A highly selective turn-on fluorescence chemosensor for Hg(II) and its application in living cell imaging

ContentslistsavailableatScienceDirect

Sensors

and

Actuators

B:

Chemical

j ou rn a l h o m ep 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

highly

selective

turn-on

fluorescence

chemosensor

for

Hg(II)

and

its

application

in

living

cell

imaging

Shiang-Yi

Yu,

Shu-Pao

Wu

∗

DepartmentofAppliedChemistry,NationalChiaoTungUniversity,Hsinchu300,Taiwan,ROC

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received4March2014

Receivedinrevisedform23April2014 Accepted23April2014

Availableonline2May2014 Keywords: Sensors Mercury Fluorescence Imagingagents Coumarin

a

b

s

t

r

a

c

t

Anewcoumarinderivative(MS1)containinganiminemoietyandahydroxylmoietyexhibitsanenhanced fluorescenceinthepresenceofHg2+_{ions.OthermetalionsAl}3+_,Ca2+_,Co2+_,Cr3+_,Cu2+_,Fe2+_,Fe3+_,Hg2+_,

K+_,Mg2+_,Mn2+_,Ni2+_,Pb2+_,andZn2+_{producedonlyminorchangesinthefluorescencevaluesofMS1.The}

bindingratioofMS1–Hg2+_{complexeswasdeterminedfromtheJobplottobe1:1.Thebindingconstant}

(Ka)ofHg2+bindingtoMS1wasfoundtobe6.85×103M−1.Themaximumfluorescenceenhancement

causedbyHg2+_{bindingtoMS1wasobservedinthepHrangeof6.5–9.0.Confocalfluorescencemicroscopy}

imagingusingRAW264.7cellsshowedthatMS1couldbeusedasaneffectivefluorescentprobefor detectingHg2+_{inlivingcells.}

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Thedevelopmentofchemosensorsthatdetectheavymetalions, suchasCd2+_,Cu2+_,Fe3+_,Hg2+_,Pb2+_,andZn2+_{,hasbeenanimportant}

researchissueonbiologicalimaging,environmentalmonitoring, andmedicaldiagnostics[1–3].Amongthem,mercuryisoneofthe mosttoxicheavymetalelementsandexistsinthreeforms: ele-mental,inorganic,andorganicmercury.Mercuryionshaveshown highaffinityforthiolgroupsinproteins,leadingtothe malfunc-tionofcells andconsequentlycausingmanyhealthproblemsin thebrain,kidney,andcentralnervoussystem.Itsaccumulation in the body resultsin a wide variety of diseases, suchas pre-natalbraindamage;seriouscognitiveandmotiondisorders;and Minamatadisease[4].Therefore,inordertospecificallydetect mer-curyionsinbiologicalandenvironmentalsamples,thedesignof highlyselective andsensitivemercury chemosensors is in high demand.

Several methods for the detection of mercury ions in vari-oussamples havebeen developed,including atomicabsorption – emission spectroscopy [5], inductively coupled plasma mass spectroscopy(ICPMS)[6],andinductivelycoupledplasma–atomic emissionspectrometry(ICP-AES)[7].Althoughthesemethodsare

∗ Correspondingauthor.Tel.:+88635712121x56506.

E-mailaddresses:[email protected],[email protected] (S.-P.Wu).

quantitative,mostofthesemethodsrequireexpensiveinstruments andarenotgoodforon-siteanalysis.Recently,moreattentionhas beenfocusedonthedevelopmentoffluorescentchemosensorsfor thedetectionofHg2+_{ionsinbiologicalandenvironmentalsamples}

[8–23].

Numerousmolecularprobesusingdifferentreceptorsand flu-orescentunitshavebeendevelopedfor Hg2+_{detection.Because}

Hg2+_{isknownasafluorescencequencherduetospin–orbit}

cou-pling[24],mostfluorescentchemosensorsdetectHg2+_througha

fluorescencequenching.Duetosensitivityconcerns,fluorescent chemosensorsdetectingmetalionsusingfluorescence enhance-ment are moreeasily monitoredthan those usingfluorescence quenching.Thispaperreportsonanewlydesigneda coumarin-basedfluorescentenhancementHg2+_chemosensor.

