Sensors and Actuators B: Chemical

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Sensors and Actuators B: Chemical

j o u r n al hom e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b

Use of molecular imprinted nanoparticles as biorecognition element on surface plasmon resonance sensor

Gulsu Sener

, Lokman Uzun

, Rıdvan Say

, Adil Denizli

aHacettepeUniversity,NanotechnologyandNanomedicineDivision,Ankara,Turkey

bHacettepeUniversity,DepartmentofChemistry,Ankara,Turkey

cAnadoluUniversity,DepartmentofChemistry,Eskis¸ehir,Turkey

a r t i c l e i n f o

Articlehistory:

Received15March2011

Receivedinrevisedform16August2011 Accepted23August2011

Available online 30 August 2011

Keywords:

Lysozyme

Molecularimprinting Nanoparticles

Surfaceplasmonresonancenanosensor Chickenegg-white

a b s t r a c t

In this study, we have prepared lysozyme imprinted poly(ethylene glycol dimethacrylate-N- methacryloyl-l-histidinemethylester) (PEDMAH)nanoparticlesandthenattachedonthesurfaceof surfaceplasmonresonance(SPR)sensor.LysozymeimprintedSPRnanosensorwascharacterizedby Fouriertransforminfraredspectroscopy,atomicforcemicroscopy,andellipsometry.Thenanosensor hasanabilitytodetectlysozymemoleculesfrombothaqueousandnaturalcomplexsource,chickenegg white,evenifithaslysozymeconcentrationaslowas32.2nM.Associationkineticsanalysis,Scatchard, Langmuir,Freundlich,Langmuir–Freundlichisothermswereappliedtodata.Thecalculateddetection limit,associationanddissociationconstantsare84pM,108.71nM⁻¹and9.20pM,respectively.Thenon- imprintednanosensorswerealso preparedtoevaluatetheselectivityoftheimprintednanosensor.

Finally,thenanosensorisusedforfiveadsorption–desorption–regenerationcycleanditgivesrepro- ducibleresponse.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Molecularimprintinghasattractedgrowingattemptstopre- paremolecularcomplementarypartsoftheinterestedmolecules intohighlycross-linkedpolymericstructure.Themethod,mainly dependsonthemolecularrecognition,isatypeofpolymerization that occursaround theinterestedmolecules calledas template [1,2]. Afterremovalof template molecules, specificcavities are createdinsidesolidpolymericmatrices[3].Thesepolymershave memoriesthatarecapableofselectivelyrebindingthetemplate molecules[4].Byusingthisapproach,recognitionsitesformany molecules including small molecules such as; metal ions [5], organicmolecules[6]andlargemoleculeslikeproteins[7–9]in asyntheticpolymercanbecreated.Thistechniqueisoneofthe mostpromising strategies toproduce artificialrecognitionsys- temsbecausemolecularlyimprintedpolymers(MIPs)usuallyhave lowcostsandhighphysical/chemicalstability.Althoughthistech- niquehasseveraladvantages,ithasalsohassomedrawbacks.First, highlycross-linkedrigidstructuresoftheMIPswithirregularshape reducetherebindingcapacity.Second,thetemplatemoleculesthat areimprintedinsidethepolymercannotberemovedeasilyfrom

∗ Correspondingauthorat:HacettepeUniversity,DepartmentofChemistry,Bio- chemistryDivision,Ankara,Turkey.Tel.:+903122977963;fax:+903122992163.

E-mailaddress:[email protected](L.Uzun).

theircavities.Therefore,somedecreaseintheanalyteadsorption anddesorptionratestotheimprintedpolymerwasobserved.To overcometheseproblems,molecularlyimprintednanosizedmate- rialswerepreferred,because,thesematerialshave highsurface tovolumeratioand morecriticallymostof theimprintedsites are locatedat thesurface. Hereby, the template moleculescan beremovedeasilyand higheradsorptionrates canbeobtained [10–14].

Surfaceplasmonresonance (SPR),anopticalphenomenon,is occurredwhenap-polarizedlightgoesthroughaprism;then,hits ametallayercoveringtheprismsurfaceataparticularangle[15].

SPRwasintroducedintheearly1990sastheunderlyingtechnology inaffinitybiosensorsforbiomolecularinteractionanalysis,anew conceptfortheanalysisofthefunctionalpropertiesofbiomolecules [16].BecauseofuniquepropertiesofSPRbiosensors,i.e.,real-time measurement,highspecificityandsensitivity,noneedtolabeling, theapplicationsofthemhavebeengettingmorepopularforinves- tigationofseveralanalytemolecules[17–20].Recently,MIPsare usedforcreationofbiorecognitivesurfacesontheSPRbiosensors [21,22].

