A sensitive and selective fluorescent sensor for Zinc(II) and its application to living cell imaging

ContentslistsavailableatScienceDirect

Sensors

and

Actuators

B:

Chemical

A

sensitive

and

selective

fluorescent

sensor

for

Zinc(II)

and

its

application

to

living

cell

imaging

Hao-Ming

Liu,

Parthiban

Venkatesan,

Shu-Pao

Wu

DepartmentofAppliedChemistry,NationalChiaoTungUniversity,Hsinchu300,Taiwan,RepublicofChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29May2014

Receivedinrevisedform10July2014 Accepted12July2014

Availableonline21July2014 Keywords: Sensors Zn(II) Fluorescence Imagingagents

a

b

s

t

r

a

c

t

Anewdipyrromethenederivative(DP1)exhibitsanenhancedfluorescenceinthepresenceofZn2+_ions

andahighselectivityforZn2+_{ionsovercompetingmetalionsinmethanol;Ag}+_,Ca2+_,Co2+_,Cr3+_,Fe2+_,Fe3+_,

Hg2+_,K+_,Mg2+_,Mn2+_,Ni2+_,andPb2+_{producedonlyminorchangesinthefluorescenceofDP1.Thebinding}

ratiooftheDP1–Zn2+_{complexeswasdeterminedfromaJobplottobe1:1.Thebindingconstant(K}

a)of

Zn2+_{bindingtoDP1wasfoundtobe6.12×10}4_M−1_{,withadetectionlimitof0.236␮M.Fluorescence}

microscopyimagingusingRAW264.7cellsshowedthatDP1couldbeusedasaneffectivefluorescent probefordetectingZn2+_{inlivingcells.}

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Thedevelopmentoffluorescentchemosensorsfordetecting bio-logicallyimportantmetalions,suchasFe3+_,Cu2+_,andZn2+_,has beenanimportantresearchtopic.Amongthetransitionmetalions, zincisthesecondmostabundantinthehumanbody,afteriron. Zincionsaremostlyboundwithinproteinsandplayvariouskey rolesinbiological systems,includingneuralsignaltransmission [1],enzymeregulation[2],apoptosis[3],andgenetranscription [4]. Disorders in Zn2+ _{metabolism have been linked to several} severeneurologicaldiseases,includingAlzheimer’sdisease(AD) [5,6],cerebralischemia[7],andepilepsy[8].Therefore,the mea-surementofZn2+_{isimportantinmonitoringbiologicalprocesses.}

Severalmethods forZn2+ _{detectionin various sampleshave} beendeveloped,suchasatomicabsorption-emissionspectroscopy [9], inductively coupled plasma-atomic emission spectrometry (ICP-AES) [10,11], andvoltammetry [12].Although these meth-ods providequantitative data, mostof them require expensive instruments,andarenotappropriatefordirectanalysis.Recently, moreattentionhasbeenfocusedonthedevelopmentof fluores-centprobesfordetectingZn2+_{ionsinbiologicalandenvironmental} samples[13–28].

Inthisstudy,afluorescentchemosensorDP1hasbeendesigned for metaliondetection. DP1consistsof a dippyrometheneand

∗ Correspondingauthor.Tel.:+88635712121x56506;fax:+88635723764. E-mailaddresses:[email protected],[email protected]

(S.-P.Wu).

a benzoimidazolemoiety(Scheme1).ThemetalionsAg+_,Ca2+_, Cd2+_,Co2+_,Cu2+_,Fe2+_,Fe3+_,Hg2+_,Mg2+_,Mn2+_,Ni2+_,andPb2+_were testedwithchemosensorDP1;Zn2+_{wastheonlyionthatcaused} strongfluorescentenhancementuponbindingwithchemosensor

DP1,producinga“turn-on”typechelation-enhancedfluorescence (CHEF)sensor.

2. Materialsandmethods

2.1. Materialsandinstrumentation

Allreagentswereobtainedfromcommercialsourcesandused as received without further purification. UV–vis spectra were recordedonanAgilent8453UV–visspectrometer.Fluorescence spectrawererecordedinaHitachiF-4500spectrometer.1_Hand13_C NMRspectrawererecordedonBrukerDRX-300NMR spectrome-terandVarianUnityInova500NMRspectrometer.Fluorescence imagingswereobtainedonaZEISSAxioScopeA1Fluorescence Microscope.