Inthisstudy,afluorescentchemosensor(MS1)containingan iminemoietyandahydroxylmoietywasdesignedformetalion detection.MS1exhibitsweakfluorescenceduetotheimine isom-erizationwhich hasbeenknowntohavea non-radiativedecay processintheexcitedstate[25].Thebindingofmetalionstothe chemosensorblockstheimineisomerizationandresultsin consid-erablefluorescenceenhancementofcoumarin.ThemetalionsAl3+_,

Ca2+_,Cd2+_,Co2+_,Cr3+_,Cu2+_,Fe2+_,Fe3+_,Hg2+_,Mg2+_,Mn2+_,Ni2+_,Pb2+_,

andZn2+_{weretestedformetalionbindingselectivitywithMS1,}

butHg2+_{wastheonlyionthatcausedablueemissionuponbinding}

withMS1.Thefluorescencemicroscopyexperimentsalso demon-stratedthatMS1canbeusedasafluorescentprobefordetecting Hg2+_{inlivingcells.}

http://dx.doi.org/10.1016/j.snb.2014.04.077 0925-4005/©2014ElsevierB.V.Allrightsreserved.

2.1. Materialsandinstrumentations

All solvents and reagents were obtained from commercial sourcesandusedasreceivedwithoutfurtherpurification.UV/vis spectrawererecordedonanAgilent8453UV/visspectrometer. NMRspectrawereobtainedonaBrukerDRX-300NMRand Var-ian Unity INOVA-500 NMR spectrometer. Fluorescence spectra measurementswereperformedonaHitachiF-7000fluorescence spectrophotometer.FluorescentimagesweretakenonaLeica TCS-SP5-XAOBSConfocalFluorescenceMicroscope.

2.2. SynthesisofchemosensorMS1

Hydrazine(50%,333␮L,10.3mmol) wasaddedtoasolution of 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde [26] (80mg, 0.42mmol)inMeOH(10mL)andthereactionmixturewasreflux forovernight.Thesolventwasremovedunderreducedpressure andthecrudesolidwastakenupintoamixtureofwaterandEtOAc. ThecombinedaqueouslayerwasextractedfourtimeswithEtOAc. Thecombinedorganiclayerwaswashedwithbrine,andthendried withNa2SO4.Thesolventwasremovedunderthereduced

pres-sureandtheresiduewaspurifiedbycolumnchromatographyusing hexane/ethylacetate(v/v=1:1)asaneluenttoaffordMS1asa yel-lowsolid.Yield:40mg(47%);mp:257–258◦C.1_HNMR(500MHz,

DMSO-d6)␦12.77(b,1H),8.38(s,1H),7.96(d,J=9.5Hz,1H),7.47(d,

J=8.5Hz,1H),7.36(s,2H),6.83(d,J=8.5Hz,1H),6.26(d,J=9.5Hz, 1H).13_{CNMR (125MHz,DMSO-d}

6)␦160.3,159.7,151.3,144.9,

134.6,128.3,113.2,111.6,110.9,106.8.MS(EI): m/z(%)=204.0 (100)[M+H]+_{,188(52),175(48),159(39).HRMS(EI):calcd.for}

C10H7NO4[M+H]+204.0535;found204.0529.

2.3. MetalionbindingstudybyUV–visandfluorescence spectroscopy

ChemosensorMS1(10.0_␮M)wasaddedwithdifferentmetal ions(60.0␮M).Allspectraweremeasuredin1.0mLmetanol–water solution(v/v=4:1,10mMHEPES,pH7.0).Thelightpathlengthof cuvettewas1.0cm.

studiedbyfluorescencespectroscopy

ChemosensorMS1(10.0␮M)wasaddedwithHg2+_(10.0␮M)

in1.0mLmetanol–watersolution(v/v=4:1,10mM buffer).The bufferswere:pH3–4,AcONa/AcOH;pH5–10,HEPES;pH11–12, Tris.