In this study, we have focused our attention oncombining of molecular imprinting into nanoparticles and SPR biosensor approachesandproducingSPRnanosensorforlysozyme,chosenas modelprotein,usinglysozymeimprintednanoparticles.Lysozyme (EC3.2.1.17),calledasbody’sownantibiotic,isarelativelysmall protein(MW:14.3kDa)consistsofonly129aminoacidresidues 0925-4005/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.snb.2011.08.064

andhasanisoelectricpointof11.0.Duetoitssmallsizeandsim- plemolecularstructure, lysozymehasbeenchosenasa unique model protein in developing of new detection methods. There areseveralbenchmarklysozymedetectionmethodsdependingon determinationoflyticactivitybyMicrococcuslysodeikticuscellsand enzymelinkedimmunosorbentassays(ELISA)[23].Formeronehas limitationssuchas lowdetection limit, impossibilityof routine analysisinaccuratequantificationdue tointerfering substances.

Inaddition,unexpectedcross-reactivityhighcastandlowshelf- lifeofELISAkitsarestill waitingforthesolution[24,25].There areseveralcase-reportsinwhichchangeoflysozymeconcentra- tioncanbeamarkerforspecifichealthproblem.Forinstance,the lysozymeconcentrationincreasedincerebrospinalfluidincaseof meningitis[26];inserum andurineincaseof leukemia[27,28]

andseveralkidneyproblems[29,30].Also,it hasbeenreported thatlysozymemaybea newprognosticfactorin patientswith breast cancer [30]. Recently, antibodiesagainst tocitrullinated variantsoflysozymewerefoundinrheumatoidarthritispatients [31]. Therefore, detectionof lysozyme hasbeen gettingimpor- tanceanddevelopingnew,rapid,cheapandeffectivebiosensors havebeenunderinvestigation.Inthiscontent,wehadprepared a QCMsensor for detection of lysozyme[10].In accordance to this purpose, we began with preparation and characterization of lysozyme imprinted poly(ethylene glycol dimethacrylate-N- methacryloyl-l-histidine methylester) (PEDMAH) nanoparticles.

Imprintednanoparticlesweresynthesizedviamini-emulsionpoly- merization;then,attachedonthesensorsurface.Here,weprepared a SPR sensor by using the same approach and compared the resultswiththatofQCMsensorpreparedbefore.Similartopre- viousstudy,lysozymedetectionstudieswerecarriedoutbyusing aqueouslysozymesolutionsandnaturallysozymesource,chicken egg-white, in different concentrations. Kinetic and isotherm parameterswerecalculatedbyapplyingassociationkineticsanal- ysis,Scatchard,Langmuir,Freundlich, and Langmuir–Freundlich isotherms.

2. Experimental

2.1. Materials

Templatemolecule lysozyme (EC 3.2.1.17), albumin (bovine serum),poly(vinylalcohol)(PVA),sodiumdodecylsulfate(SDS), ammoniumpersulfate, sodiumbicarbonateandsodiumbisulfite wereobtainedfrom SigmaChemical Co. (St.Louis, USA). Ethy- leneglycoldimethacrylatewaspurchasedfromFlukaA.G.(Buchs, Switzerland).Allotherchemicalswereofreagentgradeandpur- chasedfromMerckA.G.(Darmstadt,Germany).

2.2. SynthesisofN-methacryloyl-l-histidinemethylester(MAH) monomer

Thefollowingexperimentalprocedurewasappliedforthesyn- thesisoffunctionalmonomer,N-methacryloyl-l-histidinemethyl ester(MAH)[32].l-Histidinemethylester(5.0g)andhydroquinone (0.2g)weredissolvedin100mLofdichloromethanesolution.This solutionwascooleddownto0^◦C.Triethylamine(12.7g)wasadded tothesolution.Methacryloylchloride(5.0mL)waspouredslowly intothis solutionand stirredmagneticallyatroomtemperature for 2h. At theend of theperiod, hydroquinone and unreacted methacryloylchloride wereextracted with10% NaOHsolution.

Aqueousphasewasevaporatedinarotaryevaporator.Theresidue wascrystallizedinNaOHsolution(10%,w/w).

Functional monomer was characterized by ¹H NMR. The obtainedpeaksinspectrumarelistedas¹HNMR(400MHz,DMSO- d6,ı):1.85(t,3H,CH3),1.4(m;2H,CH2),3.42(s;3H,–OCH3),5.28

(s;1H,vinylH),5.6(s;1H,vinylH),6.6–6.9(m;5H,aromatic);7.42 (1H,NH);ı7.47(1H,NH).

2.3. PreparationoflysozymeimprintedPEDMAHnanoparticles

LysozymeimprintedPEDMAHnanoparticleswerepreparedby two-phasemini-emulsionpolymerizationmethod[10].Thefirst aqueousphasewaspreparedbydissolvingofPVA(200mg),SDS (30mg)andsodiumbicarbonate(25mg)in10mLdeionizedwater.