2.2. Synthesisof5-(pyren-1-yl)-4,6-dipyrromethane(1):

To a stirred solution of 1-pyrenecarboxyaldehyde (1.25g, 6.05mmol)in pyrrole(21mL,0.3mol)at23◦C underN2 atmo-sphere,trifluoroaceticacid(0.180mL,2.40mmol)wasadded.After 30minthereactionmixturewasevaporatedunderreduced pres-sure.Theresiduewasdissolvedindichloromethane(70mL)and washed with a 1.0M aqueous solution of NaOH (100mL). The aqueouslayerwasextractedwithdichloromethane(70mL)and http://dx.doi.org/10.1016/j.snb.2014.07.049

Scheme1. SynthesisofDP1.

thecombined organiclayers were concentrated under reduced pressure.Theresiduewaspurifiedbysilicagelchromatography (EtOAc/Hexane=1/6)togiveacompound1asawhitesolid(yield: 1.25g,60%).Meltingpoint:207–208◦C.1_HNMR(300MHz, DMSO-d6):ı10.74(s,2H),8.45(d,J=9.3Hz,1H),8.28(d,J=3.6Hz,1H), 8.26(d,J=3.3Hz,1H),8.22(d,J=8.4Hz,2H),8.16–8.13(m,2H), 8.07(t,J=7.6Hz,1H),7.69(d,J=7.8Hz,1H),6.66(d,J=1.2Hz,2H), 6.52(s,1H),5.92(d,J=2.7Hz,2H),5.62(s,2H).13_CNMR(75MHz, DMSO-d6):ı138.8,134.1,131.7,131.1,130.3,128.7,128.3,127.6, 127.0,126.9,126.0,125.7,125.6,124.9,124.8,124.3,117.8,107.9, 107.8,40.6.MS(EI):m/z(%)=346(100),278(82),HRMS(EI):calcd. forC25H18N2(M+)346.1470;found:346.1467.

2.3. Synthesisof1-formyl-5-(pyren-1-yl)-4,6-dipyrromethane (2):

Benzoylchloride(0.57mL,4.7mmol)wasaddedtoacooledDMF (2.0mL,26mmol)andstirred30min.Compound1(1.0g,2.9mmol) inDMFwereaddedinthereactionmixtureunderN2atmosphere. Themixturewasstirredat 0◦C for 2handthen another2hat roomtemperature.Thereactionmixturewasquenchedby addi-tionof Na2CO3 (2.0g) dissolved in 50% aquous EtOH (150mL). Theresulting solution wasextracted with CH2Cl2 (100mL×2). TheorganiclayerwasdriedoveranhydrousMgSO4 andthe sol-ventwasevaporatedunderreducedpressure.Thecrudeproduct waspurified bysilica gelchromatography (EtOAc/Hexane=1/6) togivea compound2 asagray solid(yield:0.61g,57%). Melt-ingpoint:177–178◦C.1_{HNMR(300MHz,CD} 3CN):ı9.39(s,1H), 8.39(d,J=9.6Hz,1H),8.28(d,J=6.3Hz,1H),8.25(d,J=5.1Hz,1H), 8.21(d,J=7.8Hz,1H),8.17(d,J=9.3Hz,1H),8.12(d,J=1Hz,2H), 8.06(t,J=7.5Hz,1H),7.69(d,J=8.1Hz,1H),6.96(dd, J=3.8Hz, J=2.4Hz, 1H), 6.73 (dd, J=4.2Hz, J=2.7Hz, 1H), 6.67 (s, 1H), 6.07 (dd, J=5.7Hz, J=2.7Hz, 1H), 5.98 (dd, J=3.6Hz, J=2.4Hz, 1H), 5.79 (s, 1H). 13_{C NMR (75MHz, CDCl} 3): ı 179.1, 143.2, 134.1,132.7,131.7,131.3,131.0,130.9,129.1,128.8,128.1,127.8, 126.6,126.6,126.0,125.8,125.5,125.4,125.1,122.9,122.7,118.3, 111.7,109.2,108.7,41.2. MS(EI):m/z(%)=374(98), 278(100), 201(41).HRMS(EI):calcd.forC26H18N2O(M+):374.1419;found: 374.1411.