2.5. Determinationofthebindingstochiometryandtheapparent associationconstantsKaofHg(II)bindinginchemosensorMS1

ThebindingstochiometryofMS1–Hg2+_complexeswas

deter-mined by Job plot experiments. The fluorescence intensity at 455nmwasplottedagainstmolarfractionofMS1undera con-stanttotalconcentration(20.0␮M).Thefluorescenceapproacheda maximumintensitywhenthemolarfractionwas0.5.Theseresults indicatethatchemosensorMS1formsa1:1complexwithHg2+_.The

associationconstant(Ka)ofMS1–Hg2+complexeswasdetermined

bytheconsequentEq.(1)[27]: 1 (_I−I0)= 1 {ka×(Imax−I0)×[Hg2+]} + 1 (Imax−I0) (1) whereIisthefluorescenceintensityat455nmatanygivenHg2+

concentration,I0isthefluorescenceintensityat455intheabsence

of Hg2+_{. The associationconstant K}

a was evaluated graphically

byplotting 1/(I−I0)against 1/[Hg2+].Typical plots{1/(I−I0)vs.

1/[Hg2+_{]}areshowninFig.5.Datawerelinearlyfittedaccordingto}

Eq.(1)andtheKavaluewasobtainedfromtheslopeoftheline.

2.6. Cellculture.

RAW264.7cellsweregrowninH-DMEM(Dulbecco’sModified Eagle’sMedium,highglucose)supplementedwith10%FBS(Fetal BovineSerum)inanatmosphereof5%CO2at37◦C.

2.7. Cytotoxicityassay

Themethylthiazolyltetrazolium(MTT)assaywasusedto mea-surethecytotoxicityofMS1inRAW264.7cells.RAW264.7cells wereseededintoa96-wellcell-cultureplate.Various concentra-tions(0,5,10,15,20,30␮M)ofMS1wereaddedtothewells.The

Fig.1.FluorescenechangeofchemosensorMS1(10␮M)uponadditionofvariousmetalions(60␮M)inmethanol–H2O(v/v=4/1,10mMHEPES,pH7.0)solutions.The

O O CHO HO O O HO N NH2 NH₂NH₂-H₂O EtOH-THF

Scheme1.SynthesisofchemosensorMS1.

450 500 550 600 0 100 200 300 400 500 0 2 4 6 8 10 0 200 400 Intensity (455 nm) [ Hg2+ ] / [ MS1 ] In tn es it y Wavelength (nm) 455 nm

Fig.2.FluorescenceresponseofMS1(10.0␮M)tovariousequivalentsofHg2+_in

methanol–water(v/v=4:1,10mMHEPES,pH7.0)solutions.Theexcitation wave-lengthwas330nm.

cellswereincubatedat37◦C under5%CO2 for24h. 10␮LMTT

(5mg/mL)wasaddedtoeachwellandincubatedat37◦Cunder 5%CO2for4h.RemovetheMTTsolutionandyellowprecipitates

(formazan)observedinplatesweredissolvedin200␮LDMSOand 25␮LSorenson’sglycinebuffer(0.1Mglycineand0.1MNaCl). Mul-tiskanGOmicroplatereaderwasusedtomeasuretheabsorbanceat 570nmforeachwell.Theviabilityofcellswascalculatedaccording tothefollowingequation:

Cellviability(%)= (mean_(meanof_ofabsorbance_absorbancevalue_valueof_oftreatment_{controlgroup)}group).

2.8. Cellimaging

ThecellsculturedinDMEMweretreatedwith10mMsolutions ofHg2+_{(2␮L;finalconcentration:20␮M)dissolvedinsterilized}

0

200

400

600

In te ns it y (4 55 nm ) MS1+ metal ions MS1+metal ions + Hg2+ Hg2+Al3+ Ca2+ Cd2+ Co2+ Cr3+Cu2+Fe2+Fe3+Mg2+Mn2+Ni2+ Pb2+Zn2+

Fig.3. FluorescenceresponseofchemosensorMS1(10.0␮M)toHg2+_(60.0␮M)or

60.0␮Mofothermetalions(blackbars)andtothemixtureofothermetalions (60.0␮M)with60.0␮MofHg2+_{(graybars)inmethanol–water(v/v=4:1,10mM}

HEPES,pH7.0)solutions. 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20

(I

-I

0

)*

X

X = { [MS1] / ( [MS1] + [Hg2+] ) }

Fig.4. JobplotoftheMS1–Hg2+_{complexesinamethanol–water(v/v=4:1,10mM}

HEPES,pH7.0)solutions.ThetotalconcentrationofMS1andHg2+_{was20.0␮M.The}

monitoredwavelengthwas455nm.

