening to our Walkmans while typingonourIBM286personal computers. Nowadaysyoucanbuyakittoclone,mutate, expressandpurifyaprotein. Well, thekitmentalitycontinuedwhenthesesameinvestigatorsgotaccesstocommerciallyavailablebiosensortech-nology.Sincetheseinstrumentsaresoeasytouse,anyonecanwalkuptothemachine, chuck in their proteins, collect some response, t the data and publishtheresults,believingthatthe resultsmustbecorrectbecause theycameoutofthisveryexpensivemachine. Unfortunately, it actuallytakessomeskill andknow-howtosetup,executeandanalyzeabiosensorexperimentproperly.This leads me to the next group of biosensor users that give the technology ablackeye. Thesepeoplearetheoneswhohavebeenusinginstrumentsforalongtimeandthinktheyareexperts. Icall themSPiRts. SPiRtsareevenmorethreateningthanPessimistsbecausetheircomplacencyoftenleadsthemtoperpetuatepoor experimental technique. AcommonSPiRt mistakepub-lished in the literature is the use of multivalent analytes in solution (e.g.monoclonal antibodies or GSTfusionproteins), whichcanproduceavidityeffects.Alltoooften,SPiRtspresentelaboratebiologicaljusticationsfortheshapeoftheirunusual bindingproleswheninfacttheresponsesaresimplyindicative of poor reagent quality and/or inadequate experimental optimizationordataprocessing. Evenworse, SPiRtsusecomplexmodelstodescribetheirpoor-quality data. It seems that the latest fad of these model surfers is to applyaconformational changemechanism. Mydatat aconformational changemodel,whichmustmeanthereisaconformationalchange,right?Wrong!Toset the recordstraight, in1994 my colleague andsoftware engineerextraordinaire, Tom Morton (who I refer to as SoftEE), developed the numeri-cal integration approach to data analysis that allows one to apply anyinteractionmodel. Beforethen, wewereinthecavemandaysoflineartrans-formation and, believe me,you dont wantto go backthere.We were therstto show that a change in conformation that stabilized a bound complex wouldinfact produce achange inresponse eventhoughthere was noadditionalchangeinmass. However, intheintervening13yearsIhaveneverneededtoapplythis model todescribe the responses obtainedfrommore than1000systemsIhaveexamined.ThereasonIamreluctanttousethismodelisthattypically a data set that ts a conformational change model can be equally welldescribed by other models such asthose for ligand and/or analyte heterogene-ity.Evenmorealarmingly,theratesforthesupposedconformationalchangesmeasuredonthebiosensorareextremelyslow, oftenwithhalf-livesof2060minutes if youtake the time tocalculate them. These rates donot makebiologicalsensetome.Aquicksearchoftheclassicalconformationalchangeliterature shows that re-organizational events which occur during bindinghappenonananosecondtomillisecondtime-scale.ThehotnewtrendwiththeSPiRtsistottheirbiosensordatawithaconformational changemodelvii Forewordandthenpresentcrystalstructuredataofunboundandboundcomplexesandsay See, this change in conformation proves it. But an objective viewer woulddisagree. The fact that yousee a change inconformationinthe structureactually may not relate to the complex binding response you are measuring onthe biosensor. Dont be fooledby these sleight-of-handarguments. (Whatwould help conrmthe conformational change suggested by SPRwouldactually be to use a time-resolved structural method such as circular dichroismor uorescence resonance energy transfer and demonstrate that the time-dependentchangesarethesame.)Thecauseofthecomplexbindingresponseon the biosensor is actually more likely due to surface aggregation, nonspecicbinding,molecularcrowding,avidityeffectsorsampleheterogeneity.ThisbringsmetomyfavoriteSPRusers,whoIrefertoasSPiRits.SPiRitsarenewusersorthosehavingsomefamiliarityofbiosensortechnologywhohave a deep desire to learn more about its features, applications and potential.They are the ones who are participating in our yearly benchmark studies, whichare geared toward calibrating users experimental technique. They are willing toput in the effort to troubleshoot their systems and want to improve the qualityofthedataandnotjustsettleforwhateverthemachinespitsout.SPiRitswillbe the users who develop novel applications and implement new technologies inthefuture.We needSPiRits because the number andtypes of SPRinstruments areexploding. AnInternet searchreveals more than20 companies developingSPR-basedbiosensor systems. Lately, biosensor advances haveoccurredontwo fronts. First, many of the recently released instruments (and otherscurrentlyunder development) are dedicatedtospecic applications rangingfromsmall-moleculedrugdiscoverytothecharacterizationof complexmix-tures in the clinicaland food sciences. CorningsEpic plate-basedsystem is anexample of targetingthe technologyfor screeningapplications. Second, wehaveseena push toincreasethethroughputofbiosensoranalyses. Inthepastfew years, the launches of BioRads ProteOn XPR36 and Biacores A100 havedramatically impacted the biosensor eld since they allow for parallelprocess-ing of multiple analytes over multiple targets simultaneously. Array-basedplatforms represent the next wave in biosensor development. Biacores FlexchipandinstrumentsbeingdevelopedbyGWCTechnologies,Lumera,IBISTech-nologies, Genoptics and Maven open up the possibility of characterizinghundredstothousandsofinteractionsatonetime.Butnotsurprisingly,thesearrayformatscomewiththeirownsetsofchallenges. Themethodsusedforspotting DNA may not be optimal for producing protein arrays. Clearly, a lotof work remains to be done before protein array systems meet their fullpotential.As biosensor applications expandandnewinstruments are released, thetechnologysuserbasealsoincreases.Iworrythathigher-throughputsystemsmayallowmoreuserssimplytogeneratemorebaddatafaster.So,weclearlyneedtoimprovetheskilllevelofbothnoviceandseasonedusers.This book is a great resource to obtain the fundamental knowledge ofbiosensor technology, and also discover recent developments in bothviii Forewordinstrumentation and applications. But in order to turn professional, rememberthatthebiosensorisjustatool.Useitwisely.Beskeptical,butkeepanopenmind. Knowwhentosaywhen(not all systems areamenabletobiosensoranalysis).GoforthandbecomeagoodShePaRdofmyfavoritetechnology.DavidG.MyszkaUniversityofUtahix ForewordPrefaceLectorisalutem,Editors, authors oftenclaimthat completionof their bookwas agiant,lonesome andtedious task. Oftenthey add, their missionwas once inalifetime. As they say in Germany with a sense of humor, Einsteinmacht nochkeinHaus1 meaningthat the cooperationof people is intheheart of big achievements. So it has been with our book: all the authors had tond time in their busy life among other important engagements fortimelywritingactivities, forwhichwecannotsayoftenenoughhowgratefulweare.This Handbook of Surface Plasmon Resonance is the product of anintensiveinteractionprocessandisintendedforawideaudience:scientistsandstudents intending to use the technology, the wider public interested inSPRasaphenomenonanditsapplication,butalsoprovidersof(partsof)thetechnology. Although the book as a whole covers many aspects of the technologyat presentspanningabridge betweentheory,instrumentationandapplications,thechaptersarewrittensoastobecomprehensibleindividuallyaswell. Itishopedthatthereadersofthisbookwillshareourenthusiasmforbiomolecularinteractionanalysis basedonSPRtechnology. We alsohope that we havesucceededinrevealingthe potential of SPRbyshowinghighlyexcitingandunique opportunities for unraveling the functional relationships of complexbiologicalprocesses.Special thanks arealsoduetothemembers of theBiochipGroupof theMESA+InstituteforNanotechnologyoftheUniversityofTwentewhohavecontributedtothebook:StefanSchlautmannandHansdeBoerfortechnicalsupport andsome of the drawings. Inaddition, we thankGeert BesselinkBianca Beusink, Angelique Lokate, Dietrich Kohlheyer, Ganesh Krishna-moorthy,DawidZalewski,RemcoVerdoold,Maykevan derPloegand BjornHarinkfortheirinput. Thisdevotedteamprovidedthewarmandinspiringatmosphere of the Biochip Group during the two-year period from the birth oftheideatocompletionofthemanuscript.1Literally: even Einstein could not build a house and the German meaning of ein stein is one stone.xTheeditorswouldalsoliketothankAnnieJacoboftheRoyal SocietyofChemistry for her clear guidance and enduring patience throughout theeditorial process. Wout vanBennekomisacknowledgedfornal readingofseveralchapters.RichardSchasfoortandAnnaTudosEnschedeandAmsterdamxi PrefaceContentsChapter1 IntroductiontoSurfacePlasmonResonanceAnnaJ.TudosandRichardB.M.Schasfoort1.1 WhatisSurfacePlasmonResonance? 11.1.1 ASimpleExperiment 11.1.2 FromDiptoReal-timeMeasurement 21.2 HowtoConstructanSPRAssay? 31.2.1 TheStepsofanAssay 41.2.2 CalibrationCurve 61.2.3 DeterminationofKineticParameters 71.2.4 BasicsofInstrumentation 81.3 HistoryofSPRBiosensors 91.3.1 EarlyHistoryofSPRBiosensors 91.3.2 HistoryofSPRBiosensorsAfter1990 111.4 HowtoReadThisBook 121.5 Questions 13References 13Chapter2 PhysicsofSurfacePlasmonResonanceRobP.H.Kooyman2.1 Introduction 152.2 TheEvanescentWave 162.3 SurfacePlasmons 172.3.1 SurfacePlasmonDispersionEquations,Resonance 172.3.2 ExcitationofSurfacePlasmons 192.3.3 SurfacePlasmonProperties 212.3.4 ChoiceofExperimentalParameters 262.4 AnalysisofMulti-layeredSystems 272.5 SPRSpectroscopy 282.5.1 Enhancement of Fluorescence and Absorbance 282.5.2 SPRandMetalNanoparticles 292.6 ConcludingRemarks 31xiii2.7 Questions 322.8 Symbols 32References 33Chapter3 SPRInstrumentationRichardB.M.SchasfoortandAlanMcWhirter3.1 Introduction 353.1.1 FromSurfacePlasmontoSPRSignal 353.2 SPROptics 383.2.1 Fan-shapedBeam 393.2.2 FixedAngle 403.2.3 AngleScanning 403.2.4 GratingCoupler 413.2.5 OtherOpticalSystems 423.2.6 SPRImagingInstruments 433.2.7 GeneralOpticalRequirementsforSPRInstruments 443.3 SPRLiquidHandlingSystems 453.3.1 FlowCellSystems 453.3.2 CuvetteSystems 483.4 SPRInstruments:StateoftheArt 503.4.1 ExamplesofFan-shapedBeamSPRInstruments 503.4.2 ExamplesofFixed-angleSPRInstruments 543.4.3 Examples of Angle Scanning SPR Instruments 593.4.4 Examples of Grating Coupler SPR Instruments 603.4.5 ExamplesofOtherSPRInstruments 603.4.6 ExamplesofSPRImagingInstruments 633.5 ProteinInteractionAnalysisSystemsofBiacore 693.5.1 Introduction 693.5.2 BiacoreT100 703.5.3 BiacoreA100 733.5.4 FLEXChip 753.6 Conclusion 773.7 Questions 78References 78Chapter4 KineticModelsDescribingBiomolecularInteractionsatSurfacesDamienHall4.1 Introduction 814.1.1 TerminologyofAdsorption 824.1.2 OpticalQuanticationofAdsorptionatanInterface 86xiv Contents4.2 DeningFactorsoftheAdsorptionEvent 874.2.1 MassTransfer 904.2.2 AdsorptionMechanisms 984.2.2.1 IdealizedPartitionProcesses 994.2.2.2 EectofCompetingReactions 1014.2.2.3 SurfaceFunctionsforDierentModesofAdsorption 1024.3 SummaryandConclusions 1154.4 Questions 1164.5 Symbols 1184.6 Acknowledgements 119References 120Chapter5 KineticandThermodynamicAnalysisofLigandReceptorInteractions:SPRApplicationsinDrugDevelopmentNicoJ.deMolandMarcelJ.E.Fischer5.1 Introduction 1235.2 AnityandKineticsofaTransport-limitedBimolecularInteractionattheSensorSurface 1255.2.1 AnityConstants DerivedfromEquilibriumSPRSignals 1265.2.1.1 CorrectionforDepletionofFreeAnalyte Concentration in the Cuvette 1285.2.2 AnityConstantsandRateConstantsDerivedfromKineticAnalysis 1295.2.2.1 kobsKineticAnalysis 1305.2.2.2 GlobalKineticAnalysiswithaSimpleBimolecularBindingModel 1315.3 DetectingMassTransportLimitation:APracticalApproach 1355.3.1 EectofViscosityChangeontheAssociationPhase 1355.3.2 TransportLimitationintheDissociationPhase 1375.3.2.1 RebindingModelforTransport-limitedDissociation 1375.3.2.2 CompetingLigandtoPreventRebindingDuringDissociation 1395.3.2.3 ExperimentalProceduretoAssayHighO-rates 1415.3.3 QuantitativeConsiderationsonMassTransportLimitation 1425.3.3.1 FloworCuvette? 