工学院大学研究報告第123号　平成29年10月

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ガラスナノピペット内に作製したセシウムイオン選択膜

西 山 北 斗＊1，高 見 知 秀＊2

Cesiumion-selectivemembranefabricatedinaglassnanopipette

HokutoNISHIYAMA＊1andTomohideTAKAMI＊2

Abstract

　　ガラス製ナノピペット中にセシウムイオン選択膜を作製した．1,3-alternatethiacalix[4]biscrown-6,6 をイオン選択リガンドとして用い，その選択性を確認するためにセシウム，カリウム，ナトリウムの各イオンについてそれぞれ 0.01mM から 100mM まで濃度を変えていったときの信号強度からイオン選択性を計測した．イオン選択膜の主成分であるポリ塩化ビニルが，ナノピペット中でゲル化する前段階において，予めナノピペット中に入れている水と混ざってしまうことを防止するために，選択膜ゲル化の前段階においてその選択膜の原料を溶かした溶媒と水との液界面にシクロヘキサンの液膜を張っておく手法を開発して，選択膜作製の確度を上げることができた．Keywords:Ion-selectiveElectrode,Nanopipette,CesiumIon

1．Introduction

　Glass nanopipettes have been used as a bridgebetweenmacroworldandnanoworld.1)Pipetteshavebeenusedasa tool to transfer liquidswithvariousvolumes,2)andmanynovelphenomenaandapplicationsare involvedasgoingdown the size ofpipettes tomicroornano-meters.3-8)Nanopipettes couldbealsousedasprobesinscanningelectrochemicalmicroscopyand scanning ion conductancemicroscopy forhighresolution imaging.9-11)Moreover,microinjection is ausefulmethodof transfection to cells,12) though theviabilityof cells after themicroinjection isnothighenoughtoobtainstatisticalandquantitativeresults.13)

　An ion-selectiveelectrodewithpolymermembraneisauniversalandknowntool for therecognitionofions in a liquid, andvarious types of ion-selectiveelectrodes have been developed.14,15) However, thetypicalsizeofaconventional ion-selectiveelectrodeisontheorderofmillimeters,andthus,suchasan ion-selectiveelectrodecannotbeusedwithananopipette.Therefore,we developed amethod to prepare anion-selectivemembrane inananopipette; furtherwe

developedananopipette-baseddetection system forsubtlesignals.16-20)The iondetectionmechanismofanion-selectiveelectrodeinananopipetteisdiscussedinourrecentpaper.21)Further,weappliedourmethodtothedeterminationoflocalconcentrationsoftargetionsinvariouskindsoflivingcells.Weappliedthismethodtodetermine the localconcentrationofpotassiumorsodiumionsinHeLacells,22)ratvascularmyocytes,andneuroncellsfromembryonicstemcells.23)

　Although thedetectionof sodium,potassium,andcalcium ions could be identifiedwith ion-selectivenanopipettes,16-23)cesiumion-selectivenanopiette,whichwouldhaveanapplicationoflocaldetectionofcesiumions ina livingcell,hasnotbeendevelopedyet.Choiet al. reported the cesium ionselective glass tubeelectrodewiththeionophoreof1,3alternatethiacalix[4]biscrown-6,6inpoly(vinylchroride)membrane.24)Theyreported the linear responsewithanearNernstianslopeof57.6mVperdecadewithintheconcentrationrangeof0.001to32mM,andthelimitofdetectionwasdeterminedas0.38µM.24)

　In this paper,we have succeeded in producingcesium ionselectivenanopiette.Wehave estimated

＊1工学院大学工学部応用化学科2017年卒業　DepartmentofAppliedChemistry,KogakuinUniversity＊2工学院大学教育推進機構ナノ化学研究室教授　NanotechnologyChemistryLaboratory,DivisionofLiberalArts,KogakuinUniversity

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the select ivity coeff ic ient and conf irmed thenegativevaluesof the selectivitycoefficientsat theconcentrationofthesamplesolutionofCsCl,KCl,andNaClfrom0.01mMto100mM.