2.4. Synthesisof1-(2-benzoimidazoyl)-5-(pyren-1-yl)-4, 6-dipyrromethane(3):

Compound 2 (150mg, 0.4mmol) and o-phenylenediamine (43mg,0.4mmol)werethoroughlymixedin5mlofEtOH.Sodium hydrogen sulfite (62mg, 0.6mmol) was added to mixture and stirred at 80◦C for 8h. The reaction mixture was cooled to room temperature, and concentrated at reduced pressure. The crude product was purified by column chromatography (ethyl acetate/hexane, 1:6) togivea red-orange solid. (Yield:130mg, 70%).Meltingpoint250–251◦C.1_{HNMR(300MHz,DMSO-d}

6):ı 12.4(s,1H),11.9(s,1H),10.8(s,1H),8.55(d,J=9.6Hz,1H),8.30 (d,J=8.7Hz,1H),8.28(d,J=6.0Hz,1H),8.26(d,J=1.5Hz,1H),8.23 (d,J=9.0Hz,1H),8.14–8.11(m,2H),8.08(t,J=7.8Hz,1H),7.77(d, J=8.1Hz,1H),7.49(d,J=3.9Hz,1H),7.40(d,J=3.9Hz,1H),7.12(d, J=2.4Hz,1H),7.09(d,J=2.4Hz,1H),6.77(t,J=3.0Hz,1H),6.71(s, 1H)6.70(d,J=1.8Hz,1H),5.95(dd,J=5.4Hz,J=3.0Hz,1H),5.81 (d,J=2.4Hz,1H),5.70(s,1H).13_{CNMR(75MHz,CDCl} 3):ı146.8, 143.8,138.1,135.1,134.0,132.0,131.6,130.9,130.7,128.6,127.9, 127.4,126.3,126.2,125.5,125.4,125.3,125.1,124.9,122.8,121.8, 121.5,121.2,117.7,117.3,114.4,110.5,110.4,108.6,108.2,40.7. MS(EI):m/z(%)=462(100),395(32),278(60),261(34).HRMS(EI): calcd.forC32H22N4(M+):462.1844;found:462.1847.

2.5. SynthesisofDP1

2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ; 110mg, 0.5mmol)dissolvedinTHF(5mL)wasaddedtoasolutionof com-pound3(130mg,0.28mmol)inTHF(10mL).Afterthesolution wasstirredfor30min,thereactionmixturewasevaporatedunder reduced pressure. The crude product was purified by column chromatography(ethylacetate/hexane,1:2)togivearedsolidDP1

(yield:87mg,67%).Meltingpoint:239–240◦C,1_HNMR(300MHz, CD3OD):ı8.37(d,J=7.5Hz,1H),8.33(d,J=7.5Hz,1H),8.24(d, J=6.9Hz,3H),8.10–8.06(m,2H),8.05–8.01(m,2H),7.71(m,2H), 7.59(s,1H),7.39(d,J=2.7Hz,1H),7.30(d,J=3.3Hz,1H),7.19(d, J=4.5Hz,1H),6.56(d,J=4.8Hz,1H),6.30(t,J=2.7Hz,1H),6.11 (d,J=3.3Hz,1H).13_{CNMR (125MHz,DMSO-d} 6):ı157.3,149.3, 147.9,144.2,140.6,135.2,134.7,133.4,132.1,131.5,131.1,130.9, 130.3,129.8,128.3,128.1,128.0,127.4,126.7,126.6,125.9,125.0,

124.1,124.0,123.7,123.6,122.9,122.1,119.6,115.8,112.6,111.8. MS(EI):m/z(%)=460(11),335(7.8),277(21).HRMS(EI):calcd. forC32H20N4(M+)460.1688;found:460.1673.

2.6. Determinationofthebindingstoichiometryandthe associationconstantsforthebindingofZn(II)toDP1

ThebindingstoichiometryofDP1–Zn2+_complexeswas deter-minedfromaJobplot.Thefluorescenceintensityat560nmwas plottedagainstthemolarfractionofDP1withatotal concentra-tionofthesensorandZn2+_{ionof80.0␮M.Themolarfractionat} maximumemissionintensityrepresentsthebinding stoichiome-tryoftheDP1–Zn2+_{complexes.Themaximumemissionintensity} wasreachedatamolarfractionof0.5.Thisresultindicatesthat chemosensorDP1formsa1:1complexwithHg2+_.Theapparent associationconstant(Ka)ofDP1–Zn2+complexeswasdetermined bytheBenesi–HildebrandEquation(1)[29].

