A pyrene-based highly selective turn-on fluorescent sensor for copper(II) ions and its application in living cell imaging

ContentslistsavailableatSciVerseScienceDirect

Sensors

and

Actuators

B:

Chemical

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

A

pyrene-based

highly

selective

turn-on

fluorescent

sensor

for

copper(II)

ions

and

its

application

in

living

cell

imaging

Hsuan-Fu

Wang,

Shu-Pao

Wu

DepartmentofAppliedChemistry,NationalChiaoTungUniversity,Hsinchu300,Taiwan,ROC

a

r

t

i

c

l

e

i

n

f

o

Received29October2012 Receivedinrevisedform

24December2012

Accepted24January2013 Available online 4 February 2013 Keywords: Sensors Copper Pyrene Imagingagents

a

b

s

t

r

a

c

t

Anewpyrenederivative(1)containingabenzothiazolenhydrazonemoietyexhibitedhighselectivityfor Cu2+_{detection.InthepresenceofCu}2+_{,chemosensor1providedsignificantfluorescenceenhancement,}

whileAg+_,Ca2+_,Cd2+_,Co2+_,Fe2+_,Fe3+_,Hg2+_,K+_,Mg2+_,Mn2+_,Ni2+_,Pb2+_,andZn2+_{metalionsproducedonly}

minorchangesinfluorescenceintensity.Theassociationconstant(Ka)forCu2+bindingto1hadavalueof

5.00×108_M−2_{.ThemaximumfluorescenceenhancementinducedbyCu}2+_{bindingtothechemosensor}

wasobservedovertherangepH2–8.5.Ourfluorescencemicroscopyexperimentsdemonstratethat chemosensor1mayhaveapplicationasafluorescentprobefordetectingCu2+_{inlivingcells.}

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Thedevelopmentoffluorescentchemosensorsthatarecapable ofdetectingbiologicallyorenvironmentallyimportantmetalions suchasCd2+_,Cu2+_,Fe3+_,Hg2+_,Pb2+_,andZn2+_{hasbeenanimportant} researchtopic[1–6].Copperisthethirdmostabundantessential transitionmetalion inthehumanbody [7].Many proteinsuse copperionsasacofactorforelectrontransport,orasacatalystin oxido-reductionreactions.Copperdistributioninthehumanbody ishighlycontrolled,becauseofitscellulartoxicity.Anexcessof copperionsinlivingcellscancatalyzetheproductionofreactive oxygenspecies(ROS),whichthatcandamagelipids,nucleicacids, andproteins.Severalseriousdiseases,includingAlzheimer’s dis-ease[8],Indianchildhoodcirrhosis (ICC)[9],priondisease[10], andMenkesandWilsondiseases[11],havebeenassociatedwith thecellulartoxicityofcopperions.Duetoitsextensiveapplications inourdailylives,copperisalsoacommonmetalpollutant.Thelimit forcopperindrinkingwater,assetbytheUSEnvironmental Pro-tectionAgency(EPA)is1.3ppm,whichisamolarconcentrationof roughly20␮M.

Thedevelopmentoffluorescentchemosensorsforthedetection ofCu2+_{ionshasattractedmuchattention[12–27].Cu}2+_isa fluo-rescencequencher,andsomostfluorescentchemosensorsdetect Cu2+_{byfluorescencequenchingprocesses,whichinvolvecharge}

∗ Correspondingauthor.Tel.:+88635712121x56506.

E-mailaddresses:[email protected],[email protected]

(S.-P.Wu).

orenergytransfermechanisms[15].However,suchprocessesoffer poorsensitivityformetaliondetection;fluorescenceenhancement ismoreeasilymonitoredthanfluorescencequenching.Thispaper reportsonanewlydesignedpyrene-basedfluorescence enhance-mentchemosensorforCu2+_{thatisbasedonphotoinducedelectron} transfer (PET). The binding of Cu2+ _{tothe chemosensor blocks} thePETmechanismandgreatlyenhancesthefluorescenceofthe pyrenemoiety.