2．Experimental methods

　The resource of ion-selectivemembranewas (i)32mg of poly(vinyl chloride) (Sigma-Aldrich, highmolecularweight,CAS9002-86-2)forthemainresourceofthemembrane,(ii)62mgof2-Nitrophenyloctylether(NPOE,Dojindo,purity>99.0%,CAS37682-29-4) fortheplasticizer, (iii) 5mgof 1,3alternate thiacalix[4]biscrown-6,6 forthe ion-selective ionophore,and (iv)1mgof tetrakis[3,5-bis(trifluoromethyl)phenyl]borate,sodiumsalt(TFPB,Dojindo,purity>99.0%,CAS79060-88-1) for theanion repeller.Theyweredissolved in1mL tetrahydrofuran (THF,Sigma-Aldrich, purity>99.9%,with250ppmdibutylhydroxytoluene(BHT)forthestabilizer,CAS109-99-9).Herewecallthesolution

“solutionA”.　Next,weprepared the sample solutions ofCsCl(Sigma-Aldrich, purity>99.5%,CAS7647-17-3),KCl(Yanagishima,specialgrade,purity>99.5%,CAS7447-40-7),andNaCl (Manac, specialgrade,purity>99.5%,CAS7447-14-5)attheconcentrationsof0.01,0.1,1,10,and100mMindistilledwaterproducedwithOrganoBB-5B/G-35PA(resistivity5MΩcm).Inthisway,wehaveprepared15kindsofsamplesolutions.

　Before thepreparationof ion-selectivenanopipette,we prepared conventional ion-selective electrodeswithboronsilicateglasstubes (7mmouterdiameter,0.8mmthickness,70mmlength).Thecutfaceoftheglass tubewaspolishedwithawaterproof abrasivepaper.Figure1 shows theprocess to fabricate theconventional ion-selectiveglass tubeelectrode.Thesolution A was poured in a glass petri dish and

dried for1daytopreparePVCmembranefilmwithionophore.The filmwas cut and attached to thebottomoftheglasstubewithanepoxy.Aftercuringtheepoxy,0.01MCsClaqwasfilledintheglasstube.Finally,a0.20mmdiametersilverwire(Niraco,purity>99.99%) coated with a Ag/AgCl ink (BAS) wasinsertedintotheglasstubetopreparetheion-selectiveelectrode.

　TheNanopipettewasfabricatedfromboronsilicatecapillary tubes (NarishigeGD-1 outerdiameter: 1.0mm, innerdiameter: 0.6mm)with apuller (SutterP-2000) at ShizuokaUniversity.Thedetails of thepreparationmethodwerewritten in the previousreports.25-28)Figure2showstheprocesstofabricatetheion-selectivenanopipette.First,purewaterwasfilledin thenanopipette to the shankendusingamicro-

Fig.1 (Color online) The process to prepare ion-selective electrode. The details of the process are described in the text.

Fig.2 (Color online) The process to prepare ion-selective nanopipette. The details of the process are described in the text.

Fig.3 (Color online) Photos of nanopiette tube during the curing process of PVC membrane inside the nanopipette. (A-1): After pouring the PVC solution (“solution A” in the text) on the distilled water covered with cyclohexane. (A-2): Five minutes later after taking the photo (A-1). (B-1): After pouring the PVC solution on the distilled water, without using cyclohexane. (B-2): Five minutes later after taking the photo (B-1).

ガラスナノピペット内に作製したセシウムイオン選択膜

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syringe.Then,wepoured ca. 0.01µL cyclohexane(Sigma-Aldrich,purity>99.0%,CAS110-82-7) on thewater.Aftermaking the cyclohexane solvent layeron thewater, 10µLof“solutionA”waspoured inthenanopipette,anddriedfor1daytocurethePVCmembrane in the nanopipette.Using cyclohexanebeforepouring thesolutionA is the improvedpointin this study.Figure3 showshow thecyclohexanesolventworksbetweenPVCsolutionandwater.Theupperphotos (A-1)and (A-2) inFig.3 indicates thatthemixing ofwater and solutionAdidnot occurevenafter5minutesofpouring(A-2)sothatthePVCmembranecanbeformedfirmlyinthenanopipette.Ontheotherhand,thelowerphotos(B-1)and(B-2)showsthemixingofwaterandsolutionAbecauseTHFcandissolve inwater.Moreover, themixedPVCsolutioninwatergotwhite to start theprecipitationafter5