ThesecondphasewaspreparedbydissolvingofPVA(100mg)and SDS(100mg)in200mLofdeionizedwater.Functionalmonomer [MAH, 5mg (≈21␮mol)] was dissolved in monomer (ethylene glycol dimethacrylate,2.1mL) toform oilphase. Theoil phase wasslowlyaddedtothefirstaqueousphase. Inordertoobtain mini-emulsion,themixturewashomogenizedat25000rpmby ahomogenizer(T10,IkaLabortechnik,Germany).Afterhomoge- nization,thetemplatemolecule[lysozyme,100mg(≈7␮mol)]was addedtomini-emulsiontoestablishtheratiobetweenmonomer andtemplateas3:1inmolebasis.Then,themixturewasslowly added tothe second aqueous phase while the phase hasbeen stirring in a sealed-cylindrical polymerizationreactor (250mL).

Thereactorwasmagneticallystirredat300rpm(RadleysCarousel 6,Essex,UK).Thepolymerizationmixturewasslowlyheatedto 40^◦C, polymerizationtemperature.After that,nitrogengas was bubbledthroughsolutionfor5mintoremovedissolvedoxygen.

Then,initiators,sodiumbisulfite(125mg)andammoniumpersul- fate(125mg),wereaddedintothesolution.Polymerizationwas continuedfor24h.Theobtainedlysozymeimprintednanoparti- cleswerewashedwithwater andwater/ethylalcoholmixtures, inordertoremoveunreactedmonomers,surfactantandinitiator.

Thesolutionswerecentrifugedat30000rpmfor30min(Allegra- 64RBeckmanCoulter,USA)foreachwashingstepandthenthe nanoparticlesweredispersedinfreshsolution.Afterlastwashing step,thenanoparticlesweredispersedindeionizedwatercontain- ing0.5%sodiumazidetopreventcontaminationandstoredat4^◦C.

Thenon-imprinted nanoparticlesweresynthesized byapplying sameprocedureexceptadditionoftemplatemolecules,lysozyme.

2.4. CharacterizationoflysozymeimprintedPEDMAH nanoparticles

Characterizationstudiesofthenanoparticleswerecarriedout byZetasizer(NanoS,MalvernInstruments,London,UK)andTEM (FEI,TecnaiG2F30,Oregon,USA).Inzeta-sizemeasurement,the lightscatteringwascarriedoutatincidenceangle90^◦ and25^◦C.

Fordataanalysis,densityandrefractionindexofdeionizedwater wereusedas0.88mPas⁻¹and1.33,respectively.ForTEManalysis, imprintednanoparticlesamplewasdroppedontocarboncoated coppergridandthendriedatroomtemperature.TEMphotographs weretakenat200kVbyTEMmicroscope.

2.5. Preparationoflysozymeimprintednanosensor

Inordertocleanthesensorsurface,thesensorwasimmersed in 20mL of acidic piranha solution (3:1 H₂SO₄:H₂O₂, v/v) for 30s. Then, it was washed with pure ethyl alcohol and dried in vacuum oven (200mmHg, 37^◦C) for 3h. Later on, the chip was immersed in ethanol/water (4:1, v/v) solution containing 3.0M3-mercaptopropenefor12h.Afterthat,analiquot(5␮L)of nanoparticlesdispersion(4.2%,v/v)wasdroppedonthegoldsur- faceoftheSPRsensortoattachlysozymeimprintednanoparticles ontotheSPRsensor.Then,thesensorwasdriedinoven(37^◦C,6h).

Finally,lysozymeimprintednanosensorwaswashedthreetimes withbothwater and ethylalcohol and driedwithnitrogengas undervacuum(200mmHg,37^◦C).

2.6. Templateremovalfromnanoparticles

Inordertoremovetemplatelysozyme,1MNaClsolution(pH 8.0, phosphate buffer) was used as desorption agent. The first removal study was carried out via batch experimental setup.

Lysozymeimprintednanoparticledispersion(10mL)wasadded intothedesorptionsolution(10mL)andstirredinshakingbath (200rpm) at room temperature. After lysozyme removal, the nanoparticleswerecentrifugedat30000rpmfor30min(Allegra- 64RBeckmanCoulter,USA);then,thenanoparticlesweredispersed in fresh stocking solution. The removal of lysozyme molecules fromimprintednanoparticleswasmonitoredbyfluorescencespec- trophotometer(Shimadzu,RF53010,Tokyo,Japan).Theemission spectrawererecordedinarangeof250–700nmwhenexcitations wereappliedat 285nm whichwasoptimized excitation wave- lengthforlysozymemolecules,respectively.Otherexperimental parameterswereappliedlikethatslitwidthwas5.0nmforboth excitationandemission,scanspeedwassuper,sensitivitywashigh andresponsetimeandshutterwereautomaticallycontrolled.

