Gold-silver-nanoclusters having cholic acid imprinted nanoshell

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ContentslistsavailableatSciVerseScienceDirect

Talanta

j ourna l h o me p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t a l a n t a

Gold–silver-nanoclusters

having

cholic

acid

imprinted

nanoshell

Aytac¸

Gültekin

a

_{, Arzu}

_Ersöz

b

_{, Adil}

_Denizli

c

_{, Rıdvan}

_Say

b,∗

a_Department_of_Energy_Systems_Engineering,_{Karamano˘glu}_Mehmetbey_University,_Karaman,_Turkey b_Department_of_Chemistry,_Anadolu_University,_Eskis¸_ehir,_Turkey

c_Department_of_Chemistry,_Hacettepe_University,_Ankara,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received26August2011

Receivedinrevisedform16February2012 Accepted23February2012

Available online 3 March 2012

Keywords:

Gold–silver-nanoclusters Molecularlyimprintedpolymers Cholicacid

Photoluminesence

a

b

s

t

r

a

c

t

Molecularimprintedpolymers(MIPs)asarecognitionelementforsensorsareincreasinglyof inter-estandMIP-nanoparticleshavestartedtoappearintheliterature.Inthisstudy,wehaveproposeda novelthiolligand-cappingmethodwithpolymerizablemethacryloylamido-cysteine(MAC)attachedto gold–silver-nanoclustersreminiscentofaself-assembledmonolayerandhavereconstructedsurfaceshell bysynthetichostpolymersbasedonmolecularimprintingmethodforcholicacidrecognition.Inthis method,methacryloylamidohistidine–Pt(II)[MAH–Pt(II)]hasusedasanewmetal-chelatingmonomer viametalcoordination–chelationinteractionsandcholicacid.Nanoshellsensorswithtemplatesgivea cavitythatisselectiveforcholicacid.ThecholicacidcansimultaneouslychelatetoPt(II)metalionand fitintotheshape-selectivecavity.Thus,theinteractionbetweenPt(II)ionandfreecoordinationspheres hasaneffectonthebindingabilityofthegold–silver-nanoclustersnanosensor.Thebindingaffinityof thecholicacidimprintednanoparticleshaveinvestigatedbyusingtheLangmuirandScatchard meth-odsanddeterminedaffinityconstant(Kaffinity)hasfoundtobe2.73×104molL−1and2.13×108molL−1,

respectively.Atthelaststepofthisprocedure,cholicacidlevelinbloodserumandurinewhichbelong toahealthypeopleweredeterminedbythepreparedgold–silver-nanoclusters.

1. Introduction

Bileacidsareimportantsteroidalcompoundswhichare syn-thesizedfromcholesterolintheliver,storedinthegallbladder, andreleasedinthesmallintestine forthedigestionoffatsand lipids.Theconcentrationofbileacidsinbodyisrelatedwith hep-atitis,gallstone and otherdiseases in liver.The qualitative and quantitativeanalysisofbileacidshavebothclinicaland pharma-ceuticalsigniﬁcance.Medically,itisfeasibletoreducecholesterol contentinbodybyremovingbileacids,especiallyforthe treat-mentofhyperlipidemia[1,2].Itisthusimportanttopreparebile acidsorbentswithhighselectivityforanalysisandpotential medi-calapplications[3].Cholicacidaswellasitsderivativesisthemain componentofbileacids.Severalmethodssuchasliquid chromatog-raphy (LC)with UV detection[4–6], LCcoupledto evaporative lightscatteringdetection(ELSD)[7,8],ultrahigh-performance liq-uidchromatography–massspectrometry(UHPLC)[9],havebeen appliedforbileacidsdetectioninserumbuttheyuselargeand costlyinstrumentsandrequiresophisticated,frequentlywide anal-ysisprocedures.

∗ Correspondingauthorat:FenFakültesi,YunusEmreKampüsü,Anadolu Univer-sitesi,26470Eskis¸ehir,Turkey.Tel.:+902223350580;fax:+903204910.

E-mailaddress:rsay@anadolu.edu.tr(R.Say).