minutes(B-2).Inthisway,wecouldpreventthemixingof PVC solutionwithwater byusing cyclohexanebetweenthePVCsolutionandwater.It isnotedthatthecyclohexane solventwas finally transpiredwithTHFonthedryingprocess.Afterthedryingprocess,0.01MCsClaqwasfilledinthenanopipette,anda0.20mmdiametersilverwirecoatedwithaAg/AgCl inkwas inserted into theglass tube toprepare the ion-selectivenanopipette.　Figure4showstheschematicsofthediagramoftheexperimental setup forconventionalglass tube ion-selectiveelectrode (a) and ion-selectivenanopipette(b).Thesignalwasamplifiedwithapatch/wholecellclampamplifier (NihonKohdenCEZ-2400)atconstantcurrentmode, and theoutputvoltagewas recodedwith adigitalmultimeter (Fluke 287) and thedatawere transferred to apersonal computer (PC).Thered lines indicate theshieldedpart in theapparatus.Weprepared the shield fromconventionalmediumdensity fiberboard, aluminum mesh, and 0.1 mmthicknessaluminumfoil, asshown inFig.5.Withoutthe shield, 100Hznoisewith 250mVpeak-to-peakfromthe flickeringof fluorescent lighthindered themeasurement.Thepreparedshieldcouldreduce thenoiselessthan5mV.

　

Hereshowsthesequenceof themeasurement.First,purewaterwaspoured in theglasspetridishandmeasuretherelativevoltagebetweentheion-selectiveelectorode/ nanopipette and reference/counterelectorode,Ag/AgClwire immersed in thedish asshowninFig.4.Therelativevoltagewasadjustedtozeroafterthemeasuringvoltagewassettled.Thenthedishwaschangedto0.01mMsolutionandmeasuredthe relativevoltage.After themeasurementof 0.01

Fig.4 (Color online) Schematic diagram of the experimental set up for conventional glass tube ion-selective electrode (a) and ion-selective nanopipette (b).

Fig.5 (Color online) Photo of the shield used for the prevention of the noise on the experiments shown in Fig. 4.

(a)

(b)

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mMsolution,thedishwaschangedto0.1mMsolution.Inthisway, thesolutionwaschangedandmeasuredfrom0.01 to 100mM, and the same sequencewasconductedonCsCl,KCl,NaClsolutions.

3．Results and discussion

　First, the resultson theconventional cesium ion-selectiveglass tube electrode are shown.Figure 6shows themeasuredrelativevoltagesonCs,K,andNa ionsat theconcentrations from0.01mMto100mM.Table 1 shows themeasured relativevoltagevaluesonCs,K,andNa ionsattheconcentrationsof0.01,0.1,1,10and100mM,respectively.Theobtainedrelativevoltagevalues forcesiumionare largerthanthoseforpotassiumandsodiumions,whichprovestheselectivityofthepreparedelectrode.

　Selectivitycoefficient wasestimatedfromtheseparatesolutionmethodusingthefollowingequation,29)

　　 (1)

whereEM andEN are the responsevoltages [V] fortheM andN ions at the same concentration,ZMandZN are thevalences ofMandN,F isFaraday

constant [Cmol−1],R isgasconstant [JK−1mol−1],Tis the temperature [K], andCM is theconcentrationof thesolution [M].Sinceall the ionsdetectedbytheion-selectiveelectrode in this studyarecations, thenegativevalueoftheselectivitycoefficientindicatestheselectivity.Table1showstheestimatedselectivitycoefficientvalues.All theestimatedvaluesarenegative,sothattheion-selectivityofthepreparedglass tube electrode has been confirmed at theconcentrationrange from0.01 to100mM.Choiet al.reportedthatthecesiumionselectivityoverpotassium

=−3.7.24) Since they measured with fixedinterferencemethod,differentfromthemethodinthisstudy,directcomparisonofthevaluesisnotavailable.Inanycase, itwasconfirmed that theselectivityofthe ion-selectiveglasstubeelectrodeprepared inthisstudyisaswellastheoneinthepreviousreport.24)

　Next, the results on the cesium ion-selectivenanopipetteareshown.Figure7showsthemeasuredrelative voltages on Cs, K, and Na ions at theconcentrations from0.01mM to 100mM.Table 2shows themeasured relativevoltagevalues onCs,K, andNa ions at the concentrations of 0.01, 0.1, 1,10and100mM, respectively.Theobtained relative

C[mM] 0.01 0.1 1 10 100

VCs[V]VK[V]VNa[V]VNa[V]