In this study, we designed a pyrene-based fluorescent chemosensor, 1, for metal ion detection. The chemosensor comprisestwoparts,apyrenemoietyasareporter,anda benzoth-iazolenhydrazonecomponentasthemetalionchelator(Scheme1). Chemosensor 1 exhibitsweak fluorescencedue tofluorescence quenchingbyphoto-inducedelectrontransferfromnitrogenlone pairsonto pyrene.Thebindingof ametalion thechemosensor blocksthePETmechanism,resultinginsignificantenhancementin pyrenefluorescence.SelectivitytestingrevealedthatCu2+_causes avisiblecolorchangein1,fromlightyelloworcolorless,anda blueemissiononligationto1;noothertestedionsproducedany significantcolorchange.

2. Materialsandmethods

2.1. Materialsandinstrumentations

All solvents and reagents were obtained from commercial sourcesandusedasreceivedwithoutfurtherpurification.UV/vis spectrawererecordedonanAgilent8453UV/visspectrometer.IR datawereobtainedonBomemDA8.3Fourier-TransformInfrared

0925-4005/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.

2.2. Synthesisofchemosensor1

1-Pyrenecarboxaldehyde (230mg, 1.0mmol) and 2-benzothiazolenhydrazone (182mg, 1.1mmol) were added to a 10mL ethanol solution. The reactionmixture was stirred for 12hatroomtemperature.Theresultingprecipitatewascollected byfiltrationandthenpurifiedbycolumnchromatography(ethyl acetate:hexane=1:1) to give 1 as a bright yellow solid. Yield: 336mg,89%;m.p.265.1–267.5◦C;MS(FAB)Found,378.1[M+H]+_; HRMS(EI)CalcdforC24H15N3S1,377.0987;Found,377.0984.1H NMR(300MHz,DMSO-d6)ı12.45(b,1H),9.15(s,1H),8.87(d, J=9.3Hz, 1H),8.53(d,J=8.1Hz,1H),8.38–8.35(m,4H),8.27(d, J=9.0Hz,1H),8.22(d,J=9.0Hz,1H),8.13(t,J=7.5Hz,1H),7.83(d, J=7.8Hz, 1H),7.49(d,J=7.5Hz, 1H),7.34(t,J=7.2Hz, 1H),7.15 (t,J=7.2Hz,1H); 13_{CNMR (125MHz, DMSO-d} 6)ı167.1,131.5, 130.9,130.7,130.2,128.7,128.2,128.1,127.4,127.3,127.1,126.6, 126.5,126.0,125.8,125.7,125.3,125.1,124.3,123.9,123.8,123.7, 122.7,121.7; FTIR(cm−1)3183,3036, 2873,2809, 1625,1578, 1445,1383,1272,1135,840,743,710;

2.3. MetalionbindingstudybyUV–visandfluorescence spectroscopy

Chemosensor1(50␮M)wasaddedwithdifferentmetalions (25␮M).Allspectraweremeasuredin1.0mLacetonitrile–water solution(v/v,3:1,5mM,HEPESbuffer,pH7.0).Thelightpathlength ofcurvetwas1.0cm.

2.4. ThepHdependenceonCu2+_{bindinginchemosensor1} studiedbyfluorescencespectroscopy

Chemosensor 1 (50␮M) was added with Cu2+ _{(25␮M) in} 1.0mL acetonitrile–water solution (v/v, 3:1, 5mM buffer). The bufferswere:pH1–2,KCl/HCl;pH3–4,CH3COOH/NaOH;pH4.5–6, MES/NaOH;pH6.5–9,HEPES;pH10–12,Tris/NaOH.

2.5. Determinationofthebindingstochiometryandtheapparent associationconstantsKaofCu(II)bindinginchemosensor1

The binding stochiometry of 1–Cu2+ _{complexes was} deter-minedbyJobplotexperiments[28].Thefluorescenceintensityat 468nmwasplottedagainstmolarfractionof1underaconstant totalconcentration.Theconcentrationofthecomplexapproached amaximum intensitywhen themolarfractionwas0.65. These resultsindicatethatchemosensor1formsa2:1complexwithCu2+_. Theapparentassociationconstants(Ka)of1–Cu2+complexeswas determinedbytheconsequentEq.(1)[29]:

˛2 1−˛ =

1

2KaCF[M], (1)

totalconcentrationofchemosensor1.Thevalue“˛”wasobtained usingEq.(2)

˛=





, (2)

whereFisthefluorescenceintensityat468nmatanygivenCu2+ concentration,F1 is thefluorescenceintensityat 468nm inthe absenceofCu2+_,F

0isthemaximafluorescenceintensityat468nm inthepresenceofCu2+_{.TheassociationconstantK}

a was evalu-atedgraphicallybyplotting˛2_{/(1−˛)against1/[Cu}2+_{]. Theplot} ˛2_{/(1−˛)vs.1/[Cu}2+_{]isshowninFig.6.Datawerelinearlyfitted} accordingtoEq.(1)andtheKavaluewasobtainedfromtheslope oftheline.