Molecularimprintingisatechnologytocreaterecognitionsites inamacromolecularmatrixusingamoleculartemplate[10].In otherwords,boththeshapeimageof thetarget andalignment ofthefunctionalmoietiestointeractwiththoseinthetarget,are memorizedinthemacromolecularmatrixfortherecognitionor separationofthetargetduringformationofthepolymericmaterials themselves[11].Molecularlyimprintedpolymers(MIPs)areeasyto prepare,stable,inexpensiveandcapableofmolecularrecognition [12].Therefore,MIPscanbeconsideredasartificialaffinitymedia. Molecularrecognition-basedseparationtechniqueshavereceived muchattentioninvariousfieldsbecauseoftheirhighselectivity fortargetmolecules.Threestepsareinvolvedintheion-imprinting process:(i)complexationoftemplate(i.e.,metalions)toa polymer-izableligand,(ii)polymerizationofthiscomplexand(iii)removal oftemplateafterpolymerization[13].

A nanoparticle which historically has included nanopow-der, nanocluster, and nanocrystal is a small particle with at least one dimension less than 100nm. A nanocluster is an amorphous/semicrystallinenanostructure. Thisdistinction is an extension of the term “cluster” which is used in inor-ganic/organometallicchemistrytoindicatesmallmolecularcages of ﬁxed sizes [14]. In last decades, great research efforts have focusedonnanoparticleswithaprospecttotheirpotential appli-cationsinnanoelectronics,sensortechnology, non-linearoptics, catalysts,hydrogenstorageandsolartechnology[15].Becauseof theirlargesurface-to-volumeratio,quantummechanicsizeeffects

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A.Gültekinetal./Talanta93 (2012) 364–370 365

andcurvature-inducedsurfaceeffectsnanoparticlesexhibitnovel properties,forexamplelowermeltingpoint,higherself-diffusion coefﬁcient,lowereffectiveDebyetemperature,highersolid–solid phasetransitionpressure,decreasedferroelectricphasetransition temperature,aswellaschanged thermophysicalproperties and size-dependentcatalytic activity [16,17]. The interest in study-inggreat withmetallic and bimetallicnanoparticles becauseof boththefundamentalunderstandingandpotentialtechnological applications.Ata fundamental level,information onthe evolu-tionofelectronicstructuresofsmallmetallicparticlesandtheir effectsontheopticalabsorptionspectracontinuetobeamajor goalofresearchonmetalclustersandnanoparticles.Ona practi-callevel,theunusualopticalpropertiesofmetallicnanoparticles areexploitedforavarietyofapplicationsincludingoptical mark-ers for biomolecules, biological sensors, optical ﬁlters, surface enhancementinRamanspectroscopyandultrafastnon-linear opti-caldevices[18–20].

In last years, the combination of nanoparticles and MIPs havebeenappliedin selectivesensing detection.Themolecular imprintingnanotechnologiesareexpectedtogreatlyenhancethe molecularaffinityofMIPmaterials,andthusprovideawiderrange of applications approaching to biological receptors. Nanostruc-tured,imprintedmaterialshaveasmalldimensionwithextremely highsurface-to-volumeratio,sothatmostoftemplatemolecules aresituatedatthesurfaceandintheproximityofmaterials sur-face.Inthecaseofnanosizedparticles,mostofimprintedsitesare situatedatthesurfaceorintheproximityofsurface.Therefore,the formsofimprintedmaterialsareexpectedtogreatlyimprovethe bindingcapacity andkineticsandsite accessibilityofimprinted materials.TheMIP nanosphereswith100-nmsizehada higher bindingcapacity,whichwas2.5foldthatofnormalbulkyparticles with5-␮msize.Ontheotherhand,thelow-dimensional nanos-tructureswithimprintedsiteshaveveryregularshapesandsizes, andthetunableflexibilityofshapesandsizes.Theimprinted nano-materialshavealsobetterdispersibilityinanalytesolutionsand thusgreatlyreducetheresistanceofmasstransfer,exhibitingafast bindingkinetics.Theimprintednanomaterialswithwell-defined morphologiescanfeasiblybeeninstalledontothesurfaceofdevices inarequiredformformanyapplicationsinnanosensorsand molec-ulardetection[21].Onlyafewapplicationofnanoclusters/MIPs havebeenreported[22–24].Inthisstudy,wehaveproposedanovel thiol ligand-exchange method using polymerizable methacry-loylamidocysteine (MAC) of gold–silver-nanoclusters to cap by organic layer, reminiscent of a self-assembled monolayer and have reconstructed surface shell by synthetic host polymers basedonmolecularimprinting methodfor cholicacid recogni-tion.Methacryloylamidohistidine–Pt(II)[MAH–Pt(II)],wasusedas anewmetal-chelatingmonomerviametalcoordination–chelation interactionsandcholicacid.Wehavecombinednanoscale materi-alswithMIPconsideringtheabilityofcholicacidtochelateofPt(II) ionofMAHmonomertocreatereminiscentligandexchange(LE) assembledbindingsites,becausethePt(II)primarilyinteractswith thecholicacid[25].Inthisstudy,synthesis,characterizationand efficiencyofthegold–silver-nanoclusterssensorbasedoncholic acidimprintedpolymerhaveinvestigated.Cholicacidlevelinblood serumandurinehasdeterminedusingthisAu–Agnanosensor.