PBS(pH 7.4)andincubatedat37◦ for30min.The treatedcells werewashedwithPBS(2mL×3)toremoveremainingmetalions. DMEM(2mL)wasaddedtothecellculture,whichwasthentreated witha10mMsolutionofchemosensorMS1(2␮L;final concentra-tion:20␮M)dissolvedinDMSO.Thesampleswereincubatedat 37◦for30min.Theculturemediumwasremoved,andthetreated cellswerewashedwithPBS(2mL×3)beforeobservation. Fluo-rescenceimaging was performedwitha LeicaTCS-SP5-X AOBS Confocalmicroscope.Thecellswereexcitedwithawhitelightlaser at350nm,andemissionwascollectedat460±25nm.

2.9. Computationalmethods

Quantumchemicalcalculationsbasedondensityfunctional the-ory(DFT)werecarriedoutusingaGaussian09program.Ground state geometry optimization of MS1 was performed using the B3LYPfunctionalandthe6-31G basisset.Groundstate geome-tryoptimizationofMS1–Hg2+_{complexeswasperformedusingthe}

B3LYPfunctionalandtheLANL2DZbasisset.

3. Resultsanddiscussion

3.1. CharacterizationofchemosensorMS1

ChemosensorMS1wassynthesizedbythereactionofhydrazine and 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde to form an

Fig.5.Benesi–HildebrandplotofMS1withHg2+ _{inmethanol/water(v/v=4:1,}

10mMHEPES,pH 7.0)solutions.Theexcitationwavelengthwas 300nmand observedwavelengthwas455nm.Thebindingconstantwas6.85×103_M−1_forHg2+

Fig.6. 1HNMR300MHzspectraofMS1(10.0mM)upontitrationwithvariousequivalentsofHg2+_inDMSO-d6.

iminebond(Scheme1).ChemosensorMS1isyellowandhasan absorptionbandcenteredat330nm.ChemosensorMS1exhibits weakfluorescence(˚=0.002)comparedtocoumarin(˚>0.5).This isduetotheimineisomerizationwhichhasbeenknowntohavea non-radiativedecayprocessintheexcitedstate[25]

3.2. Cation-sensingproperties

ThesensingabilityofMS1wastestedbymixingitwiththemetal ionsAl3+_,Ca2+_,Co2+_,Cr3+_,Cu2+_,Fe2+_,Fe3+_,Hg2+_,Mg2+_,Mn2+_,Ni2+_,

Pb2+_,andZn2+_.Hg2+_{wastheonlyionthatcausedablueemission}

fromMS1(Fig.1).DuringHg2+_{titrationwithMS1,anew}

emis-sionbandcenteredat455nmwasformed(Fig.2).Afteradding 6equivalentsofHg2+_{,theemissionintensityreachedamaximum.}

Thequantumyieldoftheemissionbandwas0.039,whichis19-fold thatofMS1at0.002.TheseobservationsindicatethatHg2+_isthe

onlymetalionthatreadilybindswithMS1,causingsignificant flu-orescenceenhancementandpermittinghighlyselectivedetection ofHg2+_.

TostudytheinfluenceofothermetalionsonHg2+_bindingwith

MS1,competitiveexperimentswereperformedwithothermetal ions(60.0␮M)inthepresenceofHg2+_{(60.0␮M)(Fig.3).It was}

foundthatfluorescenceenhancementcaused bythemixtureof Hg2+ _{withmostmetalionswas similartothat causedby Hg}2+

alone.Noneoftheothermetalionswerefoundtointerferewith thebindingofMS1withHg2+_.

InordertounderstandthebindingstoichiometryofMS1–Hg2+

complexes,Jobplotexperimentswerecarried out.InFig.4,the emissionintensityat455nmisplottedagainstmolarfractionof MS1 undera constanttotal concentration(20.0␮M).Maximum emissionintensitywasreachedwhenthemolarfractionwas0.50. Resultsindicateda1:1ratioforMS1–Hg2+_{complexes,inwhichone}

Fig.7. DFT-optimizedstructuresof(a)MS1and(b)MS1–Hg2+_{complexes.Blueatom,N;redatom,O;gray-whiteatom,Hg.(Forinterpretationofthereferencestocolorin}

4 6 8 10 0 200 400 600 MS1 + Hg2+ MS1 Intensity ( 455 nm ) pH

Fig.8. Fluoresceneresponse(455nm)offreechemosensorMS1(10␮M)andafter additionHg2+_{(60␮M)inMeOH–H}

2Osolutions(v/v=4/1,10mMbuffer).The

exci-tationwavelengthwas330nm.