2.6. Cellculture

ThecelllineRAW264.7macrophagewasprovidedbytheFood IndustryResearchandDevelopmentInstitute(Taiwan).RAW264.7 cellswereculturedinDulbecco’smodifiedEagle’smedium(DMEM) supplementedwith10%fetalbovineserum(FBS)at37◦Cunderan atmosphereof5%CO2.Cellswereplatedon18mmglasscoverslips andallowedtoadherefor24h.

2.7. Fluorescenceimaging

Experiments to assess the Cu2+ _{uptake were performed in} phosphate-bufferedsaline(PBS) with20␮MCu(BF4)2. Thecells culturedinDMEMweretreatedwith10mMsolutionsofCu(BF4)2 (2␮L; final concentration: 20␮M) dissolved in sterilized PBS (pH=7.4) and incubated at 37◦C for 30min. The treated cells werewashedwithPBS(2mL)threetimestoremoveremaining metalions.DMEM(2mL)wasaddedtothecellculture,whichwas thentreatedwitha10mMsolutionofchemosensor1(2␮L;final concentration:20␮M)dissolvedinDMSO.Thesampleswere incu-batedat37◦Cfor30min.Theculturemediumwasremoved,andthe treatedcellswerewashedwithPBS(2mL)threetimesbefore obser-vation.Fluorescence imaging wasperformed witha ZEISSAxio ScopeA1fluorescencemicroscope.Cellsloadedwith chemosen-sor1wereexcitedat350nmbyusinga50WHglamp.Anemission filterof420nmwasused.

2.8. Quantumchemicalcalculation

Quantumchemicalcalculationsbasedondensityfunctional the-ory(DFT)werecarriedoutusingaGaussian09program.Ground stategeometryoptimizationof1wasperformedusingtheB3LYP functionalandthe6-31Gbasisset.Groundstategeometry opti-mizationof 1–Cu2+ _{complexes wasperformed usingthe B3LYP} functionalandtheLANL2DZbasisset.

Fig.1.Color(a)andfluorescence(b)changesofchemosensor1(500␮M)afteradditionofvariousmetalions(500␮M)inacetonitrile–water(v/v=3/1,5mMHEPES,pH7.0) solutions.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

3. Resultsanddiscussion

3.1. Characterizationofchemosensor1

Chemosensor 1 was synthesized by reaction of 2-benzothiazolenhydrazone and 1-pyrenecarboxaldehyde toform an imine bond between benzothiazolenhydrazone and pyrene (Scheme1).Chemosensor1isyellowandhasanabsorptionband centered at 385 nm, which is red-shifted by 50nm from the typicalpyreneabsorptionbandat335nm[30,31].Thisisdueto longerconjugateddoublebondsinchemosensor1.Chemosensor 1 exhibits weak fluorescence (˚=0.013) compared to pyrene (˚=0.6–0.9). Thisresultsfromfluorescence quenching by pho-toinducedelectrontransferfromelectronlonepairsonnitrogen ontopyrene.

3.2. Cation-sensingproperties

WetestedtheselectivityofChemosensor1bymixingitwith Ag+_,Ca2+_,Cd2+_,Co2+_,Cu2+_,Fe2+_,Fe3+_,Hg2+_,K+_,Mg2+_,Mn2+_,Ni2+_, Pb2+_,andZn2+_metalions.Cu2+_{causedavisiblecolorchangein} 1,fromlightyelloworcolorless,andhadablueemission(Fig.1). DuringCu2+_{titrationwith1,theabsorbanceat385nmdecreases} inintensity,andanewbandcenteredat330nmappears.Thecolor changefromlightyellowtocolorless(Fig.2)clearlyindicatesthe 55-nmblueshift.Thenewbandat330nmisclosetothe absorp-tionbandofpyrene,335nm.ThisobservationsuggeststhatCu2+