2. Experimental

2.1. Generalmethods

Methacryloylchloride, wassupplied by Aldrichand used as received. Cholic acidand chenodeoxycholic acidwere supplied bySigmaAldrichandethyleneglycoldimethacrylate(EDMA)was obtainedfromFlukaA.G.,distilledunderreducedpressureinthe presenceofhydroquinoneinhibitorandstoredat4◦C untiluse.

Fig.1.(a)MALDI-TOF/MSspectrumofMAH-Pt(II)(2␮LofMAH-Pt(II)solutionwas mixedwith18␮Lofa10mgmL−1_solution_of_CHCA_in_{acetonitril/0.1%}_TFA._The

accelerationvoltagewassetto20kVandthedelaytimewas100nsand(b)FTIR spectrumofMAH-Pt(II).

Azobisisobutyronitrile(AIBN) wasalso supplied fromFluka. All otherchemicalswereofreagentgradeandwerepurchasedfrom MerckAG.Allwaterusedintheexperimentswaspuriﬁedusinga BarnsteadNANOpureultrapurewatersystem.

Photoluminescencespectrawereacquiredby spectroﬂuorom-eter (Varian Cary Eclipse, Australia). 1_H _and 13_C _NMR _spectra

were recorded in DMSO-d6 with TMS as theinternal standard

usingBruker500MHzNMRequipment.TheTransmissionEmission Microscopy(TEM)imagesofnanocrystalswereacquiredona FEI-TecnaiTM_G2 _Sprit_transmission_electron_microscope_(20–120_kV).

Samplepreparationwasconsistedofdropcoatingthe nanoclus-tersontocarbon-coatedcoppergridsandairdried.Ramanspectra imageswereobtainedusingaRamanSpectrophotometer(Bruker SenterraDispersiveRamanMicroscope,Germany).Fourier Trans-formInfrared(FTIR)spectraimageswereobtainedusingaFTIR (Spectrum100,PerkinElmer,USA).Forthispurposethedrybeads (about 0.1g) were thoroughlymixed withKBr (0.1g, IRGrade, Merck,Germany),pressedintoapelletandthentheFTIRspectrum wasrecorded.

2.2. Preparationoffunctionalandmetal-chelatemonomers MAC,monomer,wasusedforformingofreminiscentof self-assembled monolayer on thesurface of nanoparticlesand was synthesizedand characterizedaccordingtothepreviously pub-lishedprocedure[26].Histidine-functionalmonomer,MAH,was synthesizedaccordingtothepreviouslypublishedprocedure[27] andcharacterizedwith1_H_NMR.

DataforMAH:1_H_NMR_(CDCl

3):1.99(t;3H,J=7.08Hz,CH3),

1.42(m;2H,CH2),3.56(t;3H, OCH3),4.82–4.87(m;1H,metil),

5.26(s;1H,vinilH),5.58(s;1H,vinil),6.86(d;1H,J=7.4Hz,NH), 7.82(d;1H,J=8.4Hz,NH),6.86–7.52(m;5H,aromatic).