143xv Contents5.4 GlobalKineticAnalysisofComplexBindingModels 1435.4.1 GlobalKineticAnalysisIncludingMassTransportandaConformationalChange 1445.4.2 UnusualKinetics:IntermolecularBivalentBindingtotheSensorSurface 1475.4.3 Global Kinetic Analysis: Concluding Remarks 1525.5 AnityinSolutionVersusAnityattheSurface 1545.6 Thermodynamic vant Ho Analysis Using SPR Data 1585.6.1 vantHoThermodynamicAnalysis 1585.6.2 ComparisonofSPRThermodynamicswithCalorimetry 1615.6.3 TransitionStateAnalysisUsingEyringPlots 1635.7 SPRApplications inPharmaResearch: ConcludingRemarksandFuturePerspectives 1655.8 Questions 1675.9 Symbols 1685.10 Acknowledgements 169References 169Chapter6 SurfaceChemistryinSPRTechnologyErkT.Gedig6.1 Introduction 1736.1.1 General AspectsofSurfacesforBiomolecularInteractionAnalysis 1746.1.2 SelectionoftheOptimalSurface 1776.2 AdhesionLinkingLayersforGold,GlassandPlastics 1816.2.1 AdhesionLinkingLayersforMetalSurfaces 1826.2.2 AdhesionLinkingLayersforInorganicDielectrics 1826.2.3 AdhesionLinkingLayersforPlastics 1836.3 BioinertMatrices 1836.3.1 Non-specicAdsorptionofBiomolecules 1836.3.2 BioinertHydrogels 1856.4 ChoosingtheOptimalNanoarchitecture 1876.4.1 Two-dimensionalSurfaces 1896.4.2 Three-dimensionalHydrogels 1916.5 CouplingProceduresforLigandImmobilization 1946.5.1 AdsorptiveImmobilization 1956.5.2 PreconcentrationMethods Prior toCovalentImmobilization 1956.5.2.1 ElectrostaticPreconcentration 1956.5.2.2 DryImmobilization 197xvi Contents6.5.3 CovalentActivationChemistries 1996.5.3.1 AmineCouplingviaReactiveEsters 1996.5.3.2 AmineCouplingThroughReductiveAmination 2026.5.3.3 ThiolCoupling 2036.5.3.4 ImmobilizationofAldehydesThroughHydrazideGroups 2076.5.3.5 CouplingThroughEpoxyGroups 2086.5.4 ElectrostaticMethods 2106.5.5 DirectedImmobilization 2126.5.6 ImmobilizationofMembraneProteins 2136.6 ConclusionsandOutlook 2166.7 Questions 217References 218Chapter7 MeasurementoftheAnalysisCycle:ScanningSPRMicroarrayImagingofAutoimmuneDiseasesRichard B.M. Schasfoort, Angelique M.C. Lokate, J. BiancaBeusink,GerJ.M.PruijnandGerardH.M.Engbers7.1 Introduction 2217.2 TheAnalysisCycle 2227.3 BuerSolutionsforMeasuringtheAnalysisCycle 2247.3.1 BaselineBuer 2247.3.2 RegenerationSolution 2257.4 SPR-basedImmunoassays 2257.4.1 DirectAssay 2267.4.2 CompetitionAssay 2267.4.3 InhibitionAssay 2277.4.4 SandwichAssay 2287.5 DetectionofMultiplexAnalysisCyclesUsingScanningSPRImaging 2287.5.1 DynamicRangeofScanningSPRImaging 2307.5.2 LiquidHandlingProcedures 2327.5.3 Determination of the Limit of Detection UsingMultiplexAnalysisCycles 2327.6 MonitoringofAutoantibodiesinSerumofRheumatoidArthritisPatients 2357.6.1 ExperimentalConditionsforSerumMeasurements 2357.6.1.1 SerumSamples 2357.6.1.2 SPRMicroarrayInteractionStudies 2367.6.2 Results and Discussion of Monitoring AnalysisCyclesforAutoantibodyScreening 236xvii Contents7.7 FeaturesandBenetsofMonitoringAnalysisCycleswithSPRImaging 2417.8 Conclusion 2427.9 Questions 2437.10 Acknowledgement 243References 243Chapter8 AdvancedMethodsforSPRImagingBiosensingAlastairW.Wark,HyeJinLeeandRobertM.Corn8.1 Introduction 2468.2 AdvancesinSPRIInstrumentationandSurfaceChemistry 2478.3 SurfaceEnzymaticTransformationsforEnhancedSPRIBiosensing 2548.3.1 MeasuringSurfaceEnzymeKinetics 2548.3.2 RNaseHAmpliedDetectionofDNA 2578.3.3 FabricationofRNAMicroarrayswithRNA-DNASurfaceLigationChemistry 2598.4 Nanoparticle-ampliedSPRIBiosensing 2608.4.1 SingleNucleotidePolymorphismGenotyping 2628.4.2 MicroRNADetection 2648.5 SummaryandOutlook 2698.6 Questions 2708.7 Acknowledgements 271References 271Chapter9 SurfacePlasmonFluorescenceTechniquesforBioafnityStudiesWolfgangKnoll, Amal Kasry, JingLiu, ThomasNeumann,LifangNiu,HyeyoungPark,HaraldPaulsen,RudolfRobelek,DanfengYaoandFangYu9.1 Introduction 2759.2 SurfacePlasmonFluorescenceSpectroscopy(SPFS) 2789.3 Interface Kinetics Based on the Langmuir AdsorptionModel 2829.3.1 MassTransport-controlledKinetics 2849.3.2 Interaction-controlledKinetics 2849.3.3 EquilibriumAnalysis 2869.4 ApplicationsoftheKineticModel 2869.4.1 SurfaceHybridizationReactionsofOligonucleotides 2869.4.2 ProteinBindingStudies 2989.5 NovelApproachestoSPFS 300xviii Contents9.5.1 GratingCouplingforSPFS 3019.5.2 Long-rangeSurfacePlasmonsforSPFS 3039.5.3 FluorescenceImagingandColorMultiplexing 3069.6 Conclusions 3099.7 Questions 3099.8 Acknowledgements 310References 310Chapter10 SPRImagingforClinicalDiagnosticsElainFu,TimothyChinowsky,KjellNelsonandPaulYager10.1 Introduction 31310.2 AchievingMiniaturizationandLowCost 31410.2.1 TuninginSPRImagerDesign 31510.2.2 CompactDesignofAdditional SPRImagerOpticalElements 31510.3 OptimizingImagerPerformance 31710.3.1 RefractiveIndexResolution 31710.3.2 LateralResolutionOvertheFieldofView 32010.4 RobustOperation 32110.4.1 EffectsofTemperatureFluctuations 32110.4.2 StrategiestoAlleviatetheEffectsofTemperatureFluctuations 32210.4.3 BulkRICompensation 32310.5 SPRImagingAssays 32410.5.1 MicrouidicImmunoassayDesignforSmallMoleculeAnalytes 32410.5.2 AssayCompatibilitywithComplexSamples 32710.5.3 AssayImplementation 32910.6 Conclusion 32910.7 Questions 330References 330Chapter 11 The Benets and Scope of Surface Plasmon Resonance-basedBiosensorsinFoodAnalysisAlanMcWhirterandLennartWahlstrom11.1 Introduction 33311.1.1 WhyAnalyzeFood? 33411.1.2 FoodAnalysisStepsandSPRAssayFormats 33411.2 BiacoreQandQexKitstheWorkhorseofFoodAnalysis 33811.2.1 QexKitsforScreeningVeterinaryDrugResidues 339xix Contents11.2.2 QexKitsforQuantifyingVitaminContent 34311.2.3 AOACCerticationofQexKits 34411.3 ExamplesofApplicationsforSPR-basedBiosensorsinFoodAnalysis 34511.3.1 QuantifyingAntibioticsinHoney 34511.3.2 ScreeningforVeterinaryDrugResidues 34611.3.3 MilkTesting 34711.3.4 DetectingAntibodiestoSalmonellainMeat 35011.3.5 GeneticallyModiedOrganisms 35011.4 Conclusions 35111.5 Questions 352References 352Chapter12 FutureTrendsinSPRTechnologyRichardB.M.SchasfoortandPeterSchuck12.1 Introduction 35412.2 TrendsinSPRInstrumentation 35512.2.1 SPRImaging 35612.2.2 HyphenationSPRTechnology 35612.2.2.1 SPRMS 35712.2.2.2 OtherHyphenatedSPRTechniques 35912.2.3 NanoparticleSPR 36012.3 TrendsinFluidics 36112.3.1 MicroarraySpottingonGold 36212.3.1.1 DNACodingTechnology 36412.3.2 ProspectsforSPR-basedPointofCareDevices 36612.3.3 Implementation of Lab-on-a-Chip DevicesforSPRSystems 36712.3.3.1 PumpingLiquidsUsingElectroosmoticFlowinMicro-uidicDeviceswithGoldLayers 36712.3.4 Lab-on-a-ChipImplementationUsingFreeFlowElectrophoresisandSPRImagingforProteomics-on-a-Chip 36912.3.5 DigitalMicrouidics 37312.3.5.1 Cell Diagnosis and MonoclonalAntibodyScreeningUsingSPRImagingandDigitalMicrouidics 37512.4 TrendsinSensorSurfaces 37612.4.1 SmartPolymerBrushes 37612.4.2 PhotoactivationofSurfacesforImmobilization 37812.4.3 GradientChemistries 380xx Contents12.5 TrendsinMeasuringReliableKineticParameters 38112.5.1 Introduction 38112.5.2 The Model for Distribution Analysis of RateandEquilibriumConstants 38312.5.3 ExamplesoftheDistributionAnalysisMethod 38512.5.4 ConclusionsandPerspectivesoftheDistributionAnalysisModel 38812.6 FinalComments 39112.7 Questions 391References 392SubjectIndex 395xxi ContentsCHAPTER1IntroductiontoSurfacePlasmonResonanceANNAJ.TUDOSaANDRICHARDB.M.SCHASFOORTbaShellGlobalSolutionsInternationalBV,P.O.Box380001030BNAmsterdam,TheNetherlands;bBiochipGroup,MESA+InstituteforNanotechnology,BiomedicalTechnologyInstitute(BMTI),FacultyofScienceandEngineering,UniversityofTwente,P.O.Box217,7500AEEnschede,TheNetherlands1.1 WhatisSurfacePlasmonResonance?SinceitsrstobservationbyWoodin1902[1,2],thephysicalphenomenonofsurface plasmon resonance (SPR) has found its way into practical applications insensitivedetectors, capableofdetectingsub-monomolecularcoverage. Whatissurface plasmon resonance? Wood observed a pattern of anomalous dark andlight bands in the reected light, when he shone polarized light on a mirror with adiraction grating on its surface. Physical interpretation of the phenomenon wasinitiatedbyLordRayleigh[3],andfurtherrenedbyFano[4],butacompleteexplanation of the phenomenon was not possible until 1968, when Otto [5] and inthesameyearKretschmannandRaether[6] reportedtheexcitationofsurfaceplasmons.ApplicationofSPR-basedsensorstobiomolecularinteractionmon-itoring was rst demonstrated in 1983 by Liedberg et al. [7]. A historical overviewof the use of the phenomenon for biosensor applications is given in Section 1.3 ofthis chapter. To understand the excitation of surface plasmons, let us start with asimpleexperiment.1.1.1 ASimpleExperimentConsider the experimental set-up depicted in Figure 1.1. When polarized light isshone through a prism on a sensor chip with a thin metal lm on top, the lightwill be reected by the metal lm acting as a mirror. On changing the angle ofincidence, and