Hg2+_{ionwasboundtooneMS1.TheassociationconstantK}

awas

evaluatedgraphicallybyplotting1/(I−I0)against1/[Hg2+](Fig.5).

ThedatawaslinearlyfitandtheKavaluewasobtainedfromthe

slopeandinterceptoftheline.Theassociationconstant(Ka)ofHg2+

bindinginMS1wasfoundtobe6.85×103_M−1_{.Thedetectionlimit}

ofMS1asafluorescentsensorfortheanalysisofHg2+_was

deter-minedfromtheplotoffluorescenceintensityasafunctionofthe concentrationofHg2+_{(seeFig.S3inthesupportinginformation).}

MS1wasfoundtohaveadetectionlimitof0.193␮M,whichmeans itisabletodetectHg2+_{concentrationsinthemicro-molarrange.}

TogainaclearerunderstandingofthestructureofMS1–Hg2+

complexes,1_{HNMRspectroscopywasemployed(Fig.6).Hg}2+_isa

heavymetalionandcanaffecttheprotonsignalsthatareclose totheHg2+_{binding site.The} 1_{HNMR spectraofMS1 recorded}

withincreasingamountsofHg2+_{showthattheproton(H}

aandHb)

signalsat12.75ppmand7.38ppmdisappearasHg2+_wasadded.

AdditionofHg2+_{alsocausedtheproton(H}

c)signalat8.38ppm

shifteddownfield.TheseobservationsindicatethatHg2+_bindsto

MS1mainlythroughoneoxygenatomandonenitrogenatom. ToelucidatethestructuresofMS1andMS1–Hg2+_complexes,

density functional theory (DFT) calculations were undertaken usingtheGaussian09softwarepackage.Chemosensor MS1and

MS1–Hg2+ _{complexesweresubjectedtoenergyoptimizationby}

usingB3LYP/6-31GandB3LYP/LANL2DZ,respectively.Theglobal

0 20 40 60 80 100 15 20 30 5 0 10 V ia bi lity ( % ) [MS1], 10-6 M

Fig.9.Cellviabilityvalues(%)estimatedbyanMTTassayversusincubation concen-trationsofMS1.RAW264.7cellswereculturedinthepresenceofMS1(0–30␮M) at37◦_Cfor24h.

minimastructuresforMS1andMS1–Hg2+_{complexesareshown}

inFig.7.ThedistancesofHg2+_{fromthetwonitrogenatomswere}

3.12 ˚Aand2.15 ˚A,andfromtheoxygenatomwas2.11 ˚A.

WeperformedpHtitrationofMS1toinvestigateasuitablepH rangeforHg2+_{sensing.AsdepictedinFig.8,theemissionintensities}

ofmetal-freeMS1wereverylow.AftermixingchemosensorMS1 withHg2+_{,theemissionintensityat455nmsuddenly increased}

at pH6.5and reacheda maximumin thepHrange of 6.5–9.0. TheemissionintensitydecreasesatpH>9.0.Thisindicatespoor stability of the MS1–Hg2+ _{complexes at high pH. For pH<6.5,}

the emission intensity is very low due to the protonation on the amine group, which prevents the formation of MS1–Hg2+

complexes.

3.3. Livingcellimaging

ThepotentialofMS1forimagingHg2+_{inlivingcellswas}

investi-gatednext.First,anMTTassaywithaRAW264.7celllinewasused todeterminethecytotoxicityofMS1.InFig.9,thecellularviability wasestimatedtobegreaterthan80%after24h,whichindicates thatMS1(<30␮M)haslowcytotoxicity.Furthermore,theimages ofcellswereobtainedusingaconfocalfluorescencemicroscope. WhenRAW264.7cellswereincubatedwithMS1(10␮M),no fluo-rescencewasobserved(Fig.10a).AfterthetreatmentofHg2+_,bright

Anoverlayoffluorescenceandbright-fieldimagesshowsthatthe fluorescencesignalsare localizedintheintracellular area, indi-catingasubcellulardistributionofHg2+_{andgoodcell-membrane}

permeabilityofMS1.