300 350 400 450 500 550 0.0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 0.16 0.20 0.24 0.28 0.32 385 nm 330 nm Abs o rb anc e [Cu2+] / [1] 330 nm 385 nm Absorbance Wavelength (nm)

Fig.2.Changesintheabsorptionofchemosensor1(50␮M)inthepresenceof variousequivalentsofCu2+_{inacetonitrile–water(v/v=3/1,5mMHEPES,pH7.0)} solutions. 450 500 550 600 650 700 0 300 600 900 1200 1500 1800 0 2 4 6 8 10 0 400 800 1200 1600 In tensi ty (468 nm ) [Cu2+] / [1] 0 eq. 0.5 eq. 468 nm Intensity Wavelength (nm)

Fig.3. Fluorescenceresponseofchemosensor1(50␮M)tovariousmolar equiv-alentsofCu2+_{inacetonitrile–water(v/v=3/1,5mMHEPES,pH7.0)solutions.The} excitationwavelengthwas385nm.

bindingwithchemosensor1blocksconjugationbetweenthe dou-blebonds,resultinginashorterabsorptionwavelength.

Tofurtherevaluatetheselectivityofchemosensor1,we inves-tigatedthefluorescencespectraofchemosensor1inthepresence ofvarioustransitionmetalions.OnlytreatmentwithCu2+_resulted inasignificantblueemission(Fig.1).DuringCu2+_{titrationwith} chemosensor1,anewemissionbandcenteredat468nmformed (Fig.3).Afteradding0.5molarequivalentsofCu2+_,theemission

Cu(II)Ag(I)Ca(II)Cd(II)Co(II)Fe(II)Fe(III)Hg(II) K(I) Mg(II)Mn(II)Ni(II) Pb(II)Zn(II) 0 400 800 1200 1600 Intensity (468 nm)

Fig.4.Fluorescenceresponseofchemosensor1(25␮M)toCu2+_{(15␮M)orofother} metalions(15␮M)(blackbar),andtoamixtureofothermetalions(15␮M)with 15␮MofCu2+_{(graybars)inacetonitrile–water(v/v=3/1,5mMHEPES,pH7.0)} solutions.

0.0 0.2 0.4 0.6 0.8 1.0 0

X = { [ _{1] / (} [_{1] + [Cu}2+] ) }

Fig.5.Job plotoftheCu2+_{–1complexesinacetonitrile–water(v/v=3/1,5mM} HEPES,pH7.0)solutions.Thetotalconcentrationof1andCu2+_{was50.0␮M.The}

monitoredwavelengthwas468nm.

intensityreachedamaximum.Thequantumyieldofthenew emis-sionbandwas0.49,whichis38-foldthatofchemosensor1at0.013. Cu2+_{wastheonlymetalionofthosewetestedthatreadilybinds} withchemosensor1toyieldasignificantfluorescence enhance-ment,suggestingapplicationforthehighlyselectivedetectionof Cu2+_ion.

TostudytheinfluenceofothermetalionsonCu2+_bindingwith chemosensor1,weperformedcompetitiveexperimentswithother metalions(15.0␮M)inthepresenceofCu2+_{(15.0␮M)(Fig.4).} TheobservedfluorescenceenhancementformixturesofCu2+_with mostmetalionswassimilartothatseenforCu2+_{alone.Reduced} fluorescenceenhancementwasonlyobservedformixturesofCu2+ andCo2+_orFe2+_{,indicatingthatCo}2+_andFe2+_{competewithCu}2+ forbindingwithchemosensor1.Noothermetalionsappearedto interferewiththefluorescenceofthechemosensor1andCu2+_.

Tounderstandthebindingstoichiometryof1–Cu2+_complexes, weconductedJobplotexperiments.InFig.5,theemission inten-sityat468nmisplottedagainstthemolarfractionofchemosensor 1ataconstanttotalconcentrationof50.0␮M.Maximum emis-sionintensitywasreachedforamolarfractionof0.65,indicating that complexes comprise 1 and Cu2+ _{in a 2:1 ratio; one Cu}2+

4.0x104 8.0x104 1.2x105 1.6x105 2.0x105 0 1 2 3 2 /(1-) Y = 1.998 10-5 _{X - 0.959} R = 0.996 K_a = 5.00 108 M-2 1 / [Cu2+]

Fig.6. Abindingcurvewasgeneratedfromanalysisoffluorescenceenhancement measurements,andfittedinaccordancewithEq.(1).Theassociationconstant,Ka, forCu2+_{inchemosensor1was5.0×10}8_M−2_.