monitoring the intensity of the reected light, the intensity of the1reectedlightpassesthroughaminimum(Figure1.1,lineA).Atthisangleofincidence, the light will excite surface plasmons, inducing surface plasmonresonance, causingadipinthe intensityof the reectedlight. Photons ofp-polarized light can interact with the free electrons of the metal layer, inducingawave-likeoscillationofthefreeelectronsandtherebyreducingthereectedlightintensity.The angle at which the maximum loss of the reected light intensity occurs iscalled resonance angle or SPR angle. The SPR angle is dependent on the opticalcharacteristics of the system, e.g. on the refractive indices of the media at bothsidesofthemetal,usuallygold.Whiletherefractiveindexattheprismsideisnot changing, the refractive index in the immediate vicinity of the metal surfacewill changewhenaccumulatedmass (e.g. proteins) adsorbonit. HencethesurfaceplasmonresonanceconditionsarechangingandtheshiftoftheSPRangle is suited to provide information on the kinetics of e.g. protein adsorptiononthesurface.1.1.2 FromDiptoReal-timeMeasurementSurfaceplasmonresonanceisanexcellentmethodtomonitorchangesoftherefractiveindexinthenearvicinityofthemetal surface. Whentherefractiveindexchanges,theangleatwhichtheintensityminimumisobservedwillshiftasindicatedinFigure1.2,where(A)depictstheoriginal plotofreectedlightintensity vs. incident angle and (B) indicates the plot after the change inrefractiveindex.Surfaceplasmonresonanceisnotonlysuitedtomeasurethedierence between these two states, but can also monitor the change in time, ifone follows in time the shift of the resonance angle at which the dip is observed.A Angle () BIntensity of reflected light(%) Figure 1.1 Schematic experimental set-up of surface plasmon resonance excitation. Asensor chipwithagoldcoatingis placedonahemisphere (or prism).Polarizedlight shines fromthe light source (star) onthe sensor chip.Reectedlight intensityismeasuredinthedetector(disk). At acertainangle of incidence (j), excitation of surface plasmons occurs, resulting in adip in the intensity of the reected light (A). A change in refractive index atthesurfaceofthegoldlmwillcauseanangleshiftfromAtoB.2 Chapter1Figure1.2depictstheshiftofthedipintime, aso-calledsensorgram. Ifthischangeis duetoa biomolecularinteraction, thekineticsof theinteractioncanbestudiedinrealtime.SPRsensorsinvestigateonlyaverylimitedvicinityorxedvolumeatthemetal surface. The penetrationdepthof the electromagnetic eld(so-calledevanescenteld)atwhichasignalisobservedtypicallydoesnotexceedafewhundrednanometers,decayingexponentiallywiththedistancefromthemetallayeratthesensorsurface. Thepenetrationdepthoftheevanescenteldisafunctionofthewavelengthoftheincidentlight,asexplainedinChapter2.SPR sensors lack intrinsic selectivity: all refractive index changes in the evanes-cent eld will be reected in a change of the signal. These changes can be due torefractive index dierence of the medium, e.g. a change in the buer compositionorconcentration; also, adsorptionof material onthesensorsurfacecancauserefractive index changes. The amount of adsorbed species can be determined afterinjection of the original baseline buer, as shown in Figure 1.2. To permit selectivedetection at an SPR sensor, its surface needs to be modied with ligands suited forselective capturing of the target compounds but which are not prone to adsorbingany othercomponentspresent in thesampleorbuermedia.1.2 HowtoConstructanSPRAssay?Now we have a basic understanding of the surface plasmon resonance signal andhow to measure it in time. We know that the sensor surface needs to be modiedto allow selective capturing and thus selective measurement of a target compound.AngleABTime (s)Figure1.2 Asensorgram: theangleatwhichthedipisobservedvs. time. First, nochange occurs at the sensor and a baseline is measured with the dip at SPRangle (A). After injection of the sample (arrow) biomolecules will adsorb onthesurfaceresultingin achange in refractiveindexand ashiftof theSPRangle to position B. The adsorptiondesorption process can be followed inreal timeandtheamountof adsorbedspeciescan be determined.3 IntroductiontoSurfacePlasmonResonanceIn the following, we are going to learn more about an SPR measurement. First, thestepsofanSPRassay willbediscussedfromimmobilization through analysistoregenerationinameasurement cycle. Next, we get acquaintedwithatypicalcalibration curve, followed by examples of assay formats. Finally, a short outlookis provided on the basics of theinstrumentation.1.2.1 TheStepsofanAssayIn the simplestcase of an SPR measurement, a target component or analyte iscaptured by the capturing element or so-called ligand (Figure 1.3). The ligand ispermanentlyimmobilizedonthesensorsurfaceprevioustothemeasurement.Varioussensorsurfaceswithimmobilizedligandsarecommerciallyavailable,andmanymorecanbecustom-made,asexplainedinChapters6and7.In the simplest case, the event of capturing the analyte by the ligand gives risetoameasurable signal, this is calleddirect detection. Figure 1.4shows thesensorsignalstep-by-stepinthemeasurementcyclewithdirectdetection.Each measurement starts with conditioning the sensor surface with a suitablebuersolution(1).Itisofvitalrelevancetohaveareliablebaselinebeforethecapturing event starts. At this point, the sensor surface contains the activeligands, ready to capture the target analytes. On injecting the solution containingGlassGold 50 nmFlow of sample with analyte Bound ligand Y Figure 1.3 Schematic representation of direct detection: the analyte is captured by theligands (Y) immobilized on the sensor surface. Accumulation of theanalyteresultsina refractiveindexchange intheevanescenteldshiftingtheSPRangle.Heretheligandisimmobilizedinahydrogel.4 Chapter1the analytes (2), they are captured on the surface. Also other components of thesamplemightadheretothesensorsurface; withoutasuitableselectionoftheligand,thisadherencewill benon-specic,andthuseasy tobreak.Atthis step,adsorptionkinetics of theanalytemoleculecanbedeterminedinareal-timemeasurement.Next,buerisinjectedontothesensorandthenon-specicallybound components are ushed o (3). As indicated in the gure, the accumulatedmass can be obtained from the SPR response (DR). Also in this step, dissociationof the analyte starts, enablingthe kinetics of the dissociationprocess tobestudied. Finally, aregenerationsolutionisinjected, whichbreaks thespecicbindingbetweenanalyte andligand(4). If properlyanchoredtothe sensorsurface, the ligands remain on the sensor, whereas the target analytes arequantitativelyremoved. It isvital inordertoperformmultipletestswiththesamesensorchiptousearegenerationsolutionwhichleavestheactivityoftheligands intact, as the analysis cycle is requiredtotake place repeatedlyforhundreds, sometimes even thousands of times. Again, buer is injected tocondition the surface for the next analysis cycle. If the regeneration is incomplete,remainingaccumulatedmasscauses thebaselineleveltobeincreased.OftenSPRmeasurements are carriedout todetermine the kinetics of abindingprocess.ForrealisticresultsitisvitaltopreventimmobilizationfromStep:Time Injection RSPR-dip shiftt1 2. association 1. baseline3. dissociation 4. regeneration 1. baselineFigure1.4 Sensorgramshowingthestepsofananalysiscycle:1,buerisincontactwith the sensor (baselinestep); 2, continuous injectionof samplesolution(associationstep); 3, injectionofbuer(dissociationstep);DRindicatesthemeasuredresponseduetotheboundtargetcompound;4,removalofboundspeciesfromthesurfaceduringinjectionofregenerationsolution(regenerationstep) followedbyanewanalysis cycle. Abulkrefractiveindexshiftcanbeobservedatt1.Seealsopage222.5 IntroductiontoSurfacePlasmonResonancechanging the ligandina way that wouldinuence its strengthor anitytowards thetarget component. Inaddition, kineticexperiments canprovideinformation on the thermodynamics, e.g. on the binding energy of processes. AdescriptionofthekinetictheorycanbefoundinChapter4andexamplesofkineticstudiesinChapter5.1.2.2 CalibrationCurveApart from kinetic and thermodynamic studies, SPR measurements can also beusedfor the determinationof the concentrationof the analyte inasample(quantitativeanalysis).Inthiscase,rstdierentconcentrationsoftheanalyteareappliedinseparateanalysiscycles. Thesensorgramsmeasuredatdierentconcentrations give an overlay plot similar to that depicted in Figure 1.5, with theplateaus of the association step increasing at increasing analyte concentration [8].A calibration curve can be constructed by plotting the response (DR) after acertaintimeinterval(t1)versusconcentration.When analyzing samples with an unknown concentration of the analyte,usuallymultipledilutionsaremade,forexample10,100and1000times,orformore accurate determinations serial dilutions by a factor of 2. If the concentrationtime t1dR/dt t0R SPR-dip shift Step: 1. baseline2. association 3. dissociation4. regeneration 1. baselineInjection Figure1.5 Typical overlayplotofsensorgramsfromserial dilutedanalyteconcen-trations.Justafterinjectionatt0asamplespecicbindingoftheanalyteoccurs andmass transport tothe surface is rate limitingandlinearlydependent ontheconcentration. Fromtheslopes of apositivecontrol(dR/dt), the concentrationof anunknownsample canbe determined.Duringtheassociationphasethenumberof unboundligandmoleculesdecreasesanddissociationtakesplace.Theo-rateconstantordissocia-tionconstant(kd)canbedeterminedafterinjectingdissociationbueratt1.Seeformoredetailschapter4and5ofthisbook.6 Chapter1of the analyte in the sample is very high, the undiluted sample will yield results onthe upper plateau range of the calibration curve. The diluted solutions, however,might yield points along the lower, concentration-dependent sections of thecalibrationcurve andtheconcentrationoftheanalyte canbedetermined.As mentioned above, SPRsensing means detection of refractive indexchangesat thesensorsurface, whichinpracticetranslatestotheamount ofmassdepositedat thesensorsurface. Direct detectionisonlypossibleif thecapturing event of the analyte brings about measurable refractive indexchanges.This is easier to achieve if the molecularweight of the analyte is high(i.e. around1000 Daorhigher). However, forsmall moleculestoproduceameasurable refractive index change, largenumberswouldbe required, makingthe