4. Conclusion

In conclusion, we developed a coumarin-based fluorescent chemosensorforHg2+ _{sensing.Weobserved significant}

fluores-cenceenhancementwithMS1inthepresenceofHg2+_.However,

addingAl3+_,Ca2+_,Cr3+_,Co2+_,Fe2+_,Fe3+_,Hg2+_,K+_,Mg2+_,Mn2+_,Ni2+_,

Pb2+_,orZn2+_{tothechemosensorsolutioncaused onlyminimal}

changesinfluorescenceemission.TheoptimalpHrangeforHg2+

detectionbyMS1is6.5–9.0.Inaddition,thechemosensorMS1has lowcytotoxicityandthereforecanbeappliedfordetectingHg2+_in

livingcells.

Supplementaryinformation

1_Hand13_{CNMRspectraofMS1,calibrationcurveofMS1–Hg}2+

inawater–MeOH(v/v=1/4,10mMHEPES,pH7.0)solutions. Acknowledgements

WegratefullyacknowledgethefinancialsupportoftheNational ScienceCouncil(ROC)andNationalChiaoTungUniversity.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.snb.2014.04.077. References

[1]E.M.Nolan,S.J.Lippard,Toolsandtacticsfortheopticaldetectionofmercuric ion,Chem.Rev.108(2008)3443–3480.

[2]N.Kaur,S.Kumar,Colorimetricmetalionsensors,Tetrahedron67(2011) 9233–9264.

[3]H.N.Kim,W.X.Ren,J.S.Kim,J.Yoon,Fluorescentandcolorimetricsensors fordetectionoflead,cadmium,andmercuryions,Chem.Soc.Rev.41(2012) 3210–3244.

[4]M.Harada,Minamatadisease:methylmercurypoisoninginJapancausedby environmentalpollution,Crit.Rev.Toxicol.25(1995)1–24.

[5]Y.Gao,S.DeGalan,A.DeBrauwere,W.Baeyens,M.Leermakers,Mercury speci-ationinhairbyheadspaceinjection-gaschromatography-atomicfluorescence spectrometry(methylmercury)andcombustion-atomicabsorption spectrom-etry(totalHg),Talanta82(2010)1919–1923.

[6]F.Moreno,T.Garcia-Barrera,J.L.Gomez-Ariza,Simultaneousanalysisof mer-cury andseleniumspeciesincludingchiralformsofselenomethioninein humanurineandserumbyHPLCcolumn-switchingcoupledtoICP-MS,Analyst 135(2010)2700–2705.

[7]X.Chai,X.Chang,Z.Hu,Q.He,Z.Tu,Z.Li,Solidphaseextractionoftrace Hg(II)onsilicagelmodifiedwith2-(2-oxoethyl)hydrazinecarbothioamideand determinationbyICP-AES,Talanta82(2010)1791–1796.

[8]B.N.Ahamed,P.A.Ghosh,Chelationenhancedselectivefluorescencesensingof Hg2+_{byasimplequinolinesubstitutedtripodalamidereceptor,DaltonTrans.}

40(2011)12540–12547.

fluorescencesensingofmercuryions,Tetrahedron68(2012)5279–5282. [10]C.Li,F.Xu,Y.Li,K.Zhou,Y.Zhou,AfluorescentchemosensorforHg2+_basedon

naphthalimidederivativebyfluorescenceenhancementinaqueoussolution, Anal.Chim.Acta717(2012)122–126.

[11]X.Ma,J.Wang,Q.Shan,Z.Tan,G.Wei,D.Wei,Y.A.Du,turn-on fluores-centHg2+_{chemosensorbasedonferriercarbocyclization,Org.Lett.14(2012)}

820–823.

[12]M. Vedamalai, S. Wu, A BODIPY-based colorimetric and fluorometric chemosensorforHg(II)ionsanditsapplicationtolivingcellimaging,Org.Biom. Chem.10(2012)5410–5416.

[13]J.Fan,C.Chen,Q.Lin,N.Fu,Afluorescentprobeforthedual-channel detec-tion ofHg2+_/Ag+ _{and its Hg}2+_{-basedcomplex for detection ofmercapto}

biomoleculeswithatunablemeasuringrange,Sens.ActuatorsB173(2012) 874–881.