On the other hand, metal-chelate monomer, MAH–Pt(II), was synthesized according to the our previously published

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Fig.2. Schematicillustrationof cholicacidmolecular imprinting onMACmodiﬁedAu–Ag nanoclusters (1). Au–Agnanoclusters polymerizedwith AIBN, EDMA MAH–Pt(II)–cholicacid(2).Cholicacidremovedwith12mL(3/1,v/v)of2MNaOH/THF(3).

procedure [28]. MAH–Pt(II) was preorganized using MAH and PtCl2. MAH (0.1mmol) and PtCl2. (0.1mmol) were dissolved

in 5.0mL of aceton–water and the solution was stirred for 6h. MAH–Pt(II)metal-chelate monomerwascharacterizedwith MALDI-TOF/MS(Fig.1a).Theionpeaksat415and443m/z con-ﬁrmsthatMAH–Pt–(H2O)2metal-chelatemonomerstructurewas

producedexactly.Thissitutationcanbeexplainedinthe follow-ingway:ifPtisin198isotopeforminMAH–Pt–(H2O)2structure,

the molecularweight of MAH–Pt–(H2O)2 is 457gmol−1. When

CH2 bondis removed,thepeakis observedat 415m/z.Ifthe

192isotopeislocated,themolecularweightis451and2molof H2Oisremoved fromthestructureof MAH–Pt–(H2O)2 and the

peakisobservedat443m/z.ThisindicatesthatMAH–Pthasbeen synthesized.

Furthermore, Pt–N vibration bands at 552 and 441cm−1 in FTIRspectrumshowthatPt(II)wasincorporatedintoMAH struc-ture(Fig.1b).Ligandexchangemonomer,MAH–Pt(II)–cholicacid, was preorganized using MAH–Pt(II) and template, cholic acid. MAH–Pt(II)(0.1mmol)andcholicacid(0.1mmol)weredissolved in5.0mLofethanolandthesolutionwasstirredfor24h.

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2.3. Synthesisofgold–silver-nanoclustersandcholicacid imprintedgold–silver-nanoclusterssensor

The gold–silver-nanoclusters were prepared in a two-phase water/toluene system using a modiﬁed Brust method [29,30]. Brieﬂy,nanoclusterswerepreparedbythedropwiseadditionof 0.01MaqueousNaBH4 solutionin anequalvolumeof

ammon-ical aqueous 0.5mM HAuCl4 (pH ca. 7.8) and 1mM MAC in

ethanolundervigorousstirring.0.5mMAgNO3wasaddedto40mL

of Au–MAC nanoclusters dispersion under continuous stirring. Au–Ag–MACnanoclusterswereseparatedandwashedthoroughly several times with water and toluene and dried under nitro-gen,followedbyredispersionindimethylsulfoxide(DMSO).For thesynthesisofAu–Agnanoclustershavingcholicacidimprinted nanoshell,methacryloyl-activatednanoclusters(having␲bondfor polymerization)wereaddedintothereactionmixturecontaining themetal-chelate(MAH–Pt(II)–cholicacid)monomer(0.05mmol) inDMSO, EDMAcrosslinkingmonomer (0.4mmol) and the ini-tiator,AIBN (1mol%) for theradical polymerizationin ethanol. This solutionwas transferred into thedispersion medium and stirredmagneticallyataconstantstirringrateof600rpminaglass polymerizationtube.Thepolymerizationtubewasirradiatedwith UVlightat365nmfor 4h.Afterthepolymerization, cholicacid nanoshellshavingAu–Agnanoclusterswereseparatedfromthe polymerizationmedium.Theresiduals(e.g.,unconvertedmonomer andinitiator)wereremovedbyacleaningprocedure.Theresulting nanocrystalsweretreatedwith12mL(3/1,v/v)of2MNaOH/THF solutionfor24htoremovethetemplates(Fig.2)[3].Nonimprinted (NIP)Au–Agnanoclusterssensor,withoutusingcholicacidas tem-plate,waspreparedinasimilarwayasdescribedaboveandused asareference.