analysis intrinsically less sensitive. If the analyte is a small molecule(MWo1000 Da),oftendirectdetectionisnotviable.Detectionofsmall moleculescanbecarriedoutusingadierent strategy.Mostoften, small moleculesaredetectedinasandwich, competitionorinhi-bition assay format. In all assay formats, not only the lower detectableconcentration is limited, but also the physical number of immobilized elementson the sensor surface, which provides a maximum limiting value. Discussion ofthedierentassayformatscanbefoundinChapter7andothermethodsforconcentrationdeterminationaredescribedinChapters4and5.1.2.3 DeterminationofKineticParametersThe most prominent benet of direct detection using SPR biosensor technologyisthedeterminationof kineticsof (bio)molecularinteractions. Reactionrateandequilibriumconstantsofinteractionscanbedetermined, e.g. theinterac-tion A+B-AB can be followed in real time with SPR technology, where A istheanalyteandBistheligandimmobilizedonthesensorsurface.Table 1.1 contains the most relevant kinetic parameters, the association anddissociationconstants, for the simplest case A+B-AB. The associationconstantis the reaction rateof complex(AB) formation, giving the number ofcomplexesformedpertimeatunitconcentrationofAandB.Assoonasthecomplex AB is formed, its dissociation can commence. The dissociationrateconstant describingthisprocessexpressesthenumberof ABcomplexesTable 1.1 Denitions of the most relevant kinetic parameters: the associationanddissociationconstants.Associationrateconstant,kaDissociationrateconstant,kdDenition A+B-AB AB-A+BDescription ReactionrateofABformation:numberofABcomplexesformedperunittimeatunitconcentrationofAandBDissociationrateofAB:numberofABcomplexesdissociatingperunittimeUnits l mol1s1s1Typicalrange 1031071015 1067 IntroductiontoSurfacePlasmonResonancedissociating per unit time. Note that the unit dimensions for the association anddissociationrates are dierent andcanvarywiththe stoichiometry of thecomplex. Thetypical range of the association and dissociation constantshowslargevariationsandisdependenton,amongotherthings,thetemperature.WhenassociationofAandBstarts,noproductisyetpresentatthesensingsurface. Atthis point, therate oftheassociationreactionis highestandthat ofthe dissociation reaction is lowest. As the process progresses, more and more ofthe ABcomplex is produced, enhancing the rate of dissociation. Due todecreasing Aand Bconcentration, the rate of association might decrease.Equilibriumis reached when the rates of the association and dissociationreactionsareequal;thedenitionsandunitdimensionsaregiveninTable1.2.Ascanbeseen, theequilibriumassociationanddissociationconstants, whichrepresenttheanityofaninteraction,haveareciprocalrelationshipwitheachother. The eect of parameters such as temperature is described in later chapters.The rate constants (Table 1.1) andequilibriumconstants (Table 1.2) of(bio)molecular interactions provide information on the strength of associationandthe tendency of dissociation. Various aspects of kinetics, models andcalculationofanityconstantsaredescribedinChapters4,5and9.1.2.4 BasicsofInstrumentationStudying biomolecular interactions using SPRdoes not require a detailedunderstandingof thephysical phenomena. It issucient toknowthat SPR-based instruments use an optical method to measuretherefractive index near asensor surface (within B200 nm to the surface). SPR instruments comprise threeessential units integrated in one system: optical unit, liquid handling unit and thesensor surface. Thefeatures of thesensor chiphaveavital inuenceonthequality of the interaction measurement. The sensor chip forms a physical barrierbetweentheopticalunit(drysection)andtheowcell (wet section).SPR instrumentation can be congured in various ways to measure the shift oftheSPR-dip.Ingeneral,threedierentopticalsystems(Chapter2)areusedtoexcitesurfaceplasmons: systemswithprisms, gratingsandoptical waveguides.Most widespread are instruments with a prism coupler, also called Kretschmannconguration[9].Inthisconguration, whichisshowninFigure1.1,aprismcouples p-polarized light into the sensor coated with a thin metal lm. The light isTable 1.2 Denition of the equilibrium association and dissociation constants.Equilibriumassociationconstant,KAEquilibriumdissociationconstant,KDDenition [AB]/[A][B] ka/kd[A][B]/[AB] kd/kaDescription Anitytoassociation:high KA, high anity toassociateStabilityofAB:highKD,lowstabilityofABUnit l mol1mol l1Typicalrange 105101210510128 Chapter1reected on to a detector, measuring its intensity, using a photodiode or a camera.In instruments with a grating coupler [10], light is reected at the lower refractiveindex substrate. In practice, this means that light travels through the liquid beforephotons generate surface plasmonwaves as inellipsometric instruments [11].Besides the grating couplers, some instruments apply optical waveguide couplers[12] or measure the SPRwavelength shift as a result of the biomolecularinteractionprocess (see Chapter2 andref.[13]).All congurations share the same intrinsic phenomenon: the direct, label-freeandreal-timemeasurementofrefractiveindexchangesatthesensorsurface.SPRsensors oer the capability of measuring lowlevels of chemical andbiological compounds near the sensor surface. Sensing of a biomolecularbinding event occurs when biomolecules accumulate at the sensor surfaceand change the refractive index by replacing the background electrolyte.Protein molecules have a higher refractive index than water molecules(Dn E101). The sensitivity of most SPR instruments is in the range Dn E105or 1 pg mm2of proteinous material. Ofteninreal-timebiosensingabsolutevalues are not a prerequisite, only the change is monitoredas a result ofbiospecic interaction at the sensor surface. A detailed description of commer-cialinstrumentsisgiveninChapter3.1.3 HistoryofSPRBiosensorsThetermbiosensorwasintroducedaround1975, relatingtoexploitingtrans-ducer principles for the direct detection of biomolecules at surfaces. Currently themost prominentexample ofabiosensoris theglucose sensor, reportingglucoseconcentration as an electronic signal, e.g. based on a selective, enzymatic process.Some argued that all small devices capable of reporting parameters of the humanbody were biosensors (e.g. ion-sensitive eld-eect transistors (ISFETs) measur-ing pH). But then, a thermometer recording fever should also be called biosensor.According to the present denition, in biosensors the recognition element(ligand)ofthesensorortheanalyteshouldoriginatefromabiologicalsource.Biosensors are analytical devices comprised of abiological element (tissue,microorganism, organelle, cell receptor, enzyme, antibody) and a physicochemi-cal transducer. Specic interaction between the target analyte and the biologicalmaterial producesaphysico-chemical changedetectedbythetransducer. Thetransducerthenyieldsananalogelectronicsignal proportional totheamount(concentration)ofaspecicanalyteorgroupofanalytes.1.3.1 EarlyHistoryofSPRBiosensorsApplication of SPR-based sensors to biomolecular interaction monitoringwas rst demonstrated in 1983 by Lundstroms pursuit towards physical9 IntroductiontoSurfacePlasmonResonancemethods for label-free, real-timedetectionof biomolecules [7]. Theintrinsicproperties of themolecules, e.g. mass, refractiveindexand/or chargedistri-bution[14], wereprobedusingellipsometry, refractometry, surfaceplasmonresonance, photothermic detection methods and others. At the NationalDefense ResearchLaboratoryof Sweden, proteinproteininteractions weremonitoredinreal time, label-free, usingellipsometry. Most importantly, therefractiveindexchangeat alight-reectingsurfacewas theoperatingtrans-ducer mechanism. Althoughsuccessful inthe detectionof refractive indexchangeduetothebindingof biomoleculesonoptical transducersurfaces, adisadvantageof theellipsometeristhat light passesthroughthebulkof thesamplesolution, hencelight-absorbingorparticle-containingsamplescannoteasilybemeasured.Amongother researchlaboratories inthesameperiod, theUniversityofTwente (The Netherlands) was active in the search for nding new transductionprinciples for measuringimmunochemical reactions at eldeect transistordevices(ImmunoFET)[15]andatsurfaceswithan opticalread-out(immuno-chemical optical biosensor, IMOB). Optical transducer principles [16] includingellipsometry,surfaceplasmonresonanceandinterferometricprinciples(MachZehnder)showedpromisefordirecttransductionofthebiomolecularbindingevent.Successfulmeasurements ofimmunochemical reactionsusingSPRwerecarriedoutasearlyasinthemid-1980s[17].Pharmacia BiosensorABchoseSPRastheirplatformtechnologyfordirectsensingofbiomolecularinteractions. TheKretschmanncongurationoeredadvantagesinfreedomofdesignoftheliquidhandlingsystem.Comingfromthe higher refractive index medium (the prism), light does not pass through theliquid, but isreectedat thesensorsurfacecoveredwithathinmetal layer.Goldwas chosenas the best inert metal lmrequiredfor surface plasmonresonance, although from a physical point of view silver provides a better SPReect(seeChapter2).Studiesonthesurfacechemistryledtomodicationofthegoldwithaself-assemblinglayeroflong-chainthiolstowhichahydrogel couldbeattached.Carboxylated dextran was immobilized at the surface, which providesa subst-rate for ecient covalent immobilizationof biomolecules, inadditiontoafavorable environment for most biomolecular interactions. The thickness of thedextran hydrogel of 100 nmis perfectly compatible with the ca. 200 nmevanescenteld(seeFigure1.3).Thereliableproductionofthesehigh-qualitysensor chips was unequivocallythe basis for the successful launchof SPRinstruments.Techniques weredevelopedtoetchsilicatoformacastingmoldfor themanufactureof microuidicowchannels. Also, development proceededonoptogels for use between the prism in the optical unit of the instrument and thesensor chip. The optogel ensures optical contact with the prism, allowing simplereplacement of the sensor chip. These eorts in research and development reliedonthecombinationofthreeunrelatedelds:optics,microuidicsandsurfacechemistry, and resulted in the successful development of the instrumentalconceptofbiomolecularinteractionanalysis(BIA).10 Chapter11.3.2 HistoryofSPRBiosensorsAfter1990In 1990, Pharmacia Biosensor AB launched the rst