Fig.7. 1_{HNMR(300MHz)spectraof1inthepresenceofCu}2+_inDMSO-d 6.

Fig.8. DFT-optimizedstructuresof(a)1and(b)Cu2+_–1

2complexescalculatedusing theB3LYP/LanL2DZmethod(yellowatom,S;blueatom,N;pinkatom,Cu).(For interpretationofthereferencestocolorinthisfigurelegend,thereaderisreferred tothewebversionofthearticle.)

ionbinds withtwo chemosensor1molecules.Theformationof aCu2+_–1

2 complexwasconfirmedbyESI-MS,inwhichthepeak atm/z=815.1indicatesa 2:1stoichiometryforthe[Cu2+12–H]+ complex (Fig. S3 in the supplementary data). The association constant,Ka,wasevaluatedgraphicallybyplotting˛2/(1−˛)vs.

1 2 3 4 5 6 7 8 9 10 11 12 0 800 1600 2400 3200 Intensity (468 nm ) pH 1 + Cu( II ) 1

Fig.9. Fluorescenceresponse(468nm)offreechemosensor1(50.0␮M)()and afteradditionofCu2+_{(25.0␮M)()inCH}

3CN/H2O(v/v=3/1,5mMbuffer)solutions asafunctionofvariouspHvalues.Theexcitationwavelengthwas385nm.

Fig.10.FluorescenceimagesofRAW264.7cellstreatedwith1andCu2+_{.(Left)Brightfieldimage;(center)fluorescenceimage;and(right)overlaidimage.}

1/[Cu2+_{],where˛isdefinedas[F−F}

0]/[F1−F0](Fig.6).Thedata waslinearlyfitted,andtheKavaluewasdeterminedfromtheslope andinterceptoftheline.KaforCu2+bindinginchemosensor1was foundtobe5.0_×108_M−2_{.Thelimit ofdetectionfor} chemosen-sor1asafluorescentsensorforCu2+_{detectionwasdetermined} fromaplotof fluorescenceintensityasa functionofCu2+ con-centration(Fig.S4inthesupplementarydata).Itwasfoundthat chemosensor1hasalimitofdetectionof2.73␮M,whichis rea-sonableforthedetectionofmicromolarconcentrationsofCu2+_ion, TogainaclearerunderstandingofthestructureofCu2+_–1

2 com-plexes,weused1_{HNMRspectroscopy.Cu}2+_{isaparamagneticion} whichaffects theNMR resonancefrequencyof protonsthatare closetotheCu2+_{bindingsite.The}1_{HNMRspectraofchemosensor} 1revealedthatanamineproton(NH)signalat12.4ppmalmost completelydisappearsupontheadditionofCu2+_(Fig.7).The pro-ton(Hf)signalat9.15ppmbecamebroaderafterbindingwithCu2+, indicatingthatCu2+_{bindstonitrogenataniminebond.Otherpeaks} remainedunchanged.TheseobservationsindicatethatCu2+_binds tochemosensor1throughtwonitrogenatoms,oneofwhichisan imine.

ToelucidatethestructureoftheCu2+_–1

2complex,weemployed density functionaltheory(DFT) calculations usingthe Gaussian 09 software package. Because of the 2:1 ligand-to-metal com-plexes, as determined from the mass spectrum and Job plot measurements,weappliedtheB3LYP/LANL2DZenergy optimiza-tionroutinetodeterminethepossiblestructureoftheCu2+_–1

2 complex. The lowest energy conformations for 1 and Cu2+_–1

2 complexes are shown in Fig. 8. Cu2+ _{ligates two chemosensor} 1 molecules,and is coordinatedby fournitrogenatoms at dis-tancesof 2.00,2.01,2.12, and2.18 ˚A(Fig.S5in the supplemen-tarydata).