2.4. Evaluationofnanocluster’sluminesence

Thesensingcapabilityandspecificityofthecholicacid mem-oryhavingAu–Agnanoclusters sensor wasfurtherexploredby introducingcholicacidasatemplatemolecule.Cholicacid adsorp-tionstudieswereperformedinabatchsystem.Forthispurpose, cholicacidwasdissolvedin ethanol and 20mg of MIPclusters was placed in cholic acid solution at different concentrations (10−7 to 10−3molL−1)for a period of 5minat room tempera-ture.TheinteractionsbetweencholicacidandMIPparticleswere studied observing fluorescence measurements. The cholic acid imprintedAu–Agnanoclustersshowedahighseparationbetween the excitation and emission wavelengths, simplifying fluores-cencemeasurements,recordedphotoluminescencespectrausing spectrofluorometer. Cholic acid imprinted Au–Ag nanoclusters nanosensorswereexcitedat290nm,andemissionwasrecorded at 580nm. Au–Ag nanoclusters having cholic acid imprinted nanoshellweretestedagainstbloodserumandurine.

3. Resultsanddiscussion

3.1. TEMcharacterizationofnanoshellsensors

Fig.3showsTEMimageofAu–Ag–MIPnanoclusterswithout cholicacidtemplateafter2MNaOH/THFacid(3/1,v/v)treatment. Theshapeofthesenanoclustersisclosetosphericalandaggregated andwithaveragesizeabout30nm.

3.2. Ramancharacterizationofnanoshellsensors

Au–MIPnanoparticleswithcholicacid,andAu–MIP nanoparti-cleswithoutcholicacidtemplatewerecharacterizedwithRaman spectroscopy(Fig.4).InRamanspectrumofAu–MIPnanoparticles withcholicacidhasbandshiftsat1728cm−1becauseofcholicacid

Fig. 3.TEM image of Au–Ag–MIP nanoclusters withoutcholic acid template (120kV×87,000).

itself.Thesebandshavebeenpreviouslyassigned[31].The disap-pearanceofthatpeakindicatestheremovaloftemplatefromthe cholicacidimprintednanoparticles.

3.3. Measurementofbindinginteractionsofmolecularly imprintednanoshellsensorviaphotoluminescence

Thefunctionalmetal-chelatemonomer,[MAH–Pt(II)],was cho-sento interactcholic acid, toform metalchelate and to make metal-complexing polymeric receptors for selective binding of cholicacidandanalogues[25].Metalchelatemonomerandcholic acidmoleculeweremixedthroughpreorganisationandthis preor-ganisationcomplexdeﬁnesthesizeanddirectionofthechemical interactionsofthecholicacidimprintedcavitytopreparesynthetic cholicacidreceptorofAu–Agnanoclusters(Fig.2).

The selective binding ability and detection of cholic acid imprintedAu–Agnanoclusterssensorwerestudiedwith fluores-cencespectroscopy,andtheresultsweregivenin Fig.5.Cholic acidadditioncausedsignificantdecreasesinfluorescenceintensity becausetheyinduced photoluminescenceemissionfromAu–Ag nanoclustersthroughthespecificbindingtotherecognitionsites ofthecrosslinkednanoshellpolymermatrix.

Theanalysisof thequenchingresultshasbeenperformedin termsoftheStern–Volmerequation.Themechanismof quench-ingoffluorescencehasbeenexplainedbythis way.Amodified Stern–Volmer equation is derived togenerate a linear calibra-tion.Underoptimumconditions,thefluorescenceintensityversus cholic acid concentration gave a linear response according to Stern–Volmerequationwitha0.916correlationcoefficient(Fig.6). ThefluorescenceintensityofthecholicacidimprintedAu–Ag nanoclusterscanbequenchedbytheadditionofcholicacid.The quenching fluorescence intensity is proportional tocholic acid

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Fig.4. RamanImagesof(a)Au–Ag–MIPnanoclusterswithcholicacid(unleached);(b)Au–Ag–MIPnanoclusterswithoutcholicacid(leached).