commercial SPR product,theBiacoreinstrument[18].Theinstrumentwasthemostadvanced,sensitive,accurate, reliable, reproducible direct biosensor techniqueandSPRbecame(and still is) the golden standard of transducer principles for measuring real-time biomolecular interactions. Since the early1990s, producers have beenstruggling to meet the standards set by Biacore. Fisons Instruments1[19] madeserious attempts tocompete withBiacores technology; their cuvette-basedIAsysinstrumentusesevanescenteld-basedtechnology,essentiallynotSPR,forthestudyofbiomolecularinteractions.TheBiacore2000instrumentwasintroducedin1994withimproveddetec-tion and a dierent ow system so that the sample could interact at four spotsonthesensor. Dataofthereferencespotcouldbeusedforsignal correction.Withthe introductionof Biacore 2000it alsobecame possible tomonitordirectly interactions of small molecule analytes reacting with immobilizedproteinligands[20].In1995, thecuvettebasedSPRsystemofIBISTechnologieswaslaunched.The instrument was compatible with the Biacore sensor chip. In 1997, the IBIS II,atwo-channel cuvette-basedSPRinstrumentwithautosampleroperation, wasintroduced[21]. Followingthemerger withthesensor chipcoatingcompanySsens BV in 1999, the development of an SPR imaging instrument was initiated atIBIS Technologies. In 2007, the development of the IBIS-iSPR instrument, withthe scanning angle principle, resulted in the required reliability and accuracy formicroarrayimagingofmultiplebiomolecularinteractions(4500).Thepotencyoftheinstrumentis demonstrated in Chapter 7.BiacoreX, atwo-spotinstrumentintroducedin1996, wasfollowedbytheBiacore 3000in1998. The latter was later extendedwithrecoverytools toimprove interfacing with mass spectrometry [22]. Biacore Q was introduced forthefoodanalysismarketin2000(Chapter11).Positionedforsmallmoleculeanalysis anddrugdiscovery, theintroductionof theBiacoreS51markedatechnology shift in terms of detection, ow cell design and sample capacity: theareaofthedetectedspotwasreducedfrom1to0.01 mm2andthenumberofspots was increasedfromfour tosix. In2004, a high-endinstrument wasintroducedwithfour channels eachwithve sensor spots (Biacore A100).Combining the ow cell of the Biacore S51 instrument and the performance ofthe four-channel Biacore 3000, this instrument has 20 in-line sensors to monitorbiomolecular interactions inthe owcells. The technology is not suitable,however,toimagethesurface.InChapter3,otherBiacoreinstruments(T100andX100)aredescribed.Inordertomeasureupto400interactionssimulta-neously, in2005Biacore acquiredthe gratingcoupler SPRsystemof HTSBiosystems, co-developedwithAppliedBiosystems(8500AnityAnalyzer),whichwascapableofimagingthesensorsurface.Afterrestyling,thisproduct(namedFlexchip)waslaunchedin2006[10].1LaterAnitySensors.11 IntroductiontoSurfacePlasmonResonanceAlthoughitisimpossibletodescribeaccuratelythehistoryofthedevelop-ments of the 25 companies producing SPR (related) instruments (see Chapter 3),it isjustiedtotreat thehistoryalongtheBiacoreproduct line. DuringtheyearsfollowingtheintroductionoftherstSPRinstrument, detectionsensi-tivityhasimprovedbyroughly20-fold.Therangeofanityandkineticdatathat can be determined has been extended at least 100-fold as a consequence ofthe increased sensitivity and due to improvements in data analysis. The amountof independent sensor surfaces grew from four channels in 1990 (Biacore) to atleast 500inthenewIBISSPRimaginginstrument. Thecarboxymethylateddextransurfaceintroducedin1990[23],stilltherstchoiceformanyapplica-tions, has beencomplementedwitharange of other surfaces. Systems fordedicated applications have been introduced by various manufacturers ascomplements toall-purposeresearchinstrumentation[24]. Agoodgaugeofthesuccessofbiosensortechnologyisthatmorethan1000publicationseachyearincludedatacollectedfromcommercialbiosensors.InthepaperentitledSurvey of the 2005 commercial optical biosensor literature, Rich and Myszka[25] gaveanoutstandingoverviewof theSPRliterature, includingpracticallessons inperformingandinterpretingbiomolecular interactionanalysis ex-periments. Themajorityof thepublications(985) in2005employedBiacoretechnology (87%), indicating the relevance of Biacores technology in themarket. Anity Sensors was the runner-upcompany with40 publications(B4%), EcoChemie/WindsorScientic(distributor)totaled18publications.which was essentially fromthe same technology provider (originally IBISTechnologies), Texas Instruments scored 17 publications in 2005 and 60publications(B6%)wereattributedto13othercompanies.With the introduction of a number of new SPR instruments (Chapter 3) anda series of novel sensor surfaces and chemistries, the impact of SPR biosensorsonmolecularinteractionstudieswillcontinuetogrow.Withimprovedexper-imental design, including SPR imaging instruments and advanced data analysismethods, high-quality data for the determination of kinetic parameters ofbiomolecular interactionphenomena canbe obtained. These data promiseadditional insights into the mechanisms of molecular binding events, which willbeimportant for functionregulatoryproteininteractionstudies inorder tounraveltheexcitingprocessesinlivingspecies.1.4 HowtoReadThisBookAlthoughmost chapters canbe readas stand-alone literature ondierentaspectsofSPRtechnology, thishandbookaimstoprovidethereaderwithatotal coverageof thebasicsof thetechniqueandapplicationsandthemostrelevant developments at the time of reviewing. The book starts with adescriptionof thephysicsof surfaceplasmonsandSPRinitsoriginal formandsomenovelapplications,forexample,nanoparticleSPR.ThedescriptionofSPRinstrumentationandasurveyofcurrentlyavailablecommercial products from 25 companies follows in Chapter 3. An introductionon how to obtain kinetic information from SPR measurements can be found in12 Chapter1Chapter 4, followed by Chapter 5 illustrating kinetic and thermodynamicanalysisofligandreceptorinteractions,probingthevalidityofthisapproachin pharmaceutical applications. Chapter 6 brings the reader closer to thesurface architecture and chemical design strategies of SPR sensors. Anin-depth treatise on the analysis cycle and modern assay architecture, includingSPRmicroarray imaging, is providedinChapter 7, followedby advancedmethodsforSPRimagingbiosensinginChapter8.Specic application areas are highlighted in the last few chapters of the book,revealing Surface Plasmon Fluorescence Techniques (Chapter 9) and the futureof medical applications at the point of patient care (Chapter 10) andforfood safety (Chapter 11). Finally, Chapter 12 gives an outlook on future trendsinSPRtechnology, including lab-on-a-chip microuidics andtrends formeasuringreliablekineticparameters.1.5 Questions1. SPRtechnologyfordirectandlabel-freedetectionofbiomolecularinter-actions dominates anity biosensor technologies to a great extent and it isexpected that in 2007 more than 1000 papers regarding SPR results will bepublished.WhatarethetechnicalreasonsforthesuccessofSPR?2. InSPR,theintrinsicrefractiveindexofaproteinwhichaccumulatesonthesensorsurfaceismeasured.Explainhowwecandistinguishbetweentherefractiveindexofthebuerandthatoftheadsorbedprotein.3. WhyshouldweexpressthesensitivityofanSPRinstrumentinaccumu-latedmasspersquaresurfaceandnotinmolesperliter?4. Consider the monophasic reversible interaction A+B"AB, where A istheanalyteandBistheimmobilizedligand. Thesampleisinjectedandshows a higherbackground electrolyte refractive index.Draw the sensor-gramof twoanalysis cycles of injectionof asample withthe secondanalysiscycleatwotimesdilutedsample. Consider100%regenerationaftereachanalysiscycle.5. TheresponseDRgivesusanindicationof theamount of accumulatedmass per unit surface area. How can we determine the concentration of ananalyteinsolutionfromtheseresponses?6. Thestudyoftherateconstantsofbiomolecularinteractionsisanimpor-tantfeatureofsurfaceplasmonresonancebiosensors.Why?References1. R.W.Wood,Philos.Mag.,1902,4,396402.2. R.W.Wood,Philos.Mag.,1912,23,310317.3. LordRayleigh,Proc.R.Soc.London,Ser.A,1907,79,399.4. U.Fano,J.Opt.Soc.Am.,1941,31,213222.5. A.Otto,Z.Phys.,1968,216,398410.13 IntroductiontoSurfacePlasmonResonance6. E. Kretschmann and H. Reather, Z. Naturforsch., Teil A, 1968, 23, 21352136.7. B. Liedberg, C. Nylander andI. Lundstrom, Sens. Actuators, 1983, 4,299304.8. S. Geib, G. Sandoz, K. Mabrouk, A. Matavel, P. Marchot, T. Hoshi,M. Villaz, M. Ronjat, R. Miquelis, C. Leveque and M. de Waard,Biochem.J.,2002,364,285292.9. E.Kretschmann,Z.Phys.,1971,241,313324.10. D. Wassaf, G. Kuang, K. Kopacz, Q.L. Wu, Q. Nguyen, M. Toews,J. Cosic, J. Jacques, S. Wiltshire, J. Lambert, C.C. Pazmany, S. Hogan,R.C. Ladner, A.E. NixonandD.J. Sexton, Anal. 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R.KarlssonandR.Stahlberg,Anal.Biochem.,1995,228,274280.21. T. Wink, J. de Beer, W.E. Hennink, A. Bult and W.P. van Bennekom, AnalChem.,1999,71,801805.22. D. Nedelkov, A. Rasooly and R.W. Nelson, Int. J. Food Microbiol., 2000,60,113.23. B. Johnsson, S. Lofas and G. Lindquist, Anal. Biochem., 1991, 198, 268277.24. R.L.RichandD.G.Myszka,J.Mol.Recognit.,2006,19,478534.25. R.L.RichandD.GMyszka,Anal.Biochem.,2007,361,16.14 Chapter1CHAPTER2PhysicsofSurfacePlasmonResonanceROBP.H.KOOYMANBiophysicalEngineeringGroup,FacultyofScienceandTechnology,UniversityofTwente,P.O.Box217,7500AEEnschede,TheNetherlands2.1 IntroductionInthelasttwodecades,surfaceplasmonresonance(SPR)hasevolvedfromafairlyesotericphysical phenomenontoanoptical tool thatiswidelyusedinphysical, chemical andbiological investigationswherethecharacterizationofan interface is of interest. Recently, the eld of SPR nano-optics has been addedwheremetallicstructuresonananoscalecanbedesignedsuchthattheycanperformcertainopticalfunctions.Thischapterwillbemainlyconcernedwiththe more conventional, well-understood SPR theory used in sensor applicationsanditwilltouchuponsomeofthenewerdevelopmentsrelevantforthisarea.Essential for the generation of surface plasmons (SPs) is the presence of freeelectrons at the interface of twomaterials inpractice this almost alwaysimpliesthatoneofthesematerialsisametalwherefreeconductionelectronsareabundant. Thisconditionfollowsnaturallyfromtheanalysisofametal-dielectric interface byMaxwells equations. Fromthis analysis, the pictureemerges that surface plasmons can be considered as propagating electron densitywaves occurring at the interface between metal and dielectric. Alternatively,surfaceplasmonscanbeviewedaselectromagneticwavesstronglyboundtothis interface; it is found that the surface plasmon eld intensity at the interfacecanbemadeveryhigh,whichisthemainreasonwhySPRissuchapowerfultoolformanytypesofinterfacestudies.Experimental researchonSPsstartedwithelectronbeamexcitation; in1968,optical excitation was demonstrated by Otto [1] and Kretschmann and Raether [2].This last approach turned out to be much more versatile, so in this chapter thefocus will be on the optics of SPR. The following is by no means intended as an15in-depth treatment of surface plasmons, rather it is an attempt to provide a low-threshold introduction to the physics of SPRfor those who are actuallyinvolvedinSPRworkandwant tounderstandabit morethanmeasuringtheshiftoftheSPRdip.2.2 TheEvanescentWaveBefore we discuss SPs in more detail, it may be appropriate to provide a mathe-matical description of the evanescent wave, which is so central in the concept ofSPR sensing. This is conveniently done by considering the phenomenon of totalinternalreection.An electromagnetic plane wavethat propagates in a medium with refractiveindexncanmathematicallybedescribedbyanelectriceldE:E E0 expjot jk r E0 expjot jkxx jkyy jkzz_ _2:1whereE0istheamplitudeoftheelectriceld, oistheangularfrequency,kisthewavevector, r (x,y,z) isthepositionvectorandj O1. Notethat eq.