-0.763-1.101-1.063-1.063-5.820-5.160

-0.775-1.162-1.210-1.210-6.660-7.480

0-0.04550-0.74030-0.84600-0.8460-13.5000-15.3000

0-1.20900-0.02930-0.24420-0.2442-21.3000-25.0000

02.28000.77200.51800.518-25.90-0-30.30-0

C[mM] 0.01 0.1 1 10 100

VCs[V]VK[V]VNa[V]VNa[V]

-0.0351-0.0185-0.0190-1.0630-0.9210-0.2770

00.062100.015200.0559-1.210--0.807--0.108-

0.12500.00190.0746-0.8460--2.1220--0.8720-

0.13210.05840.0891-0.2442--1.270-0-0.741-0

0.1580.1070.1180.518-0.867--0.687-

Table 1 Measured relative voltage values on Cs, K, and Na ions with conventional ion-selective glass tube electrode and the estimated selectivity coefficients at the concentrations from 0.01 to 100 mM.

Fig.6 (Color online) Measured relative voltages on Cs, K, and Na ions with conventional ion-selective glass tube electrode at the concentrations of 0.01, 0.1, 1, 10, and 100 mM.

Fig.7 (Color online) Measured relative voltages on Cs, K, and Na ions with ion-selective nanopipette at the concentrations of 0.01, 0.1, 1, 10, and 100 mM.

Table 2 Measured relative voltage values on Cs, K, and Na ions with the ion-selective nanopipette and the estimated selectivity coefficients at the concentrations from 0.01 to 100 mM.

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voltagevalues for cesium ionare larger than thosefor potassium and sodium ions,which proves theselectivityof theprepared ion-selectivenanopipette.Also,theselectivecoefficientsareestimated,asshowninTable2.Thenegativevaluesindicatesthattheion-selectivityof theprepared ion-selectivenanopipettehasbeenconfirmedat theconcentrationrange from0.01to100mM.　In principle, the ion-selective electrode has theselectivity that the increaseof theconcentrationoftargeted ion increases the signal significantlywhilethe increase of the concentration of non-targetedions increases the signal little.29)Table3 shows theestimatedslopesand thePearsonrelativecoefficientR2. Incaseof conventionalglass tubeelectrode, theslopevalueforCsis1.65timesforKand1.95timesforNa.Ontheotherhand, theslopevalue forCs is1.07times forKand1.36times forNa,whichmeansthattheselectivitywiththeion-selectivenanopipetteisnotaswellasthatwithconventionalglasstubeelectrode.

　Thereasonof the insufficientselectivity incaseofnanopipettemight attribute to theexistence of theionicleakingpartinthePVCmembrane,whichistheassignmentof thenext study.The leakingproblemduetothemixingof thePVCmembranewithwateron thecuringprocesshasbeenmostly fixed in thisstudy,asshowninFig.3.However,werecognizedtheprecipitationofPVCinwaterevenusingcyclohexaneduringthecuringprocess,confirmedwiththeopticalmicroscope,owingtothedifferenceinsolubilityofPVCbetweenTHFandcyclohexane.Also,wesometimesobservedtheslightdetachmentofthePVCmembranefromthenanopipetteinnerwall,whichcausestheionicleakage.Thecontrolof thecuringprocess, includingthehumidityandtemperature isalsotheassignmentofthefurtherstudy.

4．Conclusion and Remarks

　We have prepared the cesium ion-selectivenanopipette with the ionophore 1 ,3 -a l ternatethiacalix[4]biscrown-6,6.Comparedtherelativevoltageof theCsCl,KCl, andNaCl solutionsat thevariousconcentrationsfrom0.01to100mM,theionselectivitywasconfirmed.Fortheapplicationof thecesiumion-selectivenanopipette to thepracticaluse, thecuringprocess of thePVCmembrane in the nanopipetteshould be reconsidered in order to obtain higherselectivitycoefficient.

Acknowledgment

　WearegratefultoProf.FutoshiIwatawhoprovidedustheglassnanopipettesusedinthisstudy.Thisworkwas supportedby JSPSKAKENHIGrantNumber15K04678.

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Electrode Ionspecies Slope R2

Conventionalglasstube

Cs+

K+

Na+

0.80700.48800.4130

0.9240.8860.844

NanopipetteCs+

K+

Na+

0.03150.02950.0231

0.9360.8600.975

Table 3 Slopes and the Pearson relative coefficient (R2) estimated from the data shown in Figs. 6 and 7.

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