Author's Accepted Manuscript
β-Cyclodextrin coated CdSe/ZnS quantum dots FOR VANILLIN SENSORING IN FOOD SAMPLES Gema M. Durán, Ana M. Contento, Ángel Ríos
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S0039-9140(14)00668-7 http://dx.doi.org/10.1016/j.talanta.2014.07.100 TAL15009
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Received date: 21 May 2014 Revised date: 28 July 2014 Accepted date: 31 July 2014 Cite this article as: Gema M. Durán, Ana M. Contento, Ángel Ríos, βCyclodextrin coated CdSe/ZnS quantum dots FOR VANILLIN SENSORING IN FOOD SAMPLES, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.07.100 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
-CYCLODEXTRIN COATED CdSe/ZnS QUANTUM DOTS FOR VANILLIN SENSORING IN FOOD SAMPLES
GemaM.Durán1,2,AnaM.Contento1,ÁngelRíos1* 1
DepartmentofAnalyticalChemistryandFoodTechnology,UniversityofCastillaLaMancha. IRICA(RegionalInstituteofAppliedScientificResearch).AvenidaCamiloJoséCela,s/n.13071, CiudadReal,Spain *Email:[email protected]
2
Abstract An optical sensor for vanillin in food samples using CdSe/ZnS quantum dots (QDs) modifiedwithcyclodextrin(CD)wasdeveloped.Thisvanillinsensorisbasedonthe selectivehostguestinteractionbetweenvanillinandcyclodextrin.Theprocedurefor the synthesis of cyclodextrinCdSe/ZnS (CDCdSe/ZnSQDs) complex was optimized, and its fluorescent characteristics are reported. It was found that the interactionbetweenvanillinandCDCdSe/ZnSQDscomplexproducedthequenching of the original fluorescence of CDCdSe/ZnSQDs according to the SternVolmer equation. The mechanism of the interaction is discussed. The analytical potential of thissensoringsystemwasdemonstratedbythedeterminationofvanillininsynthetic and food samples. Themethod wasselective for vanillin,with a limit of detection of 0.99 μg mL1, and a reproducibility of 4.1% in terms of relative standard deviation (1.2%underrepeatabilityconditions).Recoveryvalueswereinthe90105%rangefor foodsamples. Keywords: CdSe/ZnS quantum dots, cyclodextrin; Functionalization; Fluorescence; Vanillinsensoring;Foodsamples. 1
1. Introduction Theuseofquantumdots(QDs)forthedevelopmentofsensorsisoneofthemost developingfieldsofnanotechnologysofar.Theirfluorescenceefficiencyissensitiveto differentcompoundsontheirsurface.Therefore,molecularrecognitionatthesurface of QDs can be utilized in the development of fluorescentbased sensors. For this purpose, several strategies for surfacemodified QDs have been employed. Thiol ligands were used to modify quantum dots such as Lcysteine or Dcysteine for carnitineenantiomersdetermination[1],mercaptoaceticacidforLcysteinedetection [2],3mercaptopropionicacidfordetectionandquantificationofparaquat[3],among otherapplications.Othersurfacemodifiedquantumdots,suchasionicliquidmodified CdSe/ZnS QDs for trimethylamine fluorimetric determination [4], silicacoated CdSe/ZnSnanoparticlesforCu2+detection[5],calix[8]arenecoatedCdSe/ZnSquantum dotsasC60nanosensor[6],havealsobeenused. Cyclodextrins(CDs)areconsideredoneofthebesthostmolecules.Cyclodextrins are cyclic receptors consisting of seven glucose units linked one to another by 14 glycoside bonds. Their cavityshaped cyclic phenol molecules are capable to forming hostguestcomplexeswithavarietyoforganicmolecules.Thehydrophobiccavitiesof cyclodextrins were used to develop different sensors [7, 8] and separation matrices [9].Thus,CDshaveattractedgreatinterestinsupramolecularchemistry.Cyclodextrins coatingensuresthehighemissionefficiencyandthesmallersizeofQDsandprovides selectivity. Therefore, several methods for the preparation of highly fluorescent and stable CdSe/ZnS quantum dots, using cyclodextrins as surface coating agents, have recently reported. Optical sensing and chiroselective sensing of different substrates were reported using CD functionalized CdSe/ZnS QDs based on a fluorescence resonance transfer (FRET) or an energy transfer mechanism [10]. CDcoated CdSe/ZnS QDs was also applied as enantioselective fluorescent sensors for amino acids,suchastyrosineandasignificantfluorescenceenhancementwasobserved[11], whichcanbeusedfortheopticaldetectionofphenolpollutantsinwatersamples[12]. OtherCDmodifiedCdTeQDswerealsousedasananosensorforacetylsalicylicacid 2
and its metabolites [13], as fluorescent probes for polycyclic aromatic hydrocarbons (PHAs) [14], and CD modified CdSe QDs as a recognition system for tyrosine enantiomers[15]. Vanillin is one of the most popular flavoring substances and it is widely used in food, beverages, perfumery and pharmaceutical industry. Natural vanillin is obtained fromvanillapods,throughalongandexpensiveprocess.Furthermore,naturalvanillin obtainedinthiswaycansupplylessthan1%ofthemarketdemand.Therefore,most of the vanillin employed is synthesised through chemical processes from eugenol (4 allyl2methoxyphenol), guaiacol (2methoxyphenol) or lignin. The chemical synthesis leadstoacheapervanillin,butoflowerqualitywithawidevarietyofcomplexmatrices thatneedselectiveandsensitivecleanupproceduresforitsextractionand/oranalysis [16].Theyieldandpurificationofvanillin(abiomoleculerelevantforseveralpurposes) are still of major interest. Different methods for determination of vanillin in several samples have been developed. Many of these methods involve electrochemical detectionwithseveraltypesofelectrodes[1720].Othermethodsincludetheuseof supported liquid membranes with amperometric [21] or piezoelectric [22] detection. Spectrophotometric [23] detection, liquid cromatography with mass spectrometry detection[24],capillaryelectrophoresis[25]orgaschromatography[26]hasalsobeen used for determining vanillin. However, these techniques usually need complicated sample pretreatment. Nowadays, as a useful analytical technique, fluorescent detection has been extensively employed with high sensitivity and selectivity. Toour knowledge, the use of CDs functionalized QDs as selective probes for fluorescent determination of flavoring is almost unexplored. In this paper, it is reported the synthesis of water soluble and stable semiconductor CdSe/ZnS QDs using CD as surfacecoatingagentbyaverysimplesonochemicalmethod.Itspotentialapplication as a selective fluorescent sensor for vanillin in several samples has also been investigated,obtainingsatisfactoryresultsforitsdeterminationinfoodsamples. 3
2. Experimental
2.1. Reagents Allchemicalreagentswereobtainedfromcommercialsourcesofanalyticalgrade andwereusedasreceivedwithoutfurtherpurification.Cadmiumoxide(CdO,99.99% metal basis), trioctylphosphine oxide (TOPO, 99%), trioctylphosphine (TOP, 90.0%), selenium(Sepowder,100mesh,99.99%metalsbasis),diethylzincsolution(ZnEt2,1M in hexane), bis(trimethylsilyl) sulfide ((TMS)2S), anhydrous methanol, ethanol and acetonitrile
were
purchased
from
SigmaAldrich
(Steinheim,
Germany).