1 (F−F0)= 1 {Ka×(Fmax−F0)×[Zn2+]} +_(F 1 max−F0) (1)

whereFisthefluorescenceintensityat560nmatanygivenZn2+ concentration,F0 is thefluorescenceintensityat 560nm inthe absenceofZn2+_,andF

maxisthemaximafluorescenceintensityat 560nminthepresenceofZn2+_{insolution.Theassociationconstant} Kawasevaluatedgraphicallybyplotting1/(F−F0)against1/[Zn2+]. DatawerelinearlyfittedaccordingtoEquation(1)andtheKavalue wasobtainedfromtheslopeandinterceptoftheline.

2.7. Cellculture

ThecelllineRAW264.7cellswasprovidedbytheFood Indus-tryResearchandDevelopmentInstitute(Taiwan).RAW264.7cells weregrown in H-DMEM(Dulbecco’sModified Eagle’s Medium, highglucose)supplementedwith10%FBS(FetalBovineSerum) inanatmosphereof5%CO2at37◦C.

2.8. Fluorescenceimaging

ThecellsculturedinDMEMweretreatedwith10mMsolutions ofZn2+_{(2␮L;finalconcentration:20␮M)dissolvedinsterilized} PBS(pH7.4)andincubatedat37◦Cfor30min.Thetreatedcells werewashedwithPBS(2mL×3)toremoveremainingmetalions. DMEM(2mL)wasaddedtothecellculture,whichwasthentreated witha10mMsolutionofchemosensorDP1(2␮L;final concen-tration:20_␮M)dissolvedinDMSO.Thesampleswereincubated at37◦C for30min.The culturemediumwasremoved,and the treatedcellswerewashedwithPBS(2mL×3)beforeobservation. FluorescenceimagingwasperformedwithaZEISSAxioScopeA1 FluorescenceMicroscope.Thecellswereexcitedwithawhitelight laserat480nm,andemissionwascollectedat535±25nm.

2.9. Computationalmethods

Quantumchemicalcalculationsbasedondensityfunctional the-ory (DFT) were carried out using a Gaussian 09 program. The ground-statestructuresofDP1andtheDP1–Zn2+_{complexeswere} computedusingthedensityfunctionaltheory(DFT)methodwith thehybrid-generalizedgradientapproximation(HGGA)functional B3LYP.The6-31Gbasissetwasassignedtononmetalelements(C, H,andN).FortheDP1–Zn2+_{complex,theLANL2DZbasissetwas} usedforZn2+_{,whereasthe6-31Gbasissetwasusedforotheratoms.}

3. Resultanddiscussion

3.1. SynthesisofDP1

The synthesis ofchemosensor DP1 is outlinedin Scheme1. Compound 1 was prepared by the condensation of 1-pyrenecarboxaldehyde and pyrrole. Compound 2 was obtained by the Vilsmeier–Haack reaction using benzoyl chloride and DMFtoformmonoformylateddipyrromethane.Inthenextstep, condensation ofo-phenylenediamine withcompound 2yielded

Fig.1. (Top)PhotographicimagesofDP1inthepresenceofvariousmetalions.(Bottom)FluorescencechangeofDP1(20␮M)uponadditionofvariousmetalions(20␮M) inmethanol.

compound3.Furtheroxidationofcompound 3usingDDQgave theproductDP1.

3.2. MetalionsensingabilityofDP1

ThesensingabilityofDP1wastestedbymixingitwiththemetal ionsCa2+_,Cd2+_,Co2+_,Cu2+_,Fe2+_,Hg2+_,Mg2+_,Mn2+_,Ni2+_,andZn2+_.

Fig.1showstheeffectofthemetalionsontheappearanceofDP1in solution.Zn2+_{wastheonlyionthatcausedayellow-greenemission} bandat560nm.Theothermetalionsdidnotproduceagreatchange intheemissionspectra.DuringZn2+_{titrationwithchemosensor}

DP1,anewemissionbandcenteredat560nmwasformed(Fig.2). AftertheadditionofmorethanoneequivalentofZn2+_,the emis-sionintensityreachedamaximum,withaquantumyield˚=0.103, whichis20timeshigherthanthatofDP1,at˚=0.005.Forthe UV–visabsorptionspectra,theabsorbance at477nmdecreased inintensity,and anewbandcenteredat540nmappeared dur-ingZn2+_{titrationwithDP1(Fig.2).Thecolorchangefromyellow} topurpleclearlyindicatesthe63nmblueshift.Theseobservations suggestthatZn2+_{istheonlymetalioncausingsignificant} fluores-cenceenhancement,therebypermittinghighlyselectivedetection ofZn2+_.