2.7. Characterizationoflysozymeimprintednanosensor 2.7.1. FTIR-ATRspectrophotometry

Characterization studies of lysozyme imprinted nanosensor weredonebyusingFTIR-ATR,AFMandellipsometer.Lysozyme imprintednanosensorwasputinasampleholderofFTIR-ATRspec- trophotometer(Thermo FisherScientific,NicoletiS10,Waltham, MA,USA)andtotallightreflectionfromsurfacewasmeasuredin thewavenumberrange of400–4000cm⁻¹ at 2cm⁻¹ resolution.

EighteenreplicateFTIR-ATRspectrawereobtainedandbaseline correctionwasdoneduetoGewindow.

2.7.2. AFMobservation

A multimode ambient AFM (Nanomagnetics Instruments, Oxford,UK)usedforAFMobservation.LysozymeimprintedSPR nanosensorwasattachedonsampledholderbyusingdouble-side carbonstrip.Observationstudywascarriedoutviatappingmodein airatmosphere.Theexperimentalparametersappliedwereoscilla- tionfrequency(341.30kHz),vibrationamplitude(1V_RMS)andfree vibrationamplitude(2V_RMS).Sampleswerescannedwith2␮ms⁻¹ scanningrateand256×256pixelsresolution toobtainviewof 2␮m×2␮marea.

2.7.3. Ellipsometry

Ellipsometer measurement was carried out by using an auto-nullingimagingellipsometer(NanofilmEP3,Germany). All thicknessmeasurements havebeenperformed ata wavelength of532nmwithanangleofincidenceof72^◦.Inthelayerthick- nessanalysis,afour-zoneauto-nullingprocedureintegratingover asampleareaofapproximately50␮m×50␮mfollowedbyafitting algorithmhasbeenperformed.Measurementwascarriedoutthrice for6differentpointsonthenanosensorsurfaceandtheresults werereportedasmeanvalueofthemeasurementswithstandard deviations.

2.8. Kineticstudieswithlysozymeimprintednanosensor

AftercharacterizationoflysozymeimprintedSPRnanosensor, thenanosensorwasusedforreal-timedetectionoflysozymefrom aqueoussolution.Forthispurpose,aSPRsystem(GenOptics,SPRi- Lab,Orsay,France)wasused.TheSPRnanosensorwaswashedwith deionizedwater(50mL,2.0mLmin⁻¹flow-rate)andequilibration buffer(pH:7.4,phosphate,50mL,2.0mLmin⁻¹ flow-rate).Then, thelysozymesolutions(21–1400nM)wereappliedtoSPRsystem (10mLand2.0mLmin⁻¹flow-rate).Thechangesinresonancefre- quencyweremonitoredinstantlyandreachedtoplatueatabout

40min.Afterthat,desorptionwasdonebyapplying10mLof1M NaClsolution(inpH8.0phosphate buffer,20mM)attheflow- rateof1.0mLmin⁻¹.Aftertheendofdesorptionstep,lysozyme imprintedSPRnanosensorwaswashedwithdeionizedwaterand equilibrationbuffer.Adsorption–desorption–cleaningstepswere repeatedforeachlysozymesample,meanwhile,SPR1001software obtainedfromproducerusedtoanalyzethekineticdata.

Chickeneggwhitesamplespreparedfreshwereusedasnatural lysozymesource.Forthis purpose,chickeneggwhitewassepa- ratedfromfresheggsanddilutedto50%(v/v)withphosphatebuffer (100mM,pH7.4).Thedilutedegg-whitewashomogenizedinan icebathandcentrifugedat4^◦C,at10000rpmfor30min.Thesam- plesweredilutedindifferentratiosbetween1/2500and1/10000.

SPRmeasurementswerecarriedoutasmentionedbefore.Thereal- timealbuminandcytochromecdetectionstudieswerealsocarried outtoshowspecificityoflysozymeimprintedSPRnanosensoras givenabove.

3. Resultsanddiscussion

3.1. Preparationandcharacterizationofnanoparticles

LysozymeimprintedPEDMAHnanoparticleswerepreparedby mini-emulsionpolymerization.Thenanoparticleswerecharacter- izedbythezeta-sizerandtransmissionelectronmicroscopy(TEM).

AsseeninFig.1a,lysozymeimprintednanoparticleshaveaverage particlesizeof64.9nmwithapolydispersityaround0.14.Asseen fromthefigure,thenanoparticleshavenarrowsizedistribution, and43%oftheimprintednanoparticleshaveaparticlesizearound averagevalue.Therefore,wecanconcludethatthepolymerization recipeappliedwassuitableforsynthesizing monosizenanopar- ticles.AsseeninTEMphotograph,eachofthenanoparticleshas sphericalshapeandsizearound50nm(Fig.1b).Thenon-imprinted nanoparticlesalsohavesimilarphysicalandchemicalproperties reportedinsupportinginformation(Fig.S1).