Hexylphosphonicacid(HPA)wasobtainedfromAlfaAesar(Karlsruhe,Germany).These reagents were used to prepare CdSe(ZnS) QDs. 4hydroxy3methoxybenzaldehyde (Vanillin, 98%) and cyclodextrin (98%) were obtained from Fluka (Steinheim, Germany).cyclodextrinwaspurchasedfromSigmaAldrich(Steinheim,Germany)and cyclodextrin(>98%)waspurchasedfromTokyoChemicalIndustryAmerica(Portland, U.S.A). Disodium hydrogen phosphate anhydrous buffer was purchased from Panreac (Barcelona, Spain). 4hydroxy3methoxybenzyl alcohol (Vanillin alcohol, 98%), 4 hydroxybenzaldehyde(98%)and4hydroxybenzylalcohol(99%)werepurchasedfrom SigmaAldrich(Steinheim,Germany). Analyticalstandardstocksolutionsofvanillinat1mgmL1werepreparedinwater.The stock solutions were stored under refrigerator conditions (4 °C) and protected from the light. The stock solution of Se/TOP was prepared using 0.051 g of Se in 3 mL of TOP.Buffersolutionswaspreparedusingdisodiumhydrogenphosphatebufferfixing thepHto8. 2.2. Apparatus Fluorescence emission spectra were measured on a Photon Technology International(PTI)Inc.QuantaMaster40spectrofluorometerthatwasequippedwitha 75W continuous xenon arc lamp. An ASOC10 USB interface FeliXGX software was usedforfluorescencedataacquisitionandalsocontrolledthehardwareforallsystem configurations.Theslitsforexcitationandemissionwidthswereboth5and3nm.All
4
optical measurements were performed in a 10 mm quartz cell at room temperature underambientconditions.UVvisspectrawereobtainedonaSECOMAMUVILightXS2 spectrophotometerequippedwithaLabPowerV350forabsorbancedataacquisition using 10 mm quartz cuvettes. QDs were precipitated and purified using a centrifuge Centrofriger BLII model 7001669, J.P Selecta (Barcelona, Spain). The pH measurements were achieved in a Crison Basic 20 pHmeter with a combined glass electrode (Barcelona, Spain). An ultrasonic cleaning bath Ultrasons, J.P. Selecta (Barcelona, Spain) and a 254/365 nm UV lamp 230 V, E2107 model, Consort nv (Turnhout,Belgium)werealsoused. 2.3. Preparation of hydrophobic CdSe/Zn QDs
CdSecorenanocrystalswerepreparedviaamodifiedprocessreportedPengetal [27].Typically,0.06gofCdO,0.22gHPA,and7gofTOPOwereloadedina250mL threeneck flask clamped in a heating mantle and air in the system was pumped off and replaced with N2. The mixture was stirred and heated at 300310 °C for 15 min, andCdOwasdissolvedinHPAandTOPO.Thesolutionwascooleddownto270°Cand 2.5 mL of the solution of Se/TOP was swiftly injected. After the injection, the temperaturewasadjustedto250°Cfornucleusgrowthduring20minandachangein thecolorofthesolutiontoredwasobserved.TomakeZnSshellontheCdSe,3mLofa solution of Zn/S/TOP (0.58 g of ZnEt2, 0.087 mL of (TMS)2S and 3.4 mL of TOP) was addeddropwisetothemixtureundervigorousstirring.Themixturewaskeptto90C for 4 h to improve the crystallinity of ZnS shell. After cooling the solution down to roomtemperature,QDsweredilutedwith10mLofanhydrouschloroform.Finally,the synthesized QDs were purified by adding 10 mL of methanol to 10 mL of the QD solution.Then,QDswereprecipitated,collectedbyultracentrifugation(at13000rpm during 15 min), and washed with methanol four times. The purified QD nanocrystals werefinallydispersedin10mLofanhydrouschloroformandstoredindarkness. 2.4. Preparation of n-cyclodextrin capped CdSe/ZnS QDs Different cyclodextrins were studied (n=, , ). The nCDCdSe/ZnSQDs were preparedbyusingamodifiedprocedurepreviouslyreported[28].Thus,a0.5mL(200 5
mgL1)ofTOPOcappedCdSe/ZnSQDsinchloroform(0.1mg)wereaddedintoa2mL polypropylenevialandthechloroformwasdriedbynitrogenatmosphere.Then,nCD powder(3.1,3.6or4.2mgfor,orCD,respectively)wasaddedtodriedQDsand the mixture was dispersed in acetonitrile (2 mL). The mixture was placed in a high intensity ultrasound bathforabout45minatroomtemperature.When the reaction was finished, a rosy precipitate was obtained. The precipitate was separated by centrifugingat13500rpm.Theresultingsupernatantwaseliminatedandtheremained acetonitrilewasevaporated.Finally,itwaspurifiedbyfurthercyclesofcentrifugation inwater.The resultingprecipitateofthenCDCdSe/ZnSQDswasdispersedinwater (10mL)andstoredatroomtemperatureinthedarkforfurtherinvestigations. 2.5. Preparation of samples and analytical procedure
Severalcommercialfoodsamples,suchassugar,milkorcustard,werepurchased fromalocalmarket.Thesesampleswerepreparedasfollows: Sugarsamplesweregroundtoafinepowder.Then,0.5gofthispowderand2mL ofabsoluteethanolwereplaceintoatubeandshackedbyalaboratoryshakerfor10 min.Thismixturewascentrifugedat10000rpm.Theclearpartofthesolutioninthe tube was used for analysis. Ethanol was evaporated and the resulting residue was dissolvedinwater. Milksamples.1mLofmilksampleand2mLofabsoluteethanolwereplaceintoa tube and shacked by a laboratory shaker at 40 C for 10 min. This mixture was centrifugedat12000rpmfor15minforprecipitatetheproteins.Thesupernatantwas evaporatedandtheresultingsampleresiduewasdissolvedinwater. Custard samples. 0.05 g of custard powder and 2 mL of absolute ethanol were placeintoatubeandshackedbyalaboratoryshakerat40Cfor10min.Thismixture was centrifugedat6000rpmfor 15min. Thesupernatantwas usedforanalysis.The absolute ethanol was evaporated and the resulting sample residue was dissolved in water. For vanillin determination, suitable amount of these samples and 0.4 mL of CD modifiedCdSe/ZnSfixedatpH8weretransferredintoavolumetricflask.Themixture wasstirredatroomtemperatureandstoredatambientlightinthedarkfor30minfor 6
reaction. Then, this mixture was transferred into a 10 mm quartz cuvette and the emissionfluorescentspectrumwasmeasured,atanexcitationwavelengthof450nm, between 500 and 670 nm. I0/I was usedas analytical signal, where I0 and I were the fluorescence intensity at 590 nm of the systems in the absence and presence of vanillin,respectively.