Fig.5.Theeffectofconcentrationofcholicacidonthequenchingofthe ﬂuores-cenceofthecholicacidimprintedAu–Agnanoclusters(sensorspeciﬁcity-adsorption isotherm).

concentration.Thedetectionlimit,defined astheconcentration of analyte giving quenching of the fluorescence equivalent to threestandarddeviationoftheblankplusthenetblank quench-ing fluorescence, was 0.05nmolmL−1. The experiments were

Fig.6. Stern–Volmer-typedescriptionofthedata.

performedinreplicatesofthreeandthesampleswereanalyzed inreplicatesofthreeaswell.Intheliterature,thelowest detec-tionlimitshavereportedas38.3nmolmL−1fordeoxycholicacid (DCA)[32],76.4nmolmL−1forursodeoxycholicacid(UDCA)[33], 0.16nmolmL−1 forbileacid[34],5nmolmL−1 forbileacid[35], 4.27nmolmL−1forcholicacid[36].Althoughmanymethodshave lowdetectionlimits,theyareexpensive[37–39].So,thisnewcholic acidnanosensorisrapidandithasbothlowdetectionlimitandvery lowcost.

Au–Ag nanoclusters having cholic acid imprinted nanoshell weretestedagainstbloodserumandurine.Threedifferentblood serumandurinesampleswhichbelongtohealthypersonswere alsoexaminedwithAu–Agnanoclustersandaveragecholicacid levelwasfoundtobe6and2.5respectively.Theamountofcolic acidinthebloodofahealtypersonisabout6␮M[40].Therefore, theresultsobtainedshowedthatthesenanoclusterscanbeused forthedetermination.

Theafﬁnityconstantsofcholicacidcanbeestimatedfromthe thermodynamicanalysisoftheﬂuorescenceintensityasafunction ofthecholicacidconcentrationbasedonScatchardanalysis[41,42] andLangmuirisotherm[43].

Ifweconsiderabindingequilibriumsuchas: Cholicacid+(Au–Ag)nanocluster

k

cholicacid−(Au–Ag)nanocluster(1)

where,cholicacidand(Au–Ag)nanoclusterrepresentcholicacidin

thesolutionandcholicacidimprintedpolymericnanoshellhaving nanocluster,respectively,andcholicacid(Au–Ag)nanoclusteristhe

cholicacid–nanocrystalboundcomplex.AScatchardrelationship canbeobtainedusingthefollowingequation.

I Co = Imax KD −

_I KD

(2) where,KDistheequlibriumdissociationandIistheﬂuorescence

intensity.AplotofIversusI/Cgaveastraightlineandthe equlib-riumbindingconstants(Ka=1/KD)weregiveninTable1.

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Table1

ComparisonofLangmuirandScatchardanalysisforcholicacidimprintednanoparticle.

Reboundmolecules LangmuirKa(M−1) Imax(a.u.) Scatchard,Ka(M−1) Imax(a.u.)

Cholicacid 2.73×104 ₂₂₁ _2.13_×₁₀8 ₂₈₃

Table2

SelectivityofcholicacidimprintedAu–Agnanosensor.

C(molL−1₎ _Emission_(MIP) _MIP_(mol_L−1₎ _NIP_(mol_L−1₎ _k_MIP _k_NIP _k

Cholicacid 1×10−4 ₃₇₀ ₁_×₁₀−4 _1.26_×₁₀−6

Chenodeoxycholicacid 1×10−4 ₄₁₄ _1.23_×₁₀−6 _2.3_×₁₀−7 ₈₁ _5.5 _14.7

ThevalidityoftheLangmuirisothermcanbetestedby

deter-miningtheafﬁnityconstantmeasuringtheﬂuorescenceintensities

at equlibrium with different bulk concentrations (5_×10−8 to

10−3molL−1)(Fig.5).Langmuirrelationshipcanbeobtainedusing thefollowingequation;