3.2. CharacterizationofSPRnanosensors

LysozymeimprintedSPRnanosensorwascharacterizedafter attachingthe nanoparticlesonto SPR sensor surface by Fourier transforminfraredspectroscopyintheattenuatedtotalreflection mode(FTIR-ATR),atomicforcemicroscopy(AFM),andellipsometry measurements.AsseenfromFig.1c,specificbandsofthepolymeric structure havebeendetected.Aliphatic–CHbandat2937cm⁻¹ and carbonylbandat 1723cm⁻¹ were determined.Also, amide bandsoriginatedfromMAHweredeterminedat1652cm⁻¹ and 1452cm⁻¹,respectively.Themostinterestingbanddeterminedin thespectrumisthedeepestbandat1145cm⁻¹thatwasstemmed fromimidazoleringoftheMAHmonomer.Thehighintensityof thebandshowsthatthefunctionalmonomers,inotherwordthe imprintedcavities,areorientedtowardtosurface.

AFM imagesof SPR nanosensorwererepresentedin Fig.1d.

Surface depthdetermined by AFM measurements of lysozyme imprintedSPRnanosensoris60.4nm.Theresultiswellfittedtothe resultsofzeta-sizermeasurementandTEManalysis.AFMimages also showthat thenanoparticles wereattached onthesurface ofSPRsensoralmosthomogeneous.Inordertoprovethissitua- tion,ellipsometryanalysiswasalsocarriedoutandcoherencyis determinedbetweenAFMand ellipsometermeasurements.Sur- facedepthobtainedfromellipsometryoflysozymeimprintedSPR nanosensoris44.04±2.75nm.Asaconclusion,itcanbededuced thathomogeneousandmonolayerattachmentofthenanoparticles hasbeenaccomplished.

Templateremovalfromimprintednanoparticleswasmonitored byflourimeter measurements(Fig.2).AsseenfromFig.2a, the

Fig.1. CharacterizationoflysozymeimprintedPEDMAHnanoparticlesandnanosensor:(a)zetasizermeasurements;(b)TEMphotograph;(c)FTIR-ATRspectrum;(d)AFM imagesoflysozymeimprintedSPRnanosensor.

standardlysozymesolutionshavetwoemission bandsatwave- lengthsas317nmand570nm,respectively.Thelatterisalsoseen intheemissionspectrumofthedesorptionagent(Fig.2c).There- fore,wehaveplottedacalibrationcurvebyusingthedataobtained fromwavelengthat317nm(Fig.2b).ThecalibrationcurvehasanR² valueas0.99255.Theemissionspectrumofdesorptionmediawas alsomeasuredafterremovaloflysozymemoleculesfromnanopar- ticles.AsseenfromFig.2d,thesolutionhasalsoemissionbands around317nm and570nmasexpected. Thelysozymeconcen- trationcalculatedindesorptionsolutionis30.77nM.Accordingto theseresults,itcanbesaidthatdesorptionagentisappropriatefor lysozymeremovalfromimprintednanoparticleswithoutcausing denaturationandconformationalproblems.

3.3. KineticstudieswithSPRnanosensor

Lysozyme imprinted nanosensor was used for real-time lysozymedetectionfromaqueoussolutions.Thesensorwasinter- actedwithlysozymesolutionsindifferentconcentrationsinthe range of 21–1400nM (Fig. 3a). As it is seen from figure, all steps were almost completed in 45min. This duration is five timesfasterthanourQCMnanosensor’sresponsetime(∼220min) [10].Increaseinconcentrationcausedalsoincrease innanosen- sorresponse.Nanosensorresponseincreasedatthebeginningand thenreachedplateauvaluearound350nMduetosaturationof

accessible imprinted nanocavities. On the other hand, QCM nanosensor has higher saturation concentration value that is around20.98␮M.Ascomparisonof SPRand QCMnanosensors, QCMnanosensorcandetectlysozymemoleculesinawidercon- centrationrange.

Fig.3bandcshowsthatconcentrationdependencyoflysozyme imprintedSPRnanosensor.AsseeninFig.3b,imprintednanosensor reachedmaximumvalueat700nMwitharesponse9.96.Linear- ity of thenanosensor response wasgiven in Fig. 3c.Lysozyme imprinted SPR nanosensor has two different linear regions for aqueouslysozyme solutions. Accordingtothe resultslysozyme moleculesboundtolysozymeimprintednanosensorthroughtwo differentorientationswithhighaffinity.Theresultsmainlydepend on the spherical structure of imprinted nanoparticles. When imprintednanoparticleswereattachedonthesensorsurface,some oftheimprintednanocavitieswerestericallyhindered.Therefore, lysozymemoleculescannotreachthesenanocavitiesaseasilyasdo foruppernanocavities;butstillhavehighaffinitytothem(Fig.4).