3. Results and Discussion StudiesforQDsmodificationwascarriedoutusingdifferentcyclodextrins(n=, or).ThesolubilizationprocedureoftheQDswasconductedbyultrasonicirradiation of a mixture of TOPOcoated CdSe/ZnS QDs and nCD. The assayed strategy for creating nCDQDs was a chemical procedure based on the formation of a hostguest complex between the passivized ligand (TOPO) and nCD by hydrophobic interaction (Figure1A).In ordertoobtainthe optimalconditionsinthemodificationprocedure, severalparameterswerestudied. 3.1. InfluenceofexperimentalfactorsinthemodificationofnCDCdSe/ZnSQDs TheeffectofsolventreactiononthefluorescenceintensityandstabilityofQDswas testedusingabsoluteethanol,anhydrousmethanolandacetonitrile.Itwasfoundthat fluorescence intensity of nCDCdSe/ZnSQDs when it was used acetonitrile as a reaction solvent was dramatically higher thanwhen ethanol or methanol were used. Therefore,thissolventwasusedforfurtherexperiments.Figure2Ashowstheeffectof thesesolventsusingCDCdSe/ZnSQDs. The concentration of nCD was varied between 0.5 and 4.5 mM, maintaining constanttheotherparameters.ItwasobservedthatthecomplexationofTOPOwithn CDisessentialtoproducethesolubilizationofCdSe/ZnSQDsinanaqueousmedium. WhentheconcentrationofnCDistoolow,onlysmallportionsofsurfaceboundTOPO molecules on the surfaces of CdSe/ZnS QDs form complexes with nCD, which is not enoughtogiveahydrophilicpropertytothenanoparticles,andhencetoproducetheir stabilization in the aqueous phase. At relatively high concentrations of nCD, substantialamountsofTOPOmoleculesareabletoformhostguestcomplexeswithn 7
CD, which led to an increase in the stability of the QDs in the hydrophilic media. However,whenthedosageofnCDisveryhigh,althoughphasetransferwasefficiently achieved,theresultingcomplexwasfoundtobeunstableinwater,andthenCDexcess couldmaskthedeterminationoftheanalyte.Therefore,1.6mMofnCDwaschosen forfurtherexperiments,asthemaximumfluorescenceintensitywasobtainedatthis concentration.Finally,timedependentexperimentswereperformedbyexposingthe reactionofmodificationofQDsatseveraltimesbetween15and180mintoultrasonic irradiation.Thebestresultswereobtainedwhen45minofultrasonicirradiationwas used. The resulting nCDCdSe/ZnSQDs thus obtained were highly fluorescent and stable. Figure 2A shows the emission spectra of nCDCdSe/ZnSQDs in water and TOPOCdSe/ZnSQDs in chloroform. As it can be seen the maximum emission band around 590 nm (ૃexc = 450 nm) were obtained in all cases. The line width of the fluorescencespectrumisrelativelynarrow(withthefullwidthathalfmaximumof44 nm), indicating that the nCDCdSe/ZnSQDs nanoparticles have a narrow size distribution. Compared to TOPOCdSe/ZnS QDs in chloroform, nCDCdSe/ZnSQDs increased the fluorescence intensity. It was also observed no change in emission wavelength and the spectral width regarding to TOPOQDs. The UV/Vis spectrum of TOPOCdSe/ZnSQDs, CDCdSe/ZnSQDs, CDCdSe/ZnSQDs and CDCdSe/ZnS QDsarealsoillustratedinFigure2B.Asitcanbeseen,absorptionbandsatca.255and ca.585 nm were obtained in all cases. Therefore, no significant differences were observed in the modification procedure of QDs. The stability of obtained for nCD CdSe/ZnSQDs in water was estimated by measurementsof the emission intensity at roomtemperatureatseveraltimes.Fromtheresultsobtaineditwasconcludedthatn CDCdSe/ZnSQDswerestableatleastfortwoweeks,withanysignificantchangesin theirfluorescencespectra. 3.2. Effect of vanillin on the luminescence response of modified QDs In the ncyclodextrin modified QDs, the hydrophobic pockets of the cyclodextrin moleculesinteractwiththealiphaticchainsoftheTOPOpresentonthenanoparticle 8
surface from the QDs synthesis (Figure 1A). Nevertheless, the immobilized cyclodextrins retain their capability of engaging molecular recognition. In this way, it wasstudiedtheuseofnCDCdSe/ZnSQDsnanoparticlesasaselectiveluminescence sensors of vanillin. First, preliminary studies were made in order to know the best recognition of vanillin when , or CDCdSe/ZnSQDs were used. It was found that vanillinaffectsluminescenceofCDCdSe/ZnSQDsinamoredrasticway,producinga quenchingeffectontheQDsemissionband,asitcanbeseeninFigure3.Therefore, CDCdSe/ZnSQDswasselectedtodevelopthevanillinsensors. The selective hostguest interaction between vanillin and cyclodextrin, through the vanillin binds to the receptor sites, can act as an electron transfer quencher of luminescence of the particles (Figure 1B). Under these conditions the association of the vanillin to the CD cavities concentrates the analyte on the semiconductor QDs surface. The timedependent luminescence changes of the CDQDs upon their interactionwith4.2mgL1ofvanillinwereinvestigated.Fromtheresultsobtained,it can be concluded that luminescence of the CDQDs in the presence of vanillin decreased until 30 min, and remained constant after this time value. The same behaviorwasalsoobservedbyrecordingofabsorbancetimespectraofvanillinCD CdSe/ZnSQDscomplex. ThepHeffectintherecognitionofvanillinusingCDCdSe/ZnSQDsnanoparticles was studied by measurements of fluorescence intensity of the CDCdSe/ZnSQDs without(I0)andatgivenvanillinconcentration(I).ItwasfoundthatthepHsignificantly influenced the fluorescence intensity of the vanillinCDCdSe/ZnSQDs system. The maximumvalueofI/I0 wasobtainedwhenthepHwas8.0.Therefore,thisvaluewas chosenasoptimum.Asasecondpartofthisstudy,theinfluenceoftheconcentration ofbuffersolution,fixedwithNa2HPO4pH=8,wascarriedinordertoevaluatetheeffect ofthisparameterinthefluorescenceintensityofvanillinCDCdSe/ZnSQDssystem. The results showed that the maximum value I/I0 was obtained when the buffer solution concentration was 1.2 mM. Therefore, this value was chosen in all experiments.
9
According to the literature [29], the surface of the nCDCdSe/ZnSQDs affords a finite number of binding sites. Each of the binding sites could absorb one vanillin moleculefromthesolution.Therefore,accordingtoLangmuirequation[28]:
C I
§ 1 · § 1 · ¨ ¸¨ ¸C © BI max ¹ © I max ¹
[1]
whereC istheconcentrationofvanillinandIthe fluorescenceintensityobtainedfor thisconcentrationlevel.Accordingtotheliterature,iftheLangmuirdescriptionofthe bindingofvanillinonthesurfaceofCDCdSe/ZnSQDiscorrect,alinearplotofc/Ias a function of c must be obtained. In this case, a good linearity was observed throughout the entire range of vanillin concentration (2 to 20 mg L1). The binding constantBofCDCdSe/ZnSQDswithvanillinisfoundtobe0.99. 3.3. Analytical features for vanillin determination In order to develop an analytical method to determine vanillin in foods, several analytical performance characteristics were evaluated under the optimized experimental conditions. The quenching effect of the vanillin can be described using thefollowingSternVolmerequation: I0 I
1 K sv >Q @
[2]
where I0 and I are the fluorescence intensity of CDCdSe/ZnSQDs in absence and presenceofvanillin.AlinearrelationshipbetweenI0/Iandvanillinconcentrationinthe range of 220 mg L1 with a correlation coefficient of 0.9963 was obtained. Figure 4 shows the fluorescence spectra of CDCdSe/ZnSQDs at different concentrations of vanillinbetween2and20mgL1.Thecalibrationequationwas:
I0 I
1.094 0.051> vanillin @
[3]
The precision of the methodology was evaluated in terms of repeatability and reproducibility.Todeterminetherepeatabilityofthemethod,10analysisofsamples containing4.12mgL1ofvanillinwerecarriedoutandtheobtainedrelativestandard deviation (R.S.D.) was 1.2%. Then, the reproducibility was estimated for three 10