(2.1)onlyrepresentsatravelingwaveiftheexponentiscomplex.In the present context, we will mainly be concerned with the wavevector k: itsdirectionis paralleltothatofthewavepropagation;itsmagnitudeisgivenbyk k2x k2y k2z_ n2pl noc2:2where l and c are the wavelength and propagation velocity in vacuum,respectively.Nextweconsidertherefractionofsuchawaveataninterfacebetweentwomedia1and2withrefractiveindicesn1andn2, respectively(seeFigure2.1).Withoutlossofgenerality,wecanchoosethedirectionofthelightbeamsuchthat kz 0 and our problembecomes essentially two-dimensional. FromelementaryphysicsweknowthatforthissituationSnellslawholds:n1 sin a n2 sin b 2:3aor,equivalently,kx1 kx2kx2:3bBy using eqs. (2.2) and (2.3b), we can nd an expression for the component ofthewavevectorkyperpendiculartotheinterface1:k2y2 n212pl_ _2n22n21 sin2a_ _2:4Now, let us assume that n14n2. From eq. (2.4), it is seen that for sin a 4n2/n1the right part is negative, and, consequently, ky is purely imaginary. Returning to1Notethatthedirectionyisinthischapteralwaysperpendiculartothesurface.16 Chapter2eq. (2.1), we conclude that for this case in medium 2 there is only a traveling waveparallel totheinterface:E2 E0eky2yexpjot jkxx 2:5with the amplitude of the electric eld exponentially decaying along the y-directionwithacharacteristicdistance1/ky2 1/jky2. Forobviousreasons, thiseldinmedium 2 is denoted as the evanescent eld. Eq. (2.4) can be used to calculate itspenetrationdepth,whichisoftheorderofhalfawavelength.Thisexplainstheinterface sensitivity of the evanescent eld: only close tothe interface is anelectromagneticeldpresent;therefore,onlyachangingdielectricproperty(e.g.a changing refractive index) in the vicinity of the interface will inuence this eld.WewillseethatalsoinSPRanevanescenteldisgenerated.2.3 SurfacePlasmons2.3.1 SurfacePlasmonDispersionEquations,ResonanceThere are several approaches that all result in the dispersion relation for an SP,that is, a relation between the angular frequency o and the wavevector k. In hislaststandardtreatiseonSPs,Raether[3]calculatedtheSPdispersionrelationfrom rst principles, viz. Maxwells equations. A particularly elegant approachwassuggestedbyCardona[4]andwewilladoptithere.ForreasonsthatwillbecomeclearinthecourseofSection2.3.2, wewill onlydiscussp-polarized2Figure2.1 Refraction of light at an incident angle a, at an interface of two materialswith refractive indices n1 and n2. Definition of axis system and quantities.2p-Polarizedlighthasitselectriceldvectorintheplaneofincidence.17 PhysicsofSurfacePlasmonResonancelightinteractingwithaninterface. Foranyinterfacebetweentwomedia, thecomplexreectioncoefcient rpforp-polarizedincident light electriceldisdescribed by Fresnelsequations (see, e.g., ref. [5] for a derivationon the basisofMaxwellsequations):rp EiErrp ejjtana btana bejj2:6awhereEiandEraretheincidentandreectedelectricelds,respectively,andtheangles aand baredenedasshowninFigure2.1.3Ofcourse,theangles aand bareagainrelatedbySnellslaw[eq.(2.3)];inaddition, aphasechangejofthereectedeldrelativetotheincidenteldoccurs,dependingontherefractiveindicesofthematerialsinvolved.For the reectance, dened as the ratio of the reected intensities, thefollowingrelationholds:Rp rp 22:6bNow,followingCardona[4],twospecialcasesexist:if a+bp/2,thenthedenominatorofeq.(2.6a)becomesverylargeandthusRpbecomeszero.Thissituation describes the Brewster angle, where there is no reection forp-polarizedlight.Theotherspecialcaseoccurswhen abp/2: weseefromeqs. (2.6a)and(2.6b)thatRpbecomesinnite: thereisaniteErforaverysmall Ei. This circumstance corresponds to resonance. Fromthis relationbetweenaandbwe candeduce the dispersionrelationif ab p/2, thencosa sinbandtana k1x/k1y n2/n1. For thecomponents of thewave-vectork(kx,ky),wecanwritek2x k21 k2y1 k21 k2xe1e22:7kx oce1e2e1 e2_and kyi oce2ie1 e22:8where e1and e2arethedielectricconstants4ofmaterials1and2,respectively,andi 1or2. Equation(2.8) isthesought SPRdispersionequationforaninterfacebetweentwohalf-innitemedia.Next, weinvestigatethecasewheremedium2isametal. Thismediumthencontains a large number of free electrons and the consequence is that at an angularfrequency ooopitsdielectricconstant e2 willbenegative(see,e.g.,ref.[5]):e2o 1 o2po22:9aop 4pnee2=me_2:9b3NotethatinFigure2.1thedirectiony(insteadofz)isperpendiculartothesurface.4Dielectricconstantandrefractiveindexarerelatedby e n2.18 Chapter2where opistheso-calledplasmafrequency,neisthefreeelectrondensityandeandmearetheelectronchargeandmass,respectively.Generally, this implies that for ooop no electromagnetic eld can propagatein a metal [cf. eqs. (2.1) and (2.2)]. More specifically, provided that e24e1, wendfortheinterfacethatkyiisimaginary, whereaskxremainsreal. Thusanelectromagnetic wave exists, propagating strictly along the interface, withevanescent tails extendingintobothsides of theinterface[cf. eq. (2.5)]. Toget a feeling for the quantities involved, it is instructive to calculate penetrationdepths for areal case, onthe basis of eq. (2.8). We takel 700 nm, thuso2.69 1015s1andagold/waterinterface.Atthiswavelength egoldE16andewaterE1.77. Wecalculatefor thepenetrationdepths 1/ky,water 238 nmand1/ky,gold 26 nm.Nowall ingredients areavailabletoappreciatetheuseof SPRinsensorapplications. Let usassumethat wehaveasituationwheremoleculesXareallowedtoadsorbtothewater/metalinterface.Wecanviewthisasaprocesswhere water molecules are replaced by molecules X. Because, generally,eXaewater, theaveragedielectricconstant closetotheinterfacewill change.Equation(2.8) thendescribestheconcomitant changeof thewavevectorkx.BecausetheSPeldisevanescentinthedirectionperpendiculartotheinter-face,achangeofthedielectricconstant e2isonlydetectableinSPcharacter-istics if this change occurs within the penetration depth of the SP eld: an SPRsensorwillonlybesensitivetomolecularprocesses(binding,adsorption,etc.)that occur at a distance to the metal surface that is roughly half the wavelengthoftheusedlight.2.3.2 ExcitationofSurfacePlasmonsBysubstitutionofeqs. (2.9a)and(2.9b)intoeq. (2.8), weobtainagraphicalrepresentation of the SPR dispersion relation as shown in Figure 2.2 (line I). Inthesamegure,thedispersionrelationfornormallightisdepicted(linea).We immediately see that, apart from the origin, there is no point where the SPRcurveandthelight curveintersect, implyingthatinthisgeometrynormallight cannot simultaneously provide the correct wavevector and angularfrequencytoexciteasurfaceplasmon.Onewaytocircumvent thisproblemistointroduceasecondinterface, asdepicted in the inset of Figure 2.2. Here a thin metal layer (dielectric constant em)issandwichedbetweentwodielectricmaterials1and3withdifferentdielectricconstantse1and e3, withe14e3.ByapplyingFresnelsequationstothetwointerfaces, more complicateddispersionequations are foundthaneq. (2.8);howevertheessentialphysicsremainsunchanged.Wenowndtwodispersionequations for kx, one for each interface, and we see that the line representing thedispersionrelationfornormal light inmedium1(lineb) intersectstheSPdispersion line for the metal/medium3 interface. This indicates that lightincidentfrommedium1canexciteSPs:byproperadjustmentoftheincomingangle a (Figure 2.2, inset), we can tune the incoming wavevector kx kn1sina to19 PhysicsofSurfacePlasmonResonancematchthewavevectornecessaryforSPexcitation.Inthisway,anykxbetweenthe two lines, labeled a and b in Figure 2.2, can be set. As an example, one suchline, labeledc, is indicated. This so-calledattenuatedtotal reection(ATR)techniquewas rst demonstrated5byKretschmannandRaether [2] andhassincethenalmostbecomethestandardtechniqueforSPexcitation.Another wayof providinga wavevectorappropriate forSP excitation is theuse of a metal layer on which a periodic structure is prepared [6] as illustrated inFigure2.3.Whenlight withwavevectorkx 2p/l.nisinyfallsonsuchastructure, thisacts as a diffractiongrating anddiffractionorders m0, 1, 2, . . . aregeneratedinthereectedlight (see, e.g., ref. [7]). Thegeneratedwavevectorkx,netparalleltotheinterfacecanbewrittenaskx;net kx m2pL2:10Figure 2.2 Dispersion relation for surface plasmons. Curves I and II represent the SPdispersion for the interfaces e3/em and e1/em, respectively. The lines a and bare the dispersion relations for normal light in mediume3ande3,respectively, which are dependent on the angle of incidence a in theexperimental setupas indicatedinthe inset. By varyinga, any line cbetweenthelinesaandbcanberealized.5Infact,Ottowastheveryrsttodemonstratethisinasomewhatlessversatileform.20 Chapter2whereListheperiodicityofthegrating. Again, thewavevectorkx,netcanbetunedtotheSPRwavevector, givenbyeq. (2.8),6bychangingtheincidentangle.Up to now the required polarization direction of the incoming light remainedunmentioned. As already pointed out, SPs are conductivity