3.4. Analysisofkineticdata

3.4.1. Equilibriumanalysis

If the total amount of binding side [BS]_o is expressed in termsof themaximum lysozymebindingcapacityof imprinted nanoparticles,all concentrationterms can beexpressedas SPR

Fig.2. TemplateremovalfromlysozymeimprintedPEDMAHnanoparticles:(a)flourescencespectraofstandardlysozymesolution;(b)calibrationcurveat317nm;(c) spectrumofdesorptionagent;(d)spectrumofelutedsample.System:Shimadzu,RF53010;excitationwavelength:285nm;slitwidth:5.0nmforbothexcitationand emission;scanspeed:super;sensitivity:high;responsetime;shutter:automaticallycontrolled.

response signal R, eliminating mass–concentration conversion.

Underpseudo-firstorderconditionswherethefreeanalyteconcen- trationisheldconstantintheflowcell,thebindingcanbedescribed byfollowingequation:

dR

dt =kaC(Rmax−R)−kdR (1)

wheredR/dtistherateofchangeoftheSPRresponsesignal;R andRmaxarethemeasuredandmaximumresponsesignals,mea- suredviabinding;Cisthelysozymeconcentrationinjected(nM);

kaistheassociationrateconstant(nM⁻¹s⁻¹);andk_disthedisso- ciationrateconstant(s⁻¹).Thebindingconstant,i.e.,association constantsK_A,maybecalculatedasK_A=ka/k_d(nM⁻¹).Atequilib- rium,dR/dt=0andtheequationcanberewrittenas:

Req

C =KARmax−KAReq (2)

Therefore,thesteadystateassociationconstantK_Acanbeobtained fromaplotofReq/CversusReqandthedissociationconstantKD

canbecalculatedas1/K_A.

Eq.(1)mayberearrangedtoobtain:

dR

dt =kaCRmax−(kaC+kd)R (3) ThusaplotofdR/dtagainstRwilltheoreticallybeastraight linewithslope−(kaC+k_d)forinteraction-controlledkinetics.The initialbindingrate(atR=0)isdirectlyproportionaltotheanalyte concentrationandcanbeusedforconcentrationmeasurements.If

Rmaxisknown,bothkaandk_dcanbedeterminedfromasingle associationsensorgram.Rmaxis,however,oftendifficulttodeter- mineexperimentally,sinceahighanalyteconcentrationisrequired tofullysaturatethesurface.Apreferableapproachistomeasure

theassociationsensorgramforseveraldifferentanalyteconcentra- tions.Foranalysisoftheforwardandbackrates,aplotofthechange intotalsensorresponse(dR/dt)versusRgivesavalueSasthe slopethatrelatestheforwardandbackratesasfollows:

S=kaC+kd (4)

AplotofSversusCwillbeastraightlinewithslopeka.Intheory, theinterceptontheordinate(C=0)givesk_dinpractice,however, thiscannotbeusedasareliablemeasureofthedissociationrate constantifkaCk_d.Amoreaccuratewaytoobtainthisvalueisby directmeasurementofthedissociationfromsaturatedbindingsites intoabufferfeedingthatcontainsnoanalyteandthedissociation isquantifiedby:

ln



Rt



=kd(t−to) (5)

whereRoistheinitialresponselevelattoandRandtrepresent valuesobtainedalongthedissociationcurve[33].

3.4.2. Equilibriumisothermmodels

Fourdifferentequilibrium isothermmodels,Scatchard,Lang- muir, Freundlich, and Langmuir–Freundlich, were examined to describe the interaction model between lysozyme imprinted nanosensorandlysozymemolecules:

Scatchard: Rex

C =KA(Rmax−Req) (6)

Langmuir : R={RmaxC/KD+C} (7)

Freundlich: R=RmaxC^1/n (8)

Langmuir–Freundlich: R={RmaxC^1/n/KD+C^1/n} (9)

Fig.3.Real-timelysozymedetectionwithlysozymeimprintedSPRnanosensor:(a)concentrationdependencyoflysozymeimprintedSPRnanosensor;(b)concentrationvs.

nanosensorresponse;(c)linearregions.

Fig.4.LysozymeimprintednanoparticlesattachmentonSPRsensorsurface.

Additionalparametersnotdefinedpreviouslyaretheequilibrium dissociation constants (KD) and Freundlich heterogeneity index (1/n).