replicatesof4.12mgL1vanillinunderinterdayconditions(fortwoconsecutivedays), obtainingaR.S.D.of4.7%.Alimitofdetection(LOD)of0.99mgL1wasobtainedfor vanillin determination, based on the IUPAC method (blank signal plus 3 times its standard deviation). 10 measurements were used to obtain the LOD. According to theseresults,itcanbeconcludedthatthisapproachopensthepossibilitiestodevelop ananalyticalmethodusingCDCdSe/ZnSQDsforthedeterminationofvanillin. Inordertoapplythemethodtofoodsamplesaselectivitystudywasalsocarried out. For vanillin determination in food samples, the main interferences come from several colorant additives, such as curcumine or riboflavine. However, following the procedure reported in the Section 2.5, the interferences of these compounds were eliminated.Bycontrast,sucrose(glucoseandlactose)couldbepresentinthesample as principal interfering compound. For this purpose, two different level of concentrationwereusedincombinationwithvanillin.Fromtheresultsobtaineditcan concludedthatwhentheconcentrationofinterferenceswereincrease,notdifference wereobservedinthevanillinsignal,givenvaluesofR.S.Dof3.9and4.5%,respectively. Thecoexistingcompoundscausedarelativeerroroflessthan±5%inthefluorescence intensityofthevanillinCDCdSe/ZnSQDs.Thereforeitcanbeconsideredtohaveno interference with the detection of vanillin. The data revealed that the proposed methodmightbeappliedtothedetectionofvanillininfoodsamples. Ontheotherhand,severalsimilarstructurestovanillin,suchasvanillinalcohol,4 hydroxybenzaldehydeor4hydroxybenzylalcoholweretested.Forthispurpose,three different levels of concentration were used in combination with vanillin. From the resultsobtaineditcanbeconcludedthatwhentheconcentrationofinterferencewas increased,nodifferencewasobservedinthevanillinsignal,withlessof±5%ofR.S.D. when vanillin alcohol and 4hydroxybenzyl alcohol were used. However, the results obtainedinthepresenceof4hydroxybenzaldehydeindicatedthatthiscompoundcan produce interference in the vanillin determination. This fact demonstrated that the structureoftheanalyteandpHofsolutionsystemcouldplayanimportantroleinthe selectivity of vanillin determination with CDCdSe/ZnSQDs. Table 1 shows the obtained change of fluorescence intensity (%) at the three concentration levels studied. 11
4. Application To demonstrate the applicability of the proposed method, it was applied to determine vanillin in several commercial sugar and milk samples, purchased in different supermarkets. Each one of these samples was spiked with several concentrations of vanillin and were prepared according to the steps described in Section 2.5. The summary of these results are shown in Table 2. The obtained recoveriesindicatedanacceptableagreementbetweentheamountsaddedandthose foundforalltypesofsamples. Theproposedmethodwasalsousedforthequantitationofvanillinincustardwith vanilla flavor. These samples contained vanillin as flavor additive. This product was analyzed by triplicate, according the procedure described in Section 2.5. To evaluate thematrixeffect,thestandardadditionmethodwasalsousedforthedetermination ofvanillininthestudiedproduct.Theobtainedresultswere75.6±0.6and76.3±0.9 mgL1withandwithoutstandardaddition,respectively,correspondingtotheoriginal sample (samples were diluted before analyses). The application of Student statistical test for a confidence level of 95% demonstrated the statistical coincidence between theconcentrationfoundwiththosefoundbythestandardadditionmethod(n=6,tcrit =2.92>texp=0.48). 5. Conclusions In this work, an optical sensor for vanillin determination based on the selective supramolecularrecognitionofvanillinwithcyclodextrinmodifiedCdSe/ZnSQDswas developed. The procedure for the synthesis of nCDCdSe/ZnSQDs complex was simple and very effective. Different coating agents, such as , and CD, were studied, and the effect of several experimental parameters was optimized. The proposed methodology presents some advantages. Thus, the solubilization of TOPO CdSe/ZnSQD,initiallyinorganicmedia,wasallowedinaqueousmedia.Thisfactallows its compatibility with biological samples, and aqueous media in general, and in additiontheconservationinorganicmediaoverlongperiodsoftime.Inthisway,itis possible to modify only the necessary amount of QDs when required. On the other 12
hand,themethodologydemonstratedthatthesurfacecoatingofQDswithdifferentn cyclodextrinskeepstheemissionintensityofthequantumdotsandtheirdiameter.In addition, the immobilized cyclodextrins on the surface of the QDs retain their capabilityofengagingmolecularrecognition.Therefore,theuseofcyclodextrinfor QDs surface modification showed selectivity in vanillin recognition to alpha and gammacyclodextrins.Thus,thepotentialofthesensorfortheanalysisoffoodsamples was demonstrated, opening other possible alternatives for the selective fluorimetric sensingofothercompoundsthroughtheappropriatedmodificationofquantumdots surface. Acknowledgements ThisresearchwassupportedbyProjectCTQ201348411P(MINECO).GemaM.Durán thankstheSpanishMinistryofEconomyandCompetitivenessforaPredoctoralGrant.
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C.P.Han,H.B.Li,Novelcyclodextrinmodifiedquantumdotsasfluorescent probes for polycyclic aromatic hydrocarbons (PAHs), Chin. Chem. Lett. 19 (2008)215218. Y. Cao, S. Wu, Y. Liang, Y. Yu, The molecular recognition of cyclodextrin modifiedCdSequantumdotswithtyrosineenantiomers:Theoreticalcalculation andexperimentalstudy,J.Mol.Struct.1031(2013)913. S. Ramachandra Rao, G. A. Ravishankar, Vanilla flavour: production by conventionalandbiotechnologicalroutes,J.Sci.FoodAgric.80(2000)289–304. Y.Yard m,M.Gülcan,Z. entürk,Determinationofvanillinincommercialfood product by adsorptive stripping voltammetry using a borondoped diamond electrode,FoodChem.141(2013)1821–1827. D.Zheng,C.Hu,T.Gan,X.Dang,S.Hu,Preparationandapplicationofanovel vanillin sensor based on biosynthesis of Au–Ag alloy nanoparticles, Sens. Actuators,B148(2010)247–252. F. Bettazzi, I. Palchetti., S. Sisalli, M. Mascini, A disposable electrochemical sensorforvanillindetection,Anal.Chim.Acta555(2006)134–138. J. L. Hardcastle, C. J. Paterson, R. G. Compton, Biphasic Sonoelectroanalysis: SimultaneousExtractionfrom,andDeterminationofVanillininFoodFlavoring, Electroanal.13(2001)899–905. M. Luque, E. LuquePérez, A. Ríos, M. Valcárcel, Supported liquid membranes forthedeterminationofvanillininfoodsampleswithamperometricdetection, Anal.Chim.Acta410(2000)127–134. M.Avila,M.Zougagh,A.Escarpa,A.Rios,Supportedliquidmembranemodified piezoelectric flow sensor with molecularly imprinted polymer for the determinationofvanillininfoodsamples,Talanta72(2007)1362–1369. Y. Ni, G. Zhang, S. Kokot, Simultaneous spectrophotometric determination of maltol, ethyl maltol, vanillin and ethyl vanillin in foods by multivariate calibrationandartificialneuralnetworks,FoodChem.89(2005)465–473. U.Pyell,B.PletschViehmann,U.Ramus,Componentanalysisofvanillaextracts and vanilla containing commercial preparations by micellar electrokinetic chromatography or highperformance liquid chromatographyA method comparison,J.Sep.Sci.25(2002)1035–1042. M.Ohashi,H.Omae,M.Hashida,Y.Sowa,S.Imai,Determinationofvanillinand related flavor compounds in cocoa drink by capillary electrophoresis, J. Chromatogr.,A,1138(2007)262–267. A. PerezSilva, E. Odoux, P. Brat, F. Ribeyre, G. RodriguezJimenes, V. Robles Olvera, GC–MS and GC–olfactometry analysis of aroma compounds in a representative organic aroma extract from cured vanilla (Vanilla planifolia G. Jackson)beans,FoodChem.99(2006)728–735. Z.A.Peng,X.Peng,FormationofhighqualityCdTe,CdSe,andCdSnanocrystals usingCdOasprecursor,J.Am.Chem.Soc.123(2001)183184. 15
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16
FIGURESCAPTION Figure1.SchematicillustrationofsurfacemodificationofTOPOCdSe/ZnSQDswithn cyclodextrins(A).HostguestinteractionbetweenCDCdSe/ZnSQDsandvanillin(B). Figure 2. Emission (A) and absorption (B) spectra of TOPOCdSe/ZnSQDs (a), CD CdSe/ZnSQDs(b),CDCdSe/ZnSQDs(c)andCDCdSe/ZnSQDs(d). Figure 3. Effect of 4.2 mg L1 vanillin concentration over luminescence of CD CdSe/ZnSQDs(A),CDCdSe/ZnSQDs(B)andCDCdSe/ZnSQDs(C). Figure4.FluorescencespectraofCDCdSe/ZnSQDswithdifferentconcentrationsof vanillinbetween2and20mgL1.
17
HIGHLIGHTS CyclodextrinCdSe/ZnSquantumdotsweresynthesized. Compatibilitywithaqueousmedia. Thenewmaterialswereusedasselectivesensorforvanillin. cyclodextrinCdSe/ZnSwasusedforanalysisoffoodsamples.