uctuationsbroughtaboutbycollectivesurfacechargedensityoscillations. Thesechargedensitywaveshavetobeexcitedbyanexternalelectriceld.Onlyanelectriceldwithacomponent perpendicular tothe interface caninduce asurfacecharge density; only p-polarized light has a perpendicular electric eldcomponent.2.3.3 SurfacePlasmonPropertiesWithSPs,anumberofspecicpropertiesareassociatedthatareparticularlyrelevant to sensor applications: (1) the eld enhancement, (2) the phase jump ofthereectedelduponSPexcitationand(3)theSPcoherencelength.Field enhancement. A calculation of the electric eld transmission coefcienton the basis of Fresnels equations for the interface reveals that the electric eldat the low index side of the metal can be much larger than that at the other sideofthemetallayer.In Figure 2.4, the intensity enhancement is depicted as a function of the angleofincidenceofincominglightforanumberofdifferentthicknessesofagoldlayer. It is found that very close to the SPR angle the intensity can be enhancedbyafactor of more than30. This circumstance accounts for muchof theFigure2.3 Schematicviewofagratingcoupler. Bydiffractionof anincident lightbeam, the grating produces kx values larger than that of the incident light.By adjusting the incident angle a the wavevector canbe tunedtokxrequiredtoproduceasurfaceplasmon.6The dispersion equation eq. (2.8) is hardly affected if the metal surface has a shallow corrugation.21 PhysicsofSurfacePlasmonResonanceremarkable sensitivitythat the SPRconditionhas for achangingdielectricenvironment.Phase jump. As already mentioned in Section 2.3.1 and expressed in eq. (2.6),areectioneventataninterfaceisgenerallyaccompaniedbyaphasejumpofthereectedeld. This is illustratedinFigure2.5afor aprismgoldwatersystem.For comparison, the conventional SPR dip is shown in Figure 2.5b for thesamelayersystem.WeseethataroundtheSPRdipthephaseofthereectedelectriceldundergoesarelativelylargechange. Thesignificanceofthisphe-nomenonforsensingpurposesismoreclearwhenweplotthereectanceandphasechangesasafunctionofincidentangleforacertainchangeindielectricconstantofthewater. ThisisdepictedinFigure2.5candd. Inthefollowingroughcalculation,weassumethatboththechangeinreectioncoefcient DRand the phase change Dj are proportional to De. From Figure 2.5c we estimatethat DR/De E30, whereas fromFigure 2.5d we nd that Dj/De E250.Experimentally, a minimumDRE103can be measured, whereas a mini-mumDjE103is feasible, usinginterferometric techniques. The conclu-sion is that on the basis of reectance measurements a minimum DeE4 105can be detected, whereas a phase measurement provides a sensitivity ofDe E4 106. Inviewof theveryapproximatecharacterofthiscalculation,Figure2.4 Fieldenhancementforvariousvaluesofthethicknessofthegoldlayer.Wavelengthof theexcitationlight is 700 nm; thelow-indexsideof themetallayerconsistsofwater(e3 1.77).22 Chapter2theabsolutevaluesfoundareoflimitedvalidity; however, thendingthataphase measurement provides an order of magnitude better sensitivity is a hardconclusion and, indeed, this was demonstrated by Nikitin and co-workers [8,9].Theonlydrawbackofthisapproachseemstobethemuchmorecomplicatedexperimentalsetup.SPcoherencelength.Generally,themetalsdielectricconstant e2iscomplexand this circumstance results in a complex propagation constantkx kx0 0+jkx0 0[cf. eq. (2.8)], where kx0 0and kx0 0are real and imaginary parts,respectively. Forasurfaceplasmon, travelingalongtheinterfacewithwave-vector kx, this implies that the eld intensity decays with a characteristicdistance 1/2kx0 0. For gold and silver, the standard metals in sensor applications,Figure2.5 Comparisonoftheangle-dependentphasechanges(a, c)andreectancechanges (b, d) for variation of the dielectric constant at the low-index sideof themetal layer. Agoldlayerisused, SPsareexcitedatl 700 nm.(c) and (d) depict the differential phase and reectance, respectively, for achangeinthemediumsdielectricconstantof0.01.23 PhysicsofSurfacePlasmonResonancetheimaginarypart ofthedielectricconstant increaseswithdecreasingwave-lengthandtheSPpropagationlengthdecreasesaccordingly.ThisisillustratedinFigure2.6: herealayersystemwaspreparedwherea30nmSiO2stripwasdepositedona50nmsilverlayer. Foraseriesofwave-lengths the angle of incidence was chosen such that SPs were excited in the areaFigure2.6 SPRresponsetoadielectricstepatseveralwavelengths.Foreachwave-length the light angle of incidence is set such that outside the strip(extendingfrom0to125 mm) theinterrogatingkxis resonant withthesurfaceplasmonwavevector.Thesurroundingsofthestriphasdielectricconstant e3 1.24 Chapter2outside the strip and for each wavelength the whole area was illuminated with acollimatedlightbeamunderaconstantangleofincidence.Becauseofthecon-trastindielectricconstantbetweenthestripanditssurroundings(air).TheSPresonanceconditionisnotfullledintheareabelowthestripandweseethedecaying SP (increasing reectance) at the left edge of the strip. Beyond the rightedge of the strip the SPR condition is again fullled and the SP resonance buildsup. The gure nicely demonstrates that withdecreasing wavelengththe SPpropagationlengthbecomesshorter: theblurringontheleft sideof thestripbecomeslessprominentforshorterwavelengths.Itturnsoutthatinthewave-length range 50000nm the propagation length varies between o10 and 40 mm.For a quantitative description of the ndings depicted in Figure 2.6, we haveto analyze the interference between the several elds that are present in the layer.InFigure 2.7, the layer systemandthe elds involvedare indicated: theresonant SP eld, the non-resonant SP eld and the external exciting eld withamplitudes E1, E2, E3, respectively. For a resonant SP that enters the SiO2 strip-coveredlayer,thetotaleldreachingthedetectorcanbewrittenas[10,11]Etotx E1 E2 ej k01jk001_ _x E2ejk00x E3ejk00x2:11where k01 is the wavevector corresponding to resonance in the covered area andk00isthewavevectorthatexcitesSPsintheuncoveredarea.DeningAE1E2andBE2E3, theresultingintensityat thedetectorbecomesItotx B2 A2e2k001x 2ABek001xcos k01 k00_ _x 2:12When a non-resonant SP leaves the covered area, the resonant SP builds up andtheintensityatthedetectordecreasesaccordingly[11]:Itotx B A1 ek000x_ _ _ _22:13From Figure 2.6, we see that this model gives a very accurate description of theexperiments.Figure 2.7 Definition of wave vectors and elds for the systemconsisting of adielectricstripontopofametallayer.25 PhysicsofSurfacePlasmonResonanceBoth thismodelandtheexperimentsindicatethata plasmon needsroughlyfour times the propagation length Lx for a full decay or for a full build-up; thispropagationlengthcanbelooselydenedasLx 12k00x2:14ThisimpliesthatSPswithmutualdistancessignificantlylargerthanLxareindependent. This is a very important conclusion because it is the fundament ofsurface plasmon microscopy [12,13], with its many applications in SPR imagingandSPRmultisensing: onasubstrate we candene areas that inanSPRexperimentwillbehavemutuallyindependently,providedthattheseareasaresignificantly larger than Lx2. For SPs on gold, excited at l 632 nm, LxE7 mmand on a total sensor area of 1 cm2more than 104independent sensor patchesthat each have an area of somewhat smaller than 100 100 mm2can in principlebedened, ofwhichtheoptical responsescanbesimultaneouslyreadoutbyusinganimagingsystem. As practical aspects areoutsidethescopeof thischapter,theinterestedreadershouldconsultChapter7formoredetails.2.3.4 ChoiceofExperimentalParametersIt is impossible to dene a general set of optimumSPRparameters, forinstance, optimal spatial resolution in an SPRmicroscopy/imaging setuprequires values of the experimental parameters other thanthose toobtainmaximumsensitivityfor dielectric changes. Therefore, this sectionprovidesonlysomegeneral guidelines, basedonconsiderationofthepropertiesofthemetallayer.To obtain maximumsensitivity,it is advantageous to maximize thesteepnessof the reectance as a function of the angle of incidence, because this allows for amore accurate determination of the angle of minimum reectance (cf. Figure 2.8).This implies optimization of the reectance minimum Rmin and minimizing thewidthoftheresonancecurve.Rmincanbemadeveryclosetozerobyselectingthe appropriate thickness of the metal layer; as canbe seeninFigure 2.8,optimumthicknessesaresomewhatdependentontheappliedwavelengthandarebetween40and50nm. Thewidthoftheresonancecurveismainlydeter-mined by the complex value of the metals dielectric constant. Generally, a large(negative) real part, together withasmall imaginarypart, results innarrowresonance curves. In practice only two options are available for the choice of themetal layer: goldor silver. As seeninFigure2.8, silver has thebetter SPRcharacteristics in view of the larger real part of its dielectric constant; however, itis chemicallyless inert. InFigure 2.8, it is alsoseenthat the use of higherexcitationwavelengthshasanappreciableeffectonthewidthoftheresonancecurve. This is one of thereasons why(near-) infraredSPRexperiments areattractingattention[14,15]. However, itshouldberealizedthatnarrowingthereectance curve necessarily implies increasing the SPR propagation length [eq.(2.13)], which can be a disadvantage in certain SPR imaging applications. For a26 Chapter2goldlayer, it canbe calculatedthat anincrease inwavelengthfrom450to1500 nm results in a change in the propagation length from 100 to almost 1 mm.Finally, it shouldbementionedthat anincreaseinwavelengthresults inanincreaseinthepenetrationdepth1/ky[cf.eqs.(2.4)and(2.5)],withtheconse-quencethat thereectanceminimumwill becomemoresensitivetodielectricchanges relatively far fromthe metal/dielectric interface; hence the surface-sensitivecharacterofSPRbecomeslessprominent.This impliesthat for detec-tionof thegrowthof thinlayers theoptimumchoiceof wavelengthwill bedifferentfromthatinasituationwhereamorebulk-likechangeinrefractiveindexhas tobedetected[33].2.4 AnalysisofMulti-layeredSystemsIn most SPR-based sensor applications, the system of interest consists of a goldorsilverlayeronwhichoneormorethinlayersaredepositedinanaqueousenvironment. Oftenthe desiredparameters are the thicknesses of the severallayers, which can be converted into surface concentrations of the layer-composingmolecules(cf.Chapters4and5).Onewayto obtain theseparametersisa repeated applicationof theFresnelequation [eq. (2.6a)]. The following relation holds for a system consisting of NFigure2.8 TheSPRdip for46 nmofsilver(dashed)andgold(solid)withwateronthelow-indexside,forseveralexcitationwavelengths.Dielectricdataforthemetallayerobtainedfromrefs.