We performed pHtitration of chemosensor 1 toinvestigate a suitable pH range for Cu2+ _{sensing. As shown in Fig. 9, the} emission intensities of the metal-free chemosensor 1 are very low.Aftermixing chemosensor1withCu2+ _{intherange ofpH} 2–8.5, the emission intensity at 468nm rapidly increases to a maximum.ForpHvaluesgreaterthan9.0,theemissionintensity is significantly less than that for the middle pH values, indi-cating poorstability of theCu2+_–1

2 complexesat highpH. For pH<2, the enhanced emission does not occur, becauseof pro-tonationofthe aminegroups,preventingformation ofCu2+_–1

2 complexes.

3.3. Livingcellimaging

Chemosensor1wasusedforlivingcellimaging.TodetectCu2+ inlivingcells, RAW264.7cellswereculturedinDMEM supple-mentedwith10%FBSat37◦Cunder5%CO2.Cellswereplatedon 18mmglasscoverslipsandallowedtoadherefor24h.RAW264.7 cellsweretreatedwith20␮MCu(BF4)2for30minandwashedwith PBSthreetimes.Thecellswerethenincubatedwithchemosensor1 (20␮M)for30minandwashedwithPBStoremoveanyremaining sensor.TheimagesofRAW264.7cellswereobtainedusinga flu-orescencemicroscope.Fig.10showsimagesofRAW264.7cells withchemosensor1aftertreatmentwithCu2+_.Anoverlayof flu-orescence and bright-field images shows that thefluorescence signalsarelocalizedintheintracellulararea,indicating subcellu-lardistributionofCu2+_{andgoodcell-membranepermeabilityof} chemosensor1.

4. Conclusion

Inconclusion,wereportapyrene-basedfluorescent chemosen-sor for Cu2+ _{sensing. We observed significant fluorescence} enhancementwithchemosensor1inthepresenceofCu2+_. How-ever,addingAg+_,Ca2+_,Cd2+_,Co2+_,Fe2+_,Fe3+_,Hg2+_,K+_,Mg2+_,Mn2+_, Ni2+_,Pb2+_,orZn2+_{tothechemosensorsolutioncausedonly} min-imalchangesinfluorescenceemission.TheoptimalpHrangefor Cu2+ _{detection by chemosensor 1 is pH 2–8.5. Chemosensor 1} mayhaveapplicationinfluorescenceimagingoflivingcells.This pyrene-basedCu2+_{chemosensorprovidesaneffectiveprobefor} Cu2+_sensing.

Acknowledgements

WegratefullyacknowledgethefinancialsupportoftheNational ScienceCouncil(ROC)andNationalChiaoTungUniversity.

AppendixA. Supplementarydata

1_{H and} 13_{C NMR spectrum of chemosensor 1, ESI mass} spectra of Cu2+_–1

2 and DFT-optimized structure of the Cu2+_–1

2 complexes calculated with B3LYP/LanL2DZ method are included in supplementary data. Supplementary data

detectionofleadcadmium,andmercuryions,ChemicalSocietyReviews41 (2012)3210–3244.

[6]K.J.Wallace,Moleculardyesusedforthedetectionofbiologicaland environ-mentalheavymetals:highlightsfrom2004to2008,SupramolecularChemistry 21(2009)89–102.

[7]J.A.Cowan,InorganicBiochemistry:AnIntroduction,Wiley-VCH,NewYork, NY,1997,pp.133–134.

[8]K.J.Barnham,C.L.Masters,A.I.Bush,Neurodegenerativediseasesandoxidative stress,NatureReviewsDrugDiscovery3(2004)205–214.

[9]S.H.Hahn,M.S.Tanner,D.M.Danke,W.A.Gahl,Normalmetallothionein syn-thesisinfibroblastsobtainedfromchildrenwithIndianchildhoodcirrhosisor copper-associatedchildhoodcirrhosis,BiochemicalandMolecularMedicine 54(1995)142–145.

[10]D.R.Brown,Copperandpriondisease,BrainResearchBulletin55(2001) 165–173.

[11]D.J.Waggoner,T.B.Bartnikas,J.D.Gitlin,Theroleofcopperin neurodegenera-tivedisease,NeurobiologyofDisease6(1999)221–230.

[12]Y.Xiang,A.Tong,P.Jin,Y.Ju,Newfluorescentrhodaminehydrazone chemosen-sorforCu(II)withhighselectivityandsensitivity,OrganicLetters8(2006) 2863–2866.