18
Table1.Effectofcoexistingforeignsubstancesatthreedifferentconcentrationlevels withrespecttovanillinconcentration.
Foreignsubstances
Sucrose Vanillinalcohol
4hydroxybenzylalcohol
4hydroxyaldehyde
Foreignspeciesratio
Errorinvanillin determination(%)
1:10 1:20 1:0.5 1:1 1:2 1:0.5 1:1 1:2 1:0.5 1:1 1:2
+1.9 +2.5 0.1 +0.3 +0.2 2.5 +0.4 +2.9 6.5 8.2 9.7
Conditions:pH8;[vanillin]=6.65μgmL1.
19
Table2.Determinationofvanillininseveralfoodsamples(n=5). Sample
Added(mgL1)
Found(mgL1)
Recovery(%)
Sugar1
5 10 20
5.26±0.2 9.96 ±0.5 19.2±1
105±4 100±5 96±5
Sugar2
5 10 20
4.9±0.2 10.0 ±0.4 20.0±0.3
99±3 100 ±4 99±1.5
Sugar3
5 10 20
5.0 ±0.2 9.6±0.4 18.2±0.9
101 ±4 96±4 91±4.5
Milk1
5 10 20
5.2±0.2 9.0±0.4 20.0±1.0
104±4 90±4 99±5
Milk2
5 10 20
5.1±0.2 9.0±0.5 19.6±0.8
103±4 90±5 98±4
20
Figure
Figure
Figure
Figure
*Graphical Abstract (for review)
GRAPHICAL ABSTRACT
β-Cyclodextrin coated CdSe/ZnS quantum dots FOR VANILLIN SENSORING IN FOOD SAMPLES Gema M. Durán, Ana M. Contento, Ángel Ríos
www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(14)00668-7 http://dx.doi.org/10.1016/j.talanta.2014.07.100 TAL15009
To appear in:
Talanta
Received date: 21 May 2014 Revised date: 28 July 2014 Accepted date: 31 July 2014 Cite this article as: Gema M. Durán, Ana M. Contento, Ángel Ríos, βCyclodextrin coated CdSe/ZnS quantum dots FOR VANILLIN SENSORING IN FOOD SAMPLES, Talanta, http://dx.doi.org/10.1016/j.talanta.2014.07.100 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
-CYCLODEXTRIN COATED CdSe/ZnS QUANTUM DOTS FOR VANILLIN SENSORING IN FOOD SAMPLES
GemaM.Durán1,2,AnaM.Contento1,ÁngelRíos1* 1
DepartmentofAnalyticalChemistryandFoodTechnology,UniversityofCastillaLaMancha. IRICA(RegionalInstituteofAppliedScientificResearch).AvenidaCamiloJoséCela,s/n.13071, CiudadReal,Spain *Email:[email protected]
2
Abstract An optical sensor for vanillin in food samples using CdSe/ZnS quantum dots (QDs) modifiedwithcyclodextrin(CD)wasdeveloped.Thisvanillinsensorisbasedonthe selectivehostguestinteractionbetweenvanillinandcyclodextrin.Theprocedurefor the synthesis of cyclodextrinCdSe/ZnS (CDCdSe/ZnSQDs) complex was optimized, and its fluorescent characteristics are reported. It was found that the interactionbetweenvanillinandCDCdSe/ZnSQDscomplexproducedthequenching of the original fluorescence of CDCdSe/ZnSQDs according to the SternVolmer equation. The mechanism of the interaction is discussed. The analytical potential of thissensoringsystemwasdemonstratedbythedeterminationofvanillininsynthetic and food samples. Themethod wasselective for vanillin,with a limit of detection of 0.99 μg mL1, and a reproducibility of 4.1% in terms of relative standard deviation (1.2%underrepeatabilityconditions).Recoveryvalueswereinthe90105%rangefor foodsamples. Keywords: CdSe/ZnS quantum dots, cyclodextrin; Functionalization; Fluorescence; Vanillinsensoring;Foodsamples. 1
1. Introduction Theuseofquantumdots(QDs)forthedevelopmentofsensorsisoneofthemost developingfieldsofnanotechnologysofar.Theirfluorescenceefficiencyissensitiveto differentcompoundsontheirsurface.Therefore,molecularrecognitionatthesurface of QDs can be utilized in the development of fluorescentbased sensors. For this purpose, several strategies for surfacemodified QDs have been employed. Thiol ligands were used to modify quantum dots such as Lcysteine or Dcysteine for carnitineenantiomersdetermination[1],mercaptoaceticacidforLcysteinedetection [2],3mercaptopropionicacidfordetectionandquantificationofparaquat[3],among otherapplications.Othersurfacemodifiedquantumdots,suchasionicliquidmodified CdSe/ZnS QDs for trimethylamine fluorimetric determination [4], silicacoated CdSe/ZnSnanoparticlesforCu2+detection[5],calix[8]arenecoatedCdSe/ZnSquantum dotsasC60nanosensor[6],havealsobeenused. Cyclodextrins(CDs)areconsideredoneofthebesthostmolecules.Cyclodextrins are cyclic receptors consisting of seven glucose units linked one to another by 14 glycoside bonds. Their cavityshaped cyclic phenol molecules are capable to forming hostguestcomplexeswithavarietyoforganicmolecules.Thehydrophobiccavitiesof cyclodextrins were used to develop different sensors [7, 8] and separation matrices [9].Thus,CDshaveattractedgreatinterestinsupramolecularchemistry.Cyclodextrins coatingensuresthehighemissionefficiencyandthesmallersizeofQDsandprovides selectivity. Therefore, several methods for the preparation of highly fluorescent and stable CdSe/ZnS quantum dots, using cyclodextrins as surface coating agents, have recently reported. Optical sensing and chiroselective sensing of different substrates were reported using CD functionalized CdSe/ZnS QDs based on a fluorescence resonance transfer (FRET) or an energy transfer mechanism [10]. CDcoated CdSe/ZnS QDs was also applied as enantioselective fluorescent sensors for amino acids,suchastyrosineandasignificantfluorescenceenhancementwasobserved[11], whichcanbeusedfortheopticaldetectionofphenolpollutantsinwatersamples[12]. OtherCDmodifiedCdTeQDswerealsousedasananosensorforacetylsalicylicacid 2
and its metabolites [13], as fluorescent probes for polycyclic aromatic hydrocarbons (PHAs) [14], and CD modified CdSe QDs as a recognition system for tyrosine enantiomers[15]. Vanillin is one of the most popular flavoring substances and it is widely used in food, beverages, perfumery and pharmaceutical industry. Natural vanillin is obtained fromvanillapods,throughalongandexpensiveprocess.Furthermore,naturalvanillin obtainedinthiswaycansupplylessthan1%ofthemarketdemand.Therefore,most of the vanillin employed is synthesised through chemical processes from eugenol (4 allyl2methoxyphenol), guaiacol (2methoxyphenol) or lignin. The chemical synthesis leadstoacheapervanillin,butoflowerqualitywithawidevarietyofcomplexmatrices thatneedselectiveandsensitivecleanupproceduresforitsextractionand/oranalysis [16].Theyieldandpurificationofvanillin(abiomoleculerelevantforseveralpurposes) are still of major interest. Different methods for determination of vanillin in several samples have been developed. Many of these methods involve electrochemical detectionwithseveraltypesofelectrodes[1720].Othermethodsincludetheuseof supported liquid membranes with amperometric [21] or piezoelectric [22] detection. Spectrophotometric [23] detection, liquid cromatography with mass spectrometry detection[24],capillaryelectrophoresis[25]orgaschromatography[26]hasalsobeen used for determining vanillin. However, these techniques usually need complicated sample pretreatment. Nowadays, as a useful analytical technique, fluorescent detection has been extensively employed with high sensitivity and selectivity. Toour knowledge, the use of CDs functionalized QDs as selective probes for fluorescent determination of flavoring is almost unexplored. In this paper, it is reported the synthesis of water soluble and stable semiconductor CdSe/ZnS QDs using CD as surfacecoatingagentbyaverysimplesonochemicalmethod.Itspotentialapplication as a selective fluorescent sensor for vanillin in several samples has also been investigated,obtainingsatisfactoryresultsforitsdeterminationinfoodsamples. 3
2. Experimental
2.1. Reagents Allchemicalreagentswereobtainedfromcommercialsourcesofanalyticalgrade andwereusedasreceivedwithoutfurtherpurification.Cadmiumoxide(CdO,99.99% metal basis), trioctylphosphine oxide (TOPO, 99%), trioctylphosphine (TOP, 90.0%), selenium(Sepowder,100mesh,99.99%metalsbasis),diethylzincsolution(ZnEt2,1M in hexane), bis(trimethylsilyl) sulfide ((TMS)2S), anhydrous methanol, ethanol and acetonitrile
were
purchased
from
SigmaAldrich
(Steinheim,
Germany).