Co I = 1 Imax·b+ Co Imax (3) TheresultsobtainedfromthelinearizedformoftheLangmuir isotherm,byplottingCo/IasafunctionofCo,and theScatchard

analysisﬁndingsarealsogiveninTable1.Association constant, Ka,andtheapperantmaximumnumberofrecognitionsites,Imax,

valuesforthespeciﬁcinteractionbetweenthetemplateimprinted polymerofthenanoparticlesandthetemplate itselfwere com-paredinTable1.AsseenfromTable1,ingeneral,themagnitudeof Ka(2.73×104molL−1,2.13×108molL−1,basedonLangmuirand

Scatchardanalysis,respectively)isduetotheaccessibilityofcholic acidtemplate molecules.Sometemplate cavitiesformedduring imprintingprocesswerestayedinsidethepolymermatrixofthe nanoclusters.

TheKaandImaxvaluesestimatedfromScatchardanalysisare

veryclosetotheLangmuiranalysisdataandcholicacidtemplated sitesofnanocrystalsarehighlyselectivetothecholicacid recog-nitionsites.So,thebasedonScatchardanalysisforthebinding ofcholicacidtoMIPnanosensor,Ka andImaxwerefoundtobe

2.13_×108_M−1_and_283,_{respectively.}_The_value_of_K_a_suggests_that

afﬁnityofthebindingsitesisverystrong.

3.4. Recognitionselectivityofcholicacidimprintednanoshell sensor

Molecularimprintingprocesswithcholicacidgivesacavitythat isselectiveforcholicacidanditsanalogues.Cholicacidimprinted nanocrystalsweretreatedwithchenodeoxycholicacidwhichhas verysimilarmolecularstructurewithcholicacidinordertocheck whetherthenanoshellhasanyeffectonrecognitionprocess.The obtainedresultsindicatedthatMIPnanosensorhas92timesgreater selectivityforcholicacidthanthatofchenodeoxycholicacid.The investigationoffluorescenceintensityofNIPdidnotshowa signif-icantchangeinfluorescenceintensity.Theselectivitycoefficient, k,ofcholicacidandchenodeoxycholicacidwasfoundtobe81 forMIPparticlesand5.5forNIPparticlesandrelativeselectivity coefficient,k,wasdeterminedas14.7.Asseenthat,MIPsensoris 14.7timesselectivewithrespecttoNIPsensor(Table2).Obtained resultsclearlyindicatedthatthechangeoffluorescenceintensityis duetospecificbindingbetweencholicacidandcholicacidmemory siteshavingnanoparticles.

ThecholicacidcansimultaneouslychelatetoPt(II)metalion andﬁtintotheshape-selectivecavity.So,thisinteractionbetween Pt(II) ion and free coordination spheres has an effect on the bindingability oftheAu–Agnanoclusters. Experimentalresults showed that shape-selective cavity formation was occured for cholicacid.

4. Conclusion

Wehavedevelopedanovelchemicalpreparationmethodfor methacryloyl based self-assembledmonolayer and to make up imprintingpolymervialigandexchangeofcholicacidonAu–Ag nanoclusters. The cholic acidimprinted MAH–Pt(II)–cholic acid copolymerofAu–Agnanoshellisexpectedtobindcholicacid.The resultsshowedthatthechangeinfluorescencecouldbeattributed tothehighcomplexationgeometricshapeaffinity(orcholicacid memory)betweencholicacidmoleculesandcholicacidcavities occuredontheAu–Agnanoshells.In conclusion,thecholicacid imprintednanoshellsensorhasbeengainingwidespread recog-nitionasasensorforcholicacidbecausetheimprintingmethods createananoenvironmentbasedonshapeofcavitymemorial,size andpositionsoffunctionalgroupsthatrecognizestheimprinted molecule,cholicacid,basedonligand-exchangeimprinting meth-ods. Nanoshell sensor having cholic acid templates responses tocholic acid and its analogues through fluorescenceintensity decrease.Thefluorescenceintensitycorrelatestotheamountof cholic acid analogues bounded to the nanoshellhaving Au–Ag nanoclustersinthecasesofincubatingthecholicacidimprinted nanoclusterssensorwiththecholicacidaqueoussolution.The flu-orescenceintensitydecreasedwithincreaseconcentrationofcholic acid.

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