TostudytheinfluenceofothermetalionsonZn2+_bindingwith chemosensorDP1,weperformedcompetitiveexperimentswith othermetalions(20␮M)inthepresenceofZn2+_{(20␮M)(Fig.3).} ThefluorescenceenhancmentcausedbythemixtureofZn2+_with mostothermetalionswassimilartothatcausedbyZn2+_alone.A smallerfluorescenceenhancementwasobservedwhenZn2+_was

Fig.2.Absorption(Top)andfluorescence(Bottom)changesofchemosensorDP1

(20␮M)inthepresenceofvariousequivalentsofZn2+_{inmethanol.Theexcitation}

wavelengthwas535nm. 0 500 1000 DP1+Metal ions

Intensity (560 nm)

Fig.3. Fluorescence response(560nm)ofchemosensor DP1(20␮M)to Zn2+

(20␮M)or20␮Mofothermetalions(theblackbarportion)andtothemixture ofothermetalions(20␮M)with20␮MofZn2+_{(thegraybarportion).}

1.0 0.8 0.6 0.4 0.2 0.0 0 100 200 300 400

Fig.4.JobplotoftheDP1–Zn2+_{complexinmethanol.Thetotalconcentrationof}

CBSandZn2+_{was80␮M.Themonitoredwavelengthwas560nm.}

mixedwithCo2+_orHg2+_{.Fluorescencequenchingwasobserved} whenZn2+_{wasmixedwithCu}2+_{.ThisindicatesthatCo}2+_,Hg2+_, andCu2+_{cancompetewithZn}2+_{forbindingwithDP1.Mostofthe} othermetalionsdonotinterferewiththebindingofDP1withZn2+_.

Fig.5. Benesi–HildebrandplotofDP1withZn2+_{inmethanol.Theexcitation}

wave-lengthwas535nmandobservedwavelengthwas560nm.Thebindingconstantwas 6.12×104_M−1_forZn2+_{bindinginDP1.}

Fig.6.1_{HNMRspectraofDP1(5mM)upontitrationwith(a)0equiv,(b)0.2equiv,(c)1equivofZn}2+_inDMSO-d 6.

Thisindicatesthattheothermetalionsdonotinterferesignificantly withthebindingofchemosensorDP1withZn2+_.

In order to understand the binding stoichiometry of the

DP1–Zn2+_{complexes,Jobplotexperimentswerecarried out.In}

Fig.4,theemissionintensityat560nmisplottedagainstthemolar fractionofDP1underaconstanttotalconcentrationofDP1and Zn2+_{.Maximum fluorescent enhancement occurredat theratio} 0.5.Thisresultindicatesa 1:1ratioforDP1–Zn2+_complexes,in whichoneZn2+_{ionbindstoonechemosensorDP1.Inaddition,the} formationof1:1DP1–Zn2+_{complexeswasalsoconfirmedusing} ESI-MS,inwhichthepeakatm/z541.1indicatesa1:1 stoichiome-tryforDP1–Zn2+_{complexes(seeFig.S9inthesupplementarydata).} TheassociationconstantKawasevaluatedgraphicallybyplotting 1/(F−F0)against1/[Zn2+](Fig.5).Thedatawerelinearlyfitandthe

Fig.7.DFT-optimizedstructuresofDP1–Zn2+_{complexes.Blueatom,N;redatom,}

O;grayatom,Zn2+_{.(Forinterpretationofthereferencestocolorinthisfigurelegend,}

thereaderisreferredtothewebversionofthearticle.)

Kavaluewasobtainedfromtheslopeandinterceptoftheline.The associationconstant(Ka)ofZn2+bindingtochemosensorDP1was foundtobe6.12×104_M−1_{.ThedetectionlimitofchemosensorDP1} asafluorescentsensorforthedetectionofZn2+_{wasdetermined} fromaplotoffluorescenceintensityasafunctionofthe concen-trationofZn2+_{.ItwasfoundthatchemosensorDP1hasadetection} limitof0.236␮M(seeFig.S10inthesupplementarydata),which allowsforthedetectionofZn2+_{inthemicromolarconcentration} range.