Hexylphosphonicacid(HPA)wasobtainedfromAlfaAesar(Karlsruhe,Germany).These reagents were used to prepare CdSe(ZnS) QDs. 4hydroxy3methoxybenzaldehyde (Vanillin, 98%) and cyclodextrin (98%) were obtained from Fluka (Steinheim, Germany).cyclodextrinwaspurchasedfromSigmaAldrich(Steinheim,Germany)and cyclodextrin(>98%)waspurchasedfromTokyoChemicalIndustryAmerica(Portland, U.S.A). Disodium hydrogen phosphate anhydrous buffer was purchased from Panreac (Barcelona, Spain). 4hydroxy3methoxybenzyl alcohol (Vanillin alcohol, 98%), 4 hydroxybenzaldehyde(98%)and4hydroxybenzylalcohol(99%)werepurchasedfrom SigmaAldrich(Steinheim,Germany). Analyticalstandardstocksolutionsofvanillinat1mgmL1werepreparedinwater.The stock solutions were stored under refrigerator conditions (4 °C) and protected from the light. The stock solution of Se/TOP was prepared using 0.051 g of Se in 3 mL of TOP.Buffersolutionswaspreparedusingdisodiumhydrogenphosphatebufferfixing thepHto8. 2.2. Apparatus Fluorescence emission spectra were measured on a Photon Technology International(PTI)Inc.QuantaMaster40spectrofluorometerthatwasequippedwitha 75W continuous xenon arc lamp. An ASOC10 USB interface FeliXGX software was usedforfluorescencedataacquisitionandalsocontrolledthehardwareforallsystem configurations.Theslitsforexcitationandemissionwidthswereboth5and3nm.All
4
optical measurements were performed in a 10 mm quartz cell at room temperature underambientconditions.UVvisspectrawereobtainedonaSECOMAMUVILightXS2 spectrophotometerequippedwithaLabPowerV350forabsorbancedataacquisition using 10 mm quartz cuvettes. QDs were precipitated and purified using a centrifuge Centrofriger BLII model 7001669, J.P Selecta (Barcelona, Spain). The pH measurements were achieved in a Crison Basic 20 pHmeter with a combined glass electrode (Barcelona, Spain). An ultrasonic cleaning bath Ultrasons, J.P. Selecta (Barcelona, Spain) and a 254/365 nm UV lamp 230 V, E2107 model, Consort nv (Turnhout,Belgium)werealsoused. 2.3. Preparation of hydrophobic CdSe/Zn QDs
CdSecorenanocrystalswerepreparedviaamodifiedprocessreportedPengetal [27].Typically,0.06gofCdO,0.22gHPA,and7gofTOPOwereloadedina250mL threeneck flask clamped in a heating mantle and air in the system was pumped off and replaced with N2. The mixture was stirred and heated at 300310 °C for 15 min, andCdOwasdissolvedinHPAandTOPO.Thesolutionwascooleddownto270°Cand 2.5 mL of the solution of Se/TOP was swiftly injected. After the injection, the temperaturewasadjustedto250°Cfornucleusgrowthduring20minandachangein thecolorofthesolutiontoredwasobserved.TomakeZnSshellontheCdSe,3mLofa solution of Zn/S/TOP (0.58 g of ZnEt2, 0.087 mL of (TMS)2S and 3.4 mL of TOP) was addeddropwisetothemixtureundervigorousstirring.Themixturewaskeptto90C for 4 h to improve the crystallinity of ZnS shell. After cooling the solution down to roomtemperature,QDsweredilutedwith10mLofanhydrouschloroform.Finally,the synthesized QDs were purified by adding 10 mL of methanol to 10 mL of the QD solution.Then,QDswereprecipitated,collectedbyultracentrifugation(at13000rpm during 15 min), and washed with methanol four times. The purified QD nanocrystals werefinallydispersedin10mLofanhydrouschloroformandstoredindarkness. 2.4. Preparation of n-cyclodextrin capped CdSe/ZnS QDs Different cyclodextrins were studied (n=, , ). The nCDCdSe/ZnSQDs were preparedbyusingamodifiedprocedurepreviouslyreported[28].Thus,a0.5mL(200 5
mgL1)ofTOPOcappedCdSe/ZnSQDsinchloroform(0.1mg)wereaddedintoa2mL polypropylenevialandthechloroformwasdriedbynitrogenatmosphere.Then,nCD powder(3.1,3.6or4.2mgfor,orCD,respectively)wasaddedtodriedQDsand the mixture was dispersed in acetonitrile (2 mL). The mixture was placed in a high intensity ultrasound bathforabout45minatroomtemperature.When the reaction was finished, a rosy precipitate was obtained. The precipitate was separated by centrifugingat13500rpm.Theresultingsupernatantwaseliminatedandtheremained acetonitrilewasevaporated.Finally,itwaspurifiedbyfurthercyclesofcentrifugation inwater.The resultingprecipitateofthenCDCdSe/ZnSQDswasdispersedinwater (10mL)andstoredatroomtemperatureinthedarkforfurtherinvestigations. 2.5. Preparation of samples and analytical procedure
Severalcommercialfoodsamples,suchassugar,milkorcustard,werepurchased fromalocalmarket.Thesesampleswerepreparedasfollows: Sugarsamplesweregroundtoafinepowder.Then,0.5gofthispowderand2mL ofabsoluteethanolwereplaceintoatubeandshackedbyalaboratoryshakerfor10 min.Thismixturewascentrifugedat10000rpm.Theclearpartofthesolutioninthe tube was used for analysis. Ethanol was evaporated and the resulting residue was dissolvedinwater. Milksamples.1mLofmilksampleand2mLofabsoluteethanolwereplaceintoa tube and shacked by a laboratory shaker at 40 C for 10 min. This mixture was centrifugedat12000rpmfor15minforprecipitatetheproteins.Thesupernatantwas evaporatedandtheresultingsampleresiduewasdissolvedinwater. Custard samples. 0.05 g of custard powder and 2 mL of absolute ethanol were placeintoatubeandshackedbyalaboratoryshakerat40Cfor10min.Thismixture was centrifugedat6000rpmfor 15min. Thesupernatantwas usedforanalysis.The absolute ethanol was evaporated and the resulting sample residue was dissolved in water. For vanillin determination, suitable amount of these samples and 0.4 mL of CD modifiedCdSe/ZnSfixedatpH8weretransferredintoavolumetricflask.Themixture wasstirredatroomtemperatureandstoredatambientlightinthedarkfor30minfor 6
reaction. Then, this mixture was transferred into a 10 mm quartz cuvette and the emissionfluorescentspectrumwasmeasured,atanexcitationwavelengthof450nm, between 500 and 670 nm. I0/I was usedas analytical signal, where I0 and I were the fluorescence intensity at 590 nm of the systems in the absence and presence of vanillin,respectively.