[3]and[33].27 PhysicsofSurfacePlasmonResonancelayerswithdielectricconstantsandthicknesseseianddi, respectively, placedbetweenaprismwithdielectricconstantepandamedium(e.g. water) withdielectricconstant ew(see.e.g.,ref.[5]):rpa M11 M12ky;wew_ _ky;pep M21 M22ky;wew_ _M11 M12ky;wew_ _ky;pep M21 M22ky;wew_ _2:15awhereMistheso-calledtransfermatrix:M M1 M2 . . . MN2:15bwithMi cos ky;idi_ _jeiky;isinky;idi_ _jky;ieisinky;idi_ _cos ky;idi_ __ _2:15cTheangulardependenceofrpiscontainedinthewavevectorsky,i, perpen-dicular to the layer system; these can be calculated using eq. (2.4). Thereectancecanbeobtainedbyapplicationofeq.(2.6b).Providedall thicknessesdianddielectricconstants eiareknown, eq. (2.15)gives an accurate description of the SPR experiment. Of course, in practice oneis concernedwiththe inverseproblemandapriori knowledge, suchas thedielectric constant and dimensions of the molecules composing a certain layer,is required for a satisfactory analysis of experimental SPRresults. Thisprecludesanunambiguousanalysisofmorethantwoorthreelayers.Another,moreintuitive,approachistheintroductionofaneffectivedielec-tricconstant, ee. Here, theactual multi-layeredsystemisreplacedbyatwo-layer system, where e1 in eq. (2.8) is replaced by the effective dielectric constantee, given by the average of all dielectric constants in the layer system, weightedbythepenetrationdepthy0oftheSPRevanescenteld[16,17]:eeff 2y0_N0eye2y=y0dy 2:16Ofcourse, withtheuseofthisequation. wefacethesameproblemsasthosewhenweuseeq.(2.15).2.5 SPRSpectroscopy2.5.1 EnhancementofFluorescenceandAbsorbanceUptonowwehaveonlyconsidered, apartfromthemetal layer, transparentlayers, i.e. layersthatarecharacterizedbyapositive, real dielectricconstant.Whenoneormorelayerscontainalight-absorbingcompound,SPscanboostthe uorescence intensity from a thin layer more than 40-fold [18]. This effect isduesolelytothelargeeldenhancementthatoccursonthelowindexsideof28 Chapter2the metal layerwhenan SP resonance conditionis established(cf. Figure 2.4).MoreinformationonthisphenomenoncanbefoundinChapter9. Another,moresubtle, featureoftheinteractionbetweenSPsandlight-absorbingmol-ecules is the increased sensitivity for the detection of absorbances in thin layers.The additionof a light-absorbing layer results intwoeffects onthe SPRangular-dependentcurve,whicharequantitativelydescribedbyeq.(2.15):(1)the SPRdipshifts toalarger angle of incidence and(2) the value of thereectanceminimumincreases. Thislast effect canreadilybeunderstoodaslightabsorptionnecessarilyresultsinadecreasedreectance.Inaddition,theSPReldenhancementonthelow-indexsideofthemetal layerwill resultinincreased sensitivity for dielectric changes [19] and therefore also for changes inthe absorbance. The rst effect is a consequence of the KramersKronigrelation(see, e.g., ref. [20]), whichinthepresentcontextcanbeexpressedasthestatementthatanychangeintheimaginarypartofthedielectricconstantwillbeaccompaniedbyachangeintherealpart;inSPRitismainlytherealpart of the dielectric prole ontopof the metal layer that determines theangularpositionoftheSPRdip.Ithasbeendemonstrated[21]thatanSPR-assisted monolayer absorbance measurement can result in a 40-fold reectanceincrease as comparedwitha metal-lacking ATRsystem. Inaddition, it ispossibletoextractunambiguouslythethicknessandthedielectricconstantofanabsorbinglayerfromasingleSPRexperiment[22].2.5.2 SPRandMetalNanoparticlesSPRphenomenaarenotrestrictedtoplanarmultilayersasdiscussedsofar;itturns out that for metal particles withdimensions muchsmaller thanthewavelengthoftheinteractinglight, SPeffectscanbemuchmoreprominent.Generally,the netelectriceld Etot around a dielectricparticleis composed ofthe superposition of an external applied eld E0 and the induced (dipole) eld intheparticle. Forapolarizablespherical particlewithradiusrmanddielectricconstant e, placed in a mediumwith dielectric constant e1, the followingexpressionisfound(see,e.g.,ref.[5])fortheeldgainG:Go EtotoE0oE1 eo e1eo 2e1rmr rm_ _32:17It is seen that G can reach enormous values for e close to 2e1; in a normaldielectricmediumwheree140, thisconditionpointstotheuseofametal,where e can be negative; additionally, the imaginary part of e should be as smallaspossible.Itturnsoutthatthisconditioncorrespondstotheexcitationofasurfaceplasmoninthemetallicnanoparticle[23]. ParticularlyintheeldofRaman spectroscopy this can result in enormous sensitivity enhancements7(forareview,seeref.[24]).7Conventional Raman spectroscopysuffers from a very low scatteringefciencywhich can be 12ordersofmagnitudelowerthanthatofuorescence.29 PhysicsofSurfacePlasmonResonanceNowconsider a Raman-active molecule near a metal nanoparticle. ThedetectedRamanintensityI(o,osc)canbeexpressedasIRamano; osc gE2exc E2sc gG2oG2oscI0oI0;scosc 2:18where Eexc is the total excitation electric eld to which the molecule is exposedandEscisthetotal Raman-scatteredeld. Theconstantgisanexperimentalconstantthatisunimportantinthepresentdiscussion.Bychoosinganangular frequencyothat excites surfaceplasmons inthemetal (usuallygoldorsilver)anddetectingscatteringfrequencies oscnottoofar from the excitation frequency, both the excitation and the scattered eld areenhancedbythepresenceofthemetalparticle.Bysubstitutingeq.(2.17)intoeq. (2.18), weseethat thedistancedependenceof thenet Ramanscatteringintensitychangeswiththepower12of themoleculenanoparticledistance!Indeed, it has been found experimentally that a surface-enhanced Ramanspectroscopy (SERS) experiment can result in experimental Raman signals thatare enhanced 10121014times compared with those obtained from non-surface-enhancedexperiments.It should be addedthat apart fromthis SPRenhancement mechanism,another chemical enhancement effect is operational, which accounts for a10100-foldamplicationofthebareRamansignal[24].It has been demonstrated [25,26] that SERS is able to detect single molecules.Togetherwith its very high molecular specicity,this offers great promise as adetectiontool for very lowconcentrations of biomolecules, suchas DNAstrandsorproteins.In principle, these eld enhancements should also be important in thedetectionof uorescent molecules near ametal nanoparticle. However, thenearby presence of a metal layerleadsto additional non-radiativedecay pathsof the electronic excited states of a nearby molecule, with the net result that inmanycasestheuorescencewillbelargelyquenched.So far, metal nanoparticles were considered as surface plasmon-assisted eldampliers. However, these particles can also be exploited as intrinsic refractiveindexsensors, analogous tothemorefamiliar planar SPRexperiments (forreviews, see refs. [27] and [28]). The physical basis of this application is the lightextinction (absorption and scattering), which is heavily dependent on thenanoparticlesdielectricconstant,sizeandgeometryandalsoonthedielectricconstant e1 of the surrounding medium. Mie theory gives a reasonably adequatedescriptionof theextinctioncoefcient Aextandfor spherical particles withdiameterlessthanabout20 nmthefollowingexpressionisfound[23]:Aext 18pNpVe3=21lImeRee 2e1 2 Ime 22:19where Np is the number of nanoparticles, each of which has a volume V, and listhewavelengthoftheappliedlight.30 Chapter2Again we see the pronounced inuence of the occurrence of SPs: at Re(e) 2e1wendamaximumintheextinctioncoefcient,whichcanreachlargevaluesfor low values of Im(e). Hence also in this situation we are led to the use of goldorsilverasametalnanoparticle.Moresophisticatedmodels(see,e.g.,ref.[29])alsoaccountforthesizeandshapeofthenanoparticlesandcomputerprogramsareavailableinthepublicdomain that can predict the extinction spectrum of nanoparticles of any shape[30], bymodelingtheparticleas aseries of dipoles placedinanoscillatingelectric eld. However, the mainfeatures of nanoparticle extinctionremaincontainedineq.(2.19).Foronenanoparticlewithadiameteraround25 nm,excitedclosetoitsSPresonance, eq. (2.19) results in Aext of the order of 1016m2, which correspondsto the more familiar molar extinction coefcient in the order of 109l mol1cm1. This value, whichindeedwas observedexperimentally[29], ismore than three orders of magnitude larger than that of strong light-absorbingorganic dye molecules, allowing for relatively simple optical detectionandcharacterizationofindividualnanoparticles[31,32].Inanotherseriesofexper-iments, the shift of the extinction maximum as a function of the refractive indexofthe surroundingmediumwasinvestigated[28].It wasfoundexperimentallythatforsilvernanoparticlesthespectrumcouldshiftasmuchas20 nmforachange of 0.1 inthe refractive index. Because molecules that adsorbtoananoparticlechangetherefractiveindexaround theparticle,itisobviousthatthis, analogous to conventional SP resonance, can be used as a sensor principle.Indeed, it has beendemonstratedexperimentallythat thefull coverageof asilvernanoparticlewithlowmolecularmassmoleculesresultedinaspectrumshiftofapproximately40 nm.Thefullcoveragecorrespondedtoonly4 104molecules.Togetherwiththesingleparticledetectioncapability,thispromisesenormoussensitivity,allowingfornearsinglemoleculedetection[32].2.6 ConcludingRemarksThephenomenonof SPRisoneof themanyexampleswhereaninterestingphysicalphenomenonleadstoapplicationsthatarehighlyimportanttobothappliedscience andsociety. Ina planar SPRsystem, it is particularlythecombinationof eldenhancement andrelativelyshort coherencelengththatallows for a unique sensor concept that provides both multiplexing capabilitiesandvery highsensitivity. The general physical picture is well understood;however, some areas are still in vivid scientic debate (SERS, optics ofnanoparticles).Fromatechnological pointofview, theemergingeldofnanotechnologywill enableus toexploit toits full potential theSPphenomenaof tailorednanoparticles. It istheauthorsrmconvictionthat mergingof (bio-)nano-technologyandSPphenomenaofnanoparticleswillultimatelyleadtosensorconcepts andsensor realizations that will reallybe important innumerous31 PhysicsofSurfacePlasmonResonanceaspectsofsociety, varyingfromfoodsafetymonitoringa