[14]L. Shi,Y. Li, Z. Liu, T.D. James, Y. Long, Simultaneousdetermination of Hg(II) and Zn(II) usinga GFPinspiredchromophore,Talanta100 (2012) 401–404.

[15]Z.Dong,X.Tian,Y.Chen,J.Hou,J.Ma,RhodaminegroupmodifiedSBA-15 fluorescentsensorforhighlyselectivedetectionofHg2+_{anditsapplication}

asanINHIBITlogicdevice,RSCAdv.3(2013)2227–2233.

[16]H.F.Wang,S.P.Wu,Highlyselectivefluorescentsensorsformercury(II)ionsand theirapplicationsinlivingcellimaging,Tetrahedron69(2013)1965–1969. [17]A.K.Atta,S.B.Kim,J.Heo,D.G.Cho,Hg(II)-mediatedintramolecular

cyclization-reactioninaqueousmediaanditsapplicationasHg(II)selectiveindicator,Org. Lett.15(2013)1072–1075.

[18]J.Chen,W.Liu,Y.Wang,H.Zhang,J.Wu,H.Xu,W.Ju,P.Wang,Turn-on fluorescencesensor basedontheaggregationof pyrazolo[3,4-b]pyridine-basedcoumarinchromophoresinducedbyHg2+_{,TetrahedronLett.54(2013)}

6447–6449.

[19]M.G.Choi,H.G.Im,J.H.Noh,D.H.Ryu,S.Chang,SpacerdependencyoftheHg2+

-selectivefluorescentsignallingbydesulfurizationofthioamideasaturn-on switch,Sens.ActuatorsB177(2013)583–588.

[20]P.Srivastava,R.Ali,S.S.Razi,M.Shahid,A.Misra,Thioureabasedmoleculardyad (ANTU): fluorogenicHg2+_{selectivechemodosimeterexhibitingblue–green}

fluorescenceinaqueous-ethanolenvironment,Sens.ActuatorsB181(2013) 584–595.

[21]J.Zhang,Y.Zhou,W.Hu,L.Zhang,Q.Huang,T.Ma,Highlyselective fluo-rescenceenhancementchemosensorforHg2+ _{basedonrhodamineandits}

applicationinlivingcellsandaqueousmedia,Sens.ActuatorsB183(2013) 290–296.

[22]H.Ye,F.Ge,X.Chen,Y.Li,H.Zhang,B.Zhao,J.Miao,Anewprobeforfluorescent recognitionofHg2+_{inlivingcellsandcolorimetricdetectionofCu}2+_inaqueous

solution,Sens.ActuatorsB182(2013)273–279.

[23]B.Zhu,W.Wang,L.Liu,H.Jiang,B.Du,Q.Wei,Ahighlyselectivecolorimetric andlong-wavelengthfluorescentprobeforHg2+_{,Sens.ActuatorsB191(2014)}

605–611.

[24]D.S.McClure,Spinvorbitinteractioninaromaticmolecules,J.Chem.Phys.20 (1952)682–686.

[25]X.Cheng,H.Jia,T.Long,J.Feng,J.Qin,Z.Li,A“turn-on”fluorescentprobe forhypochlorousacid:convenientsynthesis,goodsensingperformance,anda newdesignstrategybytheremovalofC Nisomerization,Chem.Commun.47 (2011)11978–11980.

[26]K.S.Lee,H.J.Kim,G.H.Kim,I.Shin,J.I.Hong,Fluorescentchemodosimeterfor selectivedetectionofcyanideinwater,Org.Lett.10(2008)49–51.

[27]H.A.Benesi,J.H.Hildebrand,Aspectrophotometricinvestigationofthe inter-actionofiodinewitharomatichydrocarbons,J.Am.Chem.Soc.71(1949) 2703–2707.

Biographies

Shiang-YiYuhadMSin2013,DepartmentofAppliedChemistryatNationalChiao TungUniversity.

Dr.Shu-PaoWuhadPhDin2004,DepartmentofChemistry,TheOhioState Uni-versity,USA.Currently,heisworkingasanAssociateProfessorinDepartmentof AppliedChemistryNationalChiaoTungUniversity,Taiwan,RepublicofChina.His currentinterestsaremetalionchemosensorsandAlkB.