3. Results and Discussion StudiesforQDsmodificationwascarriedoutusingdifferentcyclodextrins(n=, or).ThesolubilizationprocedureoftheQDswasconductedbyultrasonicirradiation of a mixture of TOPOcoated CdSe/ZnS QDs and nCD. The assayed strategy for creating nCDQDs was a chemical procedure based on the formation of a hostguest complex between the passivized ligand (TOPO) and nCD by hydrophobic interaction (Figure1A).In ordertoobtainthe optimalconditionsinthemodificationprocedure, severalparameterswerestudied. 3.1. InfluenceofexperimentalfactorsinthemodificationofnCDCdSe/ZnSQDs TheeffectofsolventreactiononthefluorescenceintensityandstabilityofQDswas testedusingabsoluteethanol,anhydrousmethanolandacetonitrile.Itwasfoundthat fluorescence intensity of nCDCdSe/ZnSQDs when it was used acetonitrile as a reaction solvent was dramatically higher thanwhen ethanol or methanol were used. Therefore,thissolventwasusedforfurtherexperiments.Figure2Ashowstheeffectof thesesolventsusingCDCdSe/ZnSQDs. The concentration of nCD was varied between 0.5 and 4.5 mM, maintaining constanttheotherparameters.ItwasobservedthatthecomplexationofTOPOwithn CDisessentialtoproducethesolubilizationofCdSe/ZnSQDsinanaqueousmedium. WhentheconcentrationofnCDistoolow,onlysmallportionsofsurfaceboundTOPO molecules on the surfaces of CdSe/ZnS QDs form complexes with nCD, which is not enoughtogiveahydrophilicpropertytothenanoparticles,andhencetoproducetheir stabilization in the aqueous phase. At relatively high concentrations of nCD, substantialamountsofTOPOmoleculesareabletoformhostguestcomplexeswithn 7
CD, which led to an increase in the stability of the QDs in the hydrophilic media. However,whenthedosageofnCDisveryhigh,althoughphasetransferwasefficiently achieved,theresultingcomplexwasfoundtobeunstableinwater,andthenCDexcess couldmaskthedeterminationoftheanalyte.Therefore,1.6mMofnCDwaschosen forfurtherexperiments,asthemaximumfluorescenceintensitywasobtainedatthis concentration.Finally,timedependentexperimentswereperformedbyexposingthe reactionofmodificationofQDsatseveraltimesbetween15and180mintoultrasonic irradiation.Thebestresultswereobtainedwhen45minofultrasonicirradiationwas used. The resulting nCDCdSe/ZnSQDs thus obtained were highly fluorescent and stable. Figure 2A shows the emission spectra of nCDCdSe/ZnSQDs in water and TOPOCdSe/ZnSQDs in chloroform. As it can be seen the maximum emission band around 590 nm (ૃexc = 450 nm) were obtained in all cases. The line width of the fluorescencespectrumisrelativelynarrow(withthefullwidthathalfmaximumof44 nm), indicating that the nCDCdSe/ZnSQDs nanoparticles have a narrow size distribution. Compared to TOPOCdSe/ZnS QDs in chloroform, nCDCdSe/ZnSQDs increased the fluorescence intensity. It was also observed no change in emission wavelength and the spectral width regarding to TOPOQDs. The UV/Vis spectrum of TOPOCdSe/ZnSQDs, CDCdSe/ZnSQDs, CDCdSe/ZnSQDs and CDCdSe/ZnS QDsarealsoillustratedinFigure2B.Asitcanbeseen,absorptionbandsatca.255and ca.585 nm were obtained in all cases. Therefore, no significant differences were observed in the modification procedure of QDs. The stability of obtained for nCD CdSe/ZnSQDs in water was estimated by measurementsof the emission intensity at roomtemperatureatseveraltimes.Fromtheresultsobtaineditwasconcludedthatn CDCdSe/ZnSQDswerestableatleastfortwoweeks,withanysignificantchangesin theirfluorescencespectra. 3.2. Effect of vanillin on the luminescence response of modified QDs In the ncyclodextrin modified QDs, the hydrophobic pockets of the cyclodextrin moleculesinteractwiththealiphaticchainsoftheTOPOpresentonthenanoparticle 8
surface from the QDs synthesis (Figure 1A). Nevertheless, the immobilized cyclodextrins retain their capability of engaging molecular recognition. In this way, it wasstudiedtheuseofnCDCdSe/ZnSQDsnanoparticlesasaselectiveluminescence sensors of vanillin. First, preliminary studies were made in order to know the best recognition of vanillin when , or CDCdSe/ZnSQDs were used. It was found that vanillinaffectsluminescenceofCDCdSe/ZnSQDsinamoredrasticway,producinga quenchingeffectontheQDsemissionband,asitcanbeseeninFigure3.Therefore, CDCdSe/ZnSQDswasselectedtodevelopthevanillinsensors. The selective hostguest interaction between vanillin and cyclodextrin, through the vanillin binds to the receptor sites, can act as an electron transfer quencher of luminescence of the particles (Figure 1B). Under these conditions the association of the vanillin to the CD cavities concentrates the analyte on the semiconductor QDs surface. The timedependent luminescence changes of the CDQDs upon their interactionwith4.2mgL1ofvanillinwereinvestigated.Fromtheresultsobtained,it can be concluded that luminescence of the CDQDs in the presence of vanillin decreased until 30 min, and remained constant after this time value. The same behaviorwasalsoobservedbyrecordingofabsorbancetimespectraofvanillinCD CdSe/ZnSQDscomplex. ThepHeffectintherecognitionofvanillinusingCDCdSe/ZnSQDsnanoparticles was studied by measurements of fluorescence intensity of the CDCdSe/ZnSQDs without(I0)andatgivenvanillinconcentration(I).ItwasfoundthatthepHsignificantly influenced the fluorescence intensity of the vanillinCDCdSe/ZnSQDs system. The maximumvalueofI/I0 wasobtainedwhenthepHwas8.0.Therefore,thisvaluewas chosenasoptimum.Asasecondpartofthisstudy,theinfluenceoftheconcentration ofbuffersolution,fixedwithNa2HPO4pH=8,wascarriedinordertoevaluatetheeffect ofthisparameterinthefluorescenceintensityofvanillinCDCdSe/ZnSQDssystem. The results showed that the maximum value I/I0 was obtained when the buffer solution concentration was 1.2 mM. Therefore, this value was chosen in all experiments.