Theadsorptionmodelswereusedtodeterminesurfacehomo- geneity of the imprinted materials. Langmuir model bases on the assumptions of homogeneous distribution of interaction pointswithequalenergy andnolateralinteractions.Freundlich model is well fitted to heterogeneous surfaces. Mixed model, Langmuir–Freundlichcanbeappliedtoa systemthatisnotfull fittedtobothsystems,providesheterogeneityinformationadsorp- tionbehavioroverwideconcentrationregions.

Allisotherms,exceptLangmuir–Freundlich(R²:0.8993),have high correlation coefficients (R²>0.95). The linear fit withthe Langmuirequationwascomparablythebest,whichmeansthat the binding of lysozyme molecules onto lysozyme imprinted SPR nanosensor is monolayer,although Scatchard curve shows somesurfaceheterogeneity[34,35].Surfaceheterogeneitycanbe explainedbyaccessibilityproblemofimprintednanocavitiesdue toattachmentonsensorsurface.But,thesenanocavitiesstillshow highaffinitytolysozymemolecules.Hereby,themonolayerbinding oflysozymemoleculeswasachievedandthebindingprocesswas well-fittedtoLangmuirequation.Freundlichmodelisusedtoshow multilayerbindingofanalytemolecules.Linearregressioncoeffi- cientsofFreundlichandLangmuir–Freundlichisothermswerealso

Fig.5.Lysozymedetectionfromchickenegg-whiteindifferentdilutionratios:(a)2500×;(b)3333×;(c)5000×;(d)10000×.

high,butnothigherthanthatofLangmuirmodel.Thecalculated parametersforallmodelsweregiveninTable1.

The best fitted model to explain the interaction between lysozymeimprintedSPRnanosensorandlysozyme moleculesis Langmuirisotherm(R²=0.9911).TheRmaxvalue,calculatedby usingLangmuirmodel, wasveryclosetotheexperimentalone (9.96).Bytheresults,maximumdetectionlimitwasdeterminedas 735nM,K_AversusK_Dvaluesweredeterminedas108.71nM⁻¹and 9.20pM,respectively. Detectionlimit,defined astheconcentra- tionofanalytegivingreflectivityshiftequivalenttothreestandard deviationsoftheblank,wasdeterminedas0.084nM.

3.5. Lysozymedetectionfromnaturalsource

LysozymeimprintedSPRnanosensorwasalsousedtodetect lysozymeinnaturallysozymesource,chickeneggwhite.Chicken eggwhite,containsapproximately3.5%lysozyme,sampleswere interacted with lysozyme imprinted SPR nanosensor. For this

purpose,freshlypreparedchickeneggwhitesampleswerediluted indifferentratiosintherangeof1/2500to1/10000.Asseenin Fig.5,decreaseindilutionratio,increaseinconcentration,caused increaseinnanosensorresponseasexpected.Lysozymeimprinted SPR nanosensors showed a response when egg white samples weredilutedinhighratioas10000times,lysozymeconcentration wasapproximately32.2nM.Asaconclusion,lysozymeimprinted nanosensorhasanabilitytodetectlysozymeinanaturalcomplex mixture,chickeneggwhite.

InordertoshowselectivityoflysozymeimprintedSPRnanosen- sor,thereal-timealbuminandcytochromecdetectionswerealso carriedout(Fig.6).Thesolutionscontainingcompetitormolecules (70nM,pH:7.4,phosphatebuffer)wereappliedtoSPRnanosen- sor.AsseenfromFig.5a,SPRnanosensordidnotgiveanyresponse toalbuminsolution(R=0.0497).Incaseofcytochromecdetec- tion,lysozymeimprintedSPRnanosensorsshowlownon-specific response(R=0.587).Thisresponsewasstemmedfromthestruc- turalandphysico-chemicalsimilaritiesbetweencytochromecand

Table1

Kineticandisothermparameters.

Associationkineticsanalysis Equilibriumanalysis(Scatchard) Langmuir Freundlich Langmuir–Freundlich

ka(nMmin⁻¹) 0.308 Rmax 10.59 Rmax 10.50 Rmax 6.71 Rmax 29.97

kd(min⁻¹) 6.2×10⁻⁴ KA(nM⁻¹) 103.81 KA(nM⁻¹) 108.71 1/n 0.1893 1/n 0.1893

KA(nM⁻¹) 498.4 KD(pM) 9.63 KD(pM) 9.20 R² 0.9547 KA(nM⁻¹) 22.82

KD(pM) 2.00 R² 0.9795 R² 0.9911 KD(pM) 43.8

R² 0.9617 R² 0.8993

Fig.6.ComparisonofselectivityofSPRnanosensors.Theresponsesof(a)lysozymeimprintedand(b)non-imprintedSPRnanosensor.

Table2

ComparisonofsomeparametersofSPRandQCMnanosensors.