TogainaclearerunderstandingofthestructureofDP1–Zn2+ complexes,1_{H NMRspectroscopy(Fig.6)wasemployed.In the} 1_{HNMRspectraofDP1,theprotonsignalsat13.4and13.0ppm} decreaseduponadditionofZn2+_{.ThisindicatesthatZn}2+_binding occursmainlyatthenitrogenatomsinpyrroleandbenzoimidazole.

2

4

6

8

10

12

0

400

800

Int

ensi

ty

(560

nm

)

pH

Fig.8. Fluorescenceresponse(560nm)offreeDP1(20␮M)andafteradditionof Zn2+_{(20␮M)inmethanol–water(v/v=9:1,10mMbuffer,pH2–4:sodium}

cit-rate/citraticacid;pH4.5–6:MES;pH6.5–8.5:Hepes;pH9–12:Tris–HCl)solution asafunctionofdifferentpHvalues.Theexcitationwavelengthwas530nm.

Fig.9. FluorescenceimagesofRAW264.7cellstreatedwithDP1andZn2+_{.(Left)Brightfieldimage;(middle)fluorescenceimage;(right)mergedimage.}

TheseobservationsrevealthatZn2+_{bindingwithDP1isthrough} twonitrogensatpyrrole,andonenitrogenatbenzoimidazole.

To elucidate the structures of the DP1–Zn2+ _complexes, density functional theory (DFT) calculations were undertaken using the Gaussian 09 software package. The DP1–Zn2+ com-plexesweresubjectedtoenergyoptimizationusingB3LYP/6-31G and B3LYP/LANL2DZ. The global minima structure for the

DP1–Zn2+_{complexesisshowninFig.7.ThedistancesofZn}2+_from thethreenitrogenatomsareabout2.0and2.1 ˚A.

WeperformedpHtitrationofchemosensor1todeterminea suitablepHrangeforZn2+_{sensing.InFig.8,theemission} intensi-tiesofmetal-freechemosensor1atmostpHvaluesarelow.After mixingchemosensor1withZn2+_{,theemissionintensityat560nm} suddenlyincreasedinthepHrangeof5.0–10.0.WhenthepHwas lowerthan5,theemissionintensityat560nmdecreasedslightly, comparedtothatatpH7.0.Thisisduetoprotonationonthe nitro-genatom,preventingtheformationoftheCu2+_–1complex.

3.3. Livingcellimaging

ThepotentialofDP1forimagingZn2+_{inlivingcellswasthen} investigated.First,imagesofcellswereobtainedusinga fluores-cencemicroscope.WhenRAW264.7cellswereincubatedwithDP1

(10␮M),nofluorescencewasobserved(Fig.9a).Aftertreatment withZn2+_{,brightgreenfluorescencewasobservedintheRAW264.7} cells(Fig.9b).Anoverlayofthefluorescenceandbright-fieldimages showsthatthefluorescencesignalsarelocalizedinthe intracel-lulararea,indicatingasubcellulardistributionofZn2+_andgood cell-membranepermeabilityofDP1.

4. Conclusion

Inconclusion,wedevelopedafluorescentchemosensorforZn2+ detection.Weobservedsignificantfluorescenceenhancementin thepresenceofZn2+_{.However,othermetalions,suchasAg}+_,Ca2+_, Cd2+_,Co2+_,Cu2+_,Fe2+_,Fe3+_,K+_,Mg2+_,Mn2+_,Ni2+_,andPb2+_,barely affectedthefluorescence.Inaddition,thischemosensorDP1serves asaneffectiveprobeforZn2+_{detectioninlivingcells.}

Acknowledgments

WegratefullyacknowledgethefinancialsupportofMinistryof ScienceandTechnology(Taiwan,101-2113-M-009-016-MY2)and NationalChiaoTungUniversity.

AppendixA. Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.snb.2014.07.049.

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Biographies

Hao-MingLiuhadMSin2012,DepartmentofAppliedChemistryatNationalChiao TungUniversity.

ParthibanVenkatesanisstudyingforPh.D.intheDepartmentofAppliedChemistry atNationalChiaoTungUniversity.

Dr.Shu-PaoWuhadPh.D.in2004,DepartmentofChemistry,TheOhioState Uni-versity,USA.Currently,heisworkingasanAssociateProfessorinDepartmentof AppliedChemistryNationalChiaoTungUniversity,Taiwan,RepublicofChina. Cur-rentinterests:metalionchemosensorsandAlkB.