9
According to the literature [29], the surface of the nCDCdSe/ZnSQDs affords a finite number of binding sites. Each of the binding sites could absorb one vanillin moleculefromthesolution.Therefore,accordingtoLangmuirequation[28]:
C I
§ 1 · § 1 · ¨ ¸¨ ¸C © BI max ¹ © I max ¹
[1]
whereC istheconcentrationofvanillinandIthe fluorescenceintensityobtainedfor thisconcentrationlevel.Accordingtotheliterature,iftheLangmuirdescriptionofthe bindingofvanillinonthesurfaceofCDCdSe/ZnSQDiscorrect,alinearplotofc/Ias a function of c must be obtained. In this case, a good linearity was observed throughout the entire range of vanillin concentration (2 to 20 mg L1). The binding constantBofCDCdSe/ZnSQDswithvanillinisfoundtobe0.99. 3.3. Analytical features for vanillin determination In order to develop an analytical method to determine vanillin in foods, several analytical performance characteristics were evaluated under the optimized experimental conditions. The quenching effect of the vanillin can be described using thefollowingSternVolmerequation: I0 I
1 K sv >Q @
[2]
where I0 and I are the fluorescence intensity of CDCdSe/ZnSQDs in absence and presenceofvanillin.AlinearrelationshipbetweenI0/Iandvanillinconcentrationinthe range of 220 mg L1 with a correlation coefficient of 0.9963 was obtained. Figure 4 shows the fluorescence spectra of CDCdSe/ZnSQDs at different concentrations of vanillinbetween2and20mgL1.Thecalibrationequationwas:
I0 I
1.094 0.051> vanillin @
[3]
The precision of the methodology was evaluated in terms of repeatability and reproducibility.Todeterminetherepeatabilityofthemethod,10analysisofsamples containing4.12mgL1ofvanillinwerecarriedoutandtheobtainedrelativestandard deviation (R.S.D.) was 1.2%. Then, the reproducibility was estimated for three 10
replicatesof4.12mgL1vanillinunderinterdayconditions(fortwoconsecutivedays), obtainingaR.S.D.of4.7%.Alimitofdetection(LOD)of0.99mgL1wasobtainedfor vanillin determination, based on the IUPAC method (blank signal plus 3 times its standard deviation). 10 measurements were used to obtain the LOD. According to theseresults,itcanbeconcludedthatthisapproachopensthepossibilitiestodevelop ananalyticalmethodusingCDCdSe/ZnSQDsforthedeterminationofvanillin. Inordertoapplythemethodtofoodsamplesaselectivitystudywasalsocarried out. For vanillin determination in food samples, the main interferences come from several colorant additives, such as curcumine or riboflavine. However, following the procedure reported in the Section 2.5, the interferences of these compounds were eliminated.Bycontrast,sucrose(glucoseandlactose)couldbepresentinthesample as principal interfering compound. For this purpose, two different level of concentrationwereusedincombinationwithvanillin.Fromtheresultsobtaineditcan concludedthatwhentheconcentrationofinterferenceswereincrease,notdifference wereobservedinthevanillinsignal,givenvaluesofR.S.Dof3.9and4.5%,respectively. Thecoexistingcompoundscausedarelativeerroroflessthan±5%inthefluorescence intensityofthevanillinCDCdSe/ZnSQDs.Thereforeitcanbeconsideredtohaveno interference with the detection of vanillin. The data revealed that the proposed methodmightbeappliedtothedetectionofvanillininfoodsamples. Ontheotherhand,severalsimilarstructurestovanillin,suchasvanillinalcohol,4 hydroxybenzaldehydeor4hydroxybenzylalcoholweretested.Forthispurpose,three different levels of concentration were used in combination with vanillin. From the resultsobtaineditcanbeconcludedthatwhentheconcentrationofinterferencewas increased,nodifferencewasobservedinthevanillinsignal,withlessof±5%ofR.S.D. when vanillin alcohol and 4hydroxybenzyl alcohol were used. However, the results obtainedinthepresenceof4hydroxybenzaldehydeindicatedthatthiscompoundcan produce interference in the vanillin determination. This fact demonstrated that the structureoftheanalyteandpHofsolutionsystemcouldplayanimportantroleinthe selectivity of vanillin determination with CDCdSe/ZnSQDs. Table 1 shows the obtained change of fluorescence intensity (%) at the three concentration levels studied. 11
4. Application To demonstrate the applicability of the proposed method, it was applied to determine vanillin in several commercial sugar and milk samples, purchased in different supermarkets. Each one of these samples was spiked with several concentrations of vanillin and were prepared according to the steps described in Section 2.5. The summary of these results are shown in Table 2. The obtained recoveriesindicatedanacceptableagreementbetweentheamountsaddedandthose foundforalltypesofsamples. Theproposedmethodwasalsousedforthequantitationofvanillinincustardwith vanilla flavor. These samples contained vanillin as flavor additive. This product was analyzed by triplicate, according the procedure described in Section 2.5. To evaluate thematrixeffect,thestandardadditionmethodwasalsousedforthedetermination ofvanillininthestudiedproduct.Theobtainedresultswere75.6±0.6and76.3±0.9 mgL1withandwithoutstandardaddition,respectively,correspondingtotheoriginal sample (samples were diluted before analyses). The application of Student statistical test for a confidence level of 95% demonstrated the statistical coincidence between theconcentrationfoundwiththosefoundbythestandardadditionmethod(n=6,tcrit =2.92>texp=0.48). 5. Conclusions In this work, an optical sensor for vanillin determination based on the selective supramolecularrecognitionofvanillinwithcyclodextrinmodifiedCdSe/ZnSQDswas developed. The procedure for the synthesis of nCDCdSe/ZnSQDs complex was simple and very effective. Different coating agents, such as , and CD, were studied, and the effect of several experimental parameters was optimized. The proposed methodology presents some advantages. Thus, the solubilization of TOPO CdSe/ZnSQD,initiallyinorganicmedia,wasallowedinaqueousmedia.Thisfactallows its compatibility with biological samples, and aqueous media in general, and in additiontheconservationinorganicmediaoverlongperiodsoftime.Inthisway,itis possible to modify only the necessary amount of QDs when required. On the other 12
hand,themethodologydemonstratedthatthesurfacecoatingofQDswithdifferentn cyclodextrinskeepstheemissionintensityofthequantumdotsandtheirdiameter.In addition, the immobilized cyclodextrins on the surface of the QDs retain their capabilityofengagingmolecularrecognition.Therefore,theuseofcyclodextrinfor QDs surface modification showed selectivity in vanillin recognition to alpha and gammacyclodextrins.Thus,thepotentialofthesensorfortheanalysisoffoodsamples was demonstrated, opening other possible alternatives for the selective fluorimetric sensingofothercompoundsthroughtheappropriatedmodificationofquantumdots surface. Acknowledgements ThisresearchwassupportedbyProjectCTQ201348411P(MINECO).GemaM.Durán thankstheSpanishMinistryofEconomyandCompetitivenessforaPredoctoralGrant.
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FIGURESCAPTION Figure1.SchematicillustrationofsurfacemodificationofTOPOCdSe/ZnSQDswithn cyclodextrins(A).HostguestinteractionbetweenCDCdSe/ZnSQDsandvanillin(B). Figure 2. Emission (A) and absorption (B) spectra of TOPOCdSe/ZnSQDs (a), CD CdSe/ZnSQDs(b),CDCdSe/ZnSQDs(c)andCDCdSe/ZnSQDs(d). Figure 3. Effect of 4.2 mg L1 vanillin concentration over luminescence of CD CdSe/ZnSQDs(A),CDCdSe/ZnSQDs(B)andCDCdSe/ZnSQDs(C). Figure4.FluorescencespectraofCDCdSe/ZnSQDswithdifferentconcentrationsof vanillinbetween2and20mgL1.
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HIGHLIGHTS CyclodextrinCdSe/ZnSquantumdotsweresynthesized. Compatibilitywithaqueousmedia. Thenewmaterialswereusedasselectivesensorforvanillin. cyclodextrinCdSe/ZnSwasusedforanalysisoffoodsamples.
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Table1.Effectofcoexistingforeignsubstancesatthreedifferentconcentrationlevels withrespecttovanillinconcentration.
Foreignsubstances
Sucrose Vanillinalcohol
4hydroxybenzylalcohol
4hydroxyaldehyde
Foreignspeciesratio
Errorinvanillin determination(%)
1:10 1:20 1:0.5 1:1 1:2 1:0.5 1:1 1:2 1:0.5 1:1 1:2
+1.9 +2.5 0.1 +0.3 +0.2 2.5 +0.4 +2.9 6.5 8.2 9.7
Conditions:pH8;[vanillin]=6.65μgmL1.
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Table2.Determinationofvanillininseveralfoodsamples(n=5). Sample
Added(mgL1)
Found(mgL1)
Recovery(%)
Sugar1
5 10 20
5.26±0.2 9.96 ±0.5 19.2±1
105±4 100±5 96±5
Sugar2
5 10 20
4.9±0.2 10.0 ±0.4 20.0±0.3
99±3 100 ±4 99±1.5
Sugar3
5 10 20
5.0 ±0.2 9.6±0.4 18.2±0.9
101 ±4 96±4 91±4.5
Milk1
5 10 20
5.2±0.2 9.0±0.4 20.0±1.0
104±4 90±4 99±5
Milk2
5 10 20
5.1±0.2 9.0±0.5 19.6±0.8
103±4 90±5 98±4
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Figure
Figure
Figure
Figure
*Graphical Abstract (for review)
GRAPHICAL ABSTRACT