[13]X. Zhang, Y. Shiraishi, T. Hirai, Cu(II)-selective green fluorescence

of a rhodamine–diacetic acid conjugate, Organic Letters 9 (2007)

5039–5042.

[14]G. Li, Z. Xu,C. Chen,Z. Huang, Ahighlyefficient and selectiveturn-on fluorescentsensorforCu2+_{ionbasedoncalix[4]arenebearingfour}

imino-quinolinesubunitsontheupperrim,ChemicalCommunication177(2008)

4–177,6.

[15]H.S.Jung,P.S.Kwon,J.W.Lee,J.Kim,C.S.Hong,J.W.Kim,S.Yan,J.Y.Lee,J.W.Lee, T.Joo,S.Kim,Coumarin-derivedCu2+_{-selectivefluorescencesensor:synthesis,} mechanisms,andapplicationsinlivingcells,JournaloftheAmericanChemical Society131(2009)2008–2012.

[16]H.S.Jung,M.Park,D.Y.Han,E.Kim,C.Lee,S.Ham,J.S.Kim,Cu2+_ion-induced self-assemblyofpyrenylquinolinewithapyrenylexcimerformation,Organic Letters11(2009)3378–3381.

[17] S.Wu,S.Liu,Anewwater-solublefluorescentCu(II)chemosensorbasedon tetrapeptidehistidyl-glycyl-glycyl-glycine(HGGG),SensorsandActuatorsB 141(2009)187–191.

[24] J.Jo,H.Y.Lee,W.Liu,A.Olasz,C.Chen,D.Lee,Reactivity-baseddetectionof copper(II)ioninwater:oxidativecyclizationofazoaromaticsasfluorescence turn-onsignalingmechanism,JournaloftheAmericanChemicalSociety134

(2012)16000–16007.

[25]N.R.Chereddy,S.Thennarasu,A.B.Mandal,Anewtriazoleappendedrhodamine chemosensorforselectivedetectionofCu2+_{ionsandlive-cellimaging,Sensors} andActuatorsB171–172(2012)294–301.

[26]D.Zhang,M.Wang,M.Chai,X.Chen,Y.Ye,Y.Zhao,Threehighlysensitiveand selectivecolorimetricandoff–onfluorescentchemosensorsforCu2+_inaqueous solution,SensorsandActuatorsB168(2012)200–206.

[27]B.Hu,Q.Su,P.Lu,Y.Wang,BODIPYmodified9-cycloheptatrienylidenefluorene derivatives:fluorescentturn-onfordetectingCu2+_{withacidityindependence,} SensorsandActuatorsB168(2012)310–317.

[28]A.Senthilvelan,I.Ho,K.Chang,G.Lee,Y.Liu,W.Chung,Cooperativerecognition ofacoppercationandanionbyacalix[4]arenesubstitutedatthelowerrimby a␤-amino-␣,␤-unsaturatedketone,Chemistry–AEuropeanJournal15(2009) 6152–6160.

[29]V.S.Jisha,A.J.Thomas,D.Ramaiah,Fluorescenceratiometricselective recogni-tionofCu2+_{Ionsbydansyl-naphthalimidedyads,JournalofOrganicChemistry} 74(2009)6667–6673.

[30]E.Manandhar,K.J.Wallace,Host–guestchemistryofpyrene-basedmolecular receptors,InorganicaChimicaActa381(2012)15–43.

[31] E.Manandhar,J.H.Broome,J.Myrick,W.Lagrone,P.J.Cragg,K.J.Wallace,A pyrene-basedfluorescentsensorforZn2+_{ions:amolecular‘butterfly’,Chemical}

Communications47(2011)8796–8798.

Biographies

Hsuan-FuWangisstudyingforMSintheDepartmentofAppliedChemistryat NationalChiaoTungUniversity.

Dr.Shu-PaoWuhadPh.D.in2004,DepartmentofChemistry,TheOhioState University,USA;Adviser:J.A.Cowan,PostdoctoralFellow,2004–2006,Collegeof Chemistry,UniversityofCalifornia,Berkeley,USA;Adviser:J.P.Klinman,Assistant Professor,2006,NationalChiaoTungUniversity,Taiwan,RepublicofChina.Current interests:metalionchemosensorsandAlkB.