SPR QCM

Responsetime(min) 45 220

Lowestdetectedconcentration(nM) 21 14

Measurementrange 21nMto1.4␮M 14nMto105␮M

lysozymemolecules. But,thespecificresponse of thenanosen- sortolysozymemolecules,R=6.421,isexcessivelyhigherthan thattocytochromec.Theselectivityratiocalculatedbydividing theSPRresponse data ofthecompetitormolecules is 10.94.In ordertoconfirmbothselectivityandspecificityofthelysozyme imprinted SPR nanosensor, the non-imprinted nanosensor was preparedandusedforreal-timealbuminandcytochromecdetec- tion studies (Fig. 6b). The non-imprinted SPR nanosensor did not give any response to albumin solution (R=0.0986). The responsesofthenon-imprintednanosensortocytochromecand lysozymemoleculesweredeterminedas0.734and1.57,respec- tively. The selectivityratio between lysozyme and cytochrome cmoleculesis 2.13, respectively.Accordingtoboth nanosensor responses,itisclearlydeducedthatimprintingprocessbringsa 3D-recognitionmemory and specificity for SPR nanosensor. By thisway, lysozymeimprintednanosensor specificallyrecognize anddetectthelysozymemoleculesinbothnaturalsourcesuchas chickenegg-whiteandaqueoussolutions.

Fig.7.ReproducibilityoflysozymeimprintedSPRnanosensor.

3.6. Reproducibility

InordertoshowthereproducibilityoflysozymeimprintedSPR nanosensorresponse, fiveequilibration-adsorption-regeneration cycles were repeated using aqueous lysozyme solution with concentrationof35nM (Fig.7).Asseeninthefigure,lysozyme imprinted nanosensor has shown reproducible reflectivity responseduringfivecycles(Table2).

4. Conclusions

Here, we reported theusability of the molecular imprinted nanoparticlesasbiorecognitionelementonSPRnanosensor.The nanotechnologyservesanovelapproachasimprintinginto/onto nanoparticles to solve problems occurred during conventional imprintingprocess.By thisapproach,morehomogeneouslydis- tributedimprintedcavitiescanbeobtainedsurfaceornearinsideof thenanoparticles.Thatcausesregular,rapid,homogenousadsorp- tiondynamics[13,14].Hereby,wehavefocusedourattentionon combining of molecular imprinting into nanoparticlesand SPR biosensorapproachesandpreparedSPRnanosensorforreal-time lysozymedetectionusinglysozymeimprintednanoparticlesand compareSPRnanosensorwithQCMnanosensor.Asconclusion,we cansaythatlysozymeimprintedSPRnanosensorhasapotential useforlysozymedetectionfrombothmediaaqueoussolutionsand complexnaturalsamples.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.snb.2011.08.064.

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Biographies

GulsuSenerreceivedherBSdegree(2007)inDepartmentofChemistryandher MScdegree(2009)inNanotechnologyandNanomedicineDivisionfromHacettepe UniversityinTurkey.SheiscurrentlypursuingherPhDdegreeatthesamedivision.

Herresearchinterestsaremolecularimprintednanoparticlesandtheirapplications onbiosensors.

LokmanUzundefendedhisPhDthesisaboutmolecularimprintedbiosensors at2008.Now,heisassistantprofessoratHacettepeUniversity,Departmentof Chemistry,BiochemistryDivision.Hisresearchinterestismainlypreparationof biosensorsdependingonaffinityinteractionsandmolecularimprintedpolymers.In additiontotheseareas,hemakesresearchonproductionofnovelpolymerstosep- arateandpurifyimportantbiologicalmolecules,removeordepletetoxicmolecules suchasheavymetalions,bilirubinandunwishedproteinsfromserumandaqueous solutions.

RıdvanSayjoinedDepartmentofChemistryatAnadoluUniversity,Turkeyafter a PhD at Hacettepe University in 1998. Currently, he is a professorat this university.Hisresearchinterestsareintheareasofsyntheticreceptorsbased onmolecular imprinting and nanosystems,new imprinting approaches using supramolecularchemistry,practiceofself-assembly,biochromatographybasedon molecularlyimprintedpolymers,biosensorsbasedonsyntheticreceptortechnology andbiomimickingcatalysis.

AdilDenizliisaprofessoratHacettepeUniversity,Ankara,Turkey.Hereceived PhDdegreefromthesameuniversityin1992.Hismainresearchfieldsaremolecu- larimprintingtechnologies,hemoperfusion-removaloftoxicmaterialsfromblood, purificationofenzymesandproteinsbychromatographicmethods,biosensors basedonsyntheticreceptortechnology,productionofpolymershavedifferentsur- faceandbulkproperties,shapeandgeometries,applicationofthesepolymersin medicineandbiology.