ion specific liquid ion exchanger microelectrodes

Advisory Panel INSTRUMENTATION I Jonathan W. Amy Donald R. Johnson H a r r y L. Pardue Jack W. Frazer Charles E . Klopfenstein Ralph E . Thiers G Phillip Hicks Marv in Margoshes Wi l l iam F. Ul r ich

I

Ion Specific Liquid Ion Exchanger Microelectrodes

Miniature ion specific electrodes with liquid ion exchanger membranes provide a convenient means of measuring intracellular ionic activities in living cells

JOHN L. WALKER, JR., Department of Physiology, University of Utah College of Medicine, Salt Lake City, Utah 84112

Since the dc~dopmenr of ion specific e3 for hydrogen nnd the alkali ?:it- microelectrodes have lieen iabri-

caret1 f r o m tlieae gln-scs ( 4 . 3 ) . Chlo- ride microdecrrode? l i n w lieell i ~ x t l c by coating :I plntiiiiim n- irom the cntl of :I r.1 with -lg--lgCl ( 4 ) or li!. clepo-iting silver cliloricle in-icic the tip of n micro- 1)lpeT (6). The-c electrockc a11 h v c thr tlr:in.ixick of linviiig :I large ion ,5eiili-

tivc ,.iirfaec. which I n i i s r lie entirelj. within the cell n n d their ii>e is, tliere- forc. limited to cellb the size of skeletn! mnsclc or larger. Vliilc these electrotlcs m n ~ - linl-c :L t ip dinmeter of oiic niirron o r h 5 , tlic scnsitii-e nren is on The order of ten microns i n length n i th n t l inm- eter of five microns or more 3t the to11 of thi.5 nren, In :iddiTion t o the problem of size, the!. :irr tlifficiilt t o fnhricntc.

Liquid Ion Exchangers

Liqiiitl ioii esclinnr.cr~- h i - c liccn i i w l iii lirliiitl-liquid ioii crtr:iction processe. i n intiiiqtry for sonic time n i i d , from timc to time, 3s niotleli for liioiopicnl nicnil)r;iiic's ( 7 , 8 1 . In the p n s t decndr, T I F ~ C . ~ : i q reiicwetl intrrcst in liyiiid ioii cscli:iiigcri on the p i r t of hiok)gi~~T,5, :iiid thc thcor!- of tlicir lx4invior n~ ion sclcc t ivc m em1 r,i n c K : L ~ clewlo pcd ( 9, 1 0 ) . .\i the m n c time. :I practicnl ~ i l - c i u m clcctrotlc ~vn: dewloped n-hirli iitilizcs :I liqiiid ion csclinnpcr mcm- 1ir:inc tlic w i ~ i t i ~ - r clcmcni of tlic clectrncle ( I 1 ) . Since TllPIl, m - c r d

other ion specific liquid ion erclinnger clccrrodcs I inr-e lxcome coiiimerciall!- :iv:iilalile, This piper dimisses the 1 i i i i i i : i t~ i r i~~ i t io I i of tliccc electrodc,~ for i l i r c>spi-c+ purpose of iiicnwring in- trncellu1:ir ionic activities i n living cells.

-4 I i c I u i c l ion erclinnger i- cornpoaed of :in organic electrolyte tlisdo!vecl in :I

n-ntc'r-iiiiIni-cii)!(' .solI.ciit, ii-ii:illj . :in o r g x i i c d v c n t n i th :i low dielectric Co1i~t:lliT. The organic ion -1iould have ;i Ion- vxter 5olubility :inti is often liiglily hra~iclied to prmwit iiiicelle for- m:ition ( I ? J . Owing i o The low dielrc- tric coiict:iiit of the esclinnger, inorganic ion.. l i aw :I w r y low di i l i i l i ty i n the c~scl1:inger n i i t l . convqi ient l ! , , n nieni- 1)r:ine made of :I liqiiitl ion cschnnger i - m ~ i c f i nigre perincnhle to ions n.liosc v:iIcmce s ign is opposite t o t l int of The org;miie ion 11i:in to ions of tlir -rime

\-nlcncc ;ign liccnuse of ion p i r formn- t ion ivi th The orgnnic ion. For eznm- ple, negatively clinrgctl 1ilioqdi:itc es- ter- :irr cniion cscliaiigcr~~ while posi- tively cli:irgetl nminr. :ire miion cz- c l i a n p c r ~ . Fnri hermore. some csclin11g- rr,< csliii)it mnrkccl sclcc,tivity 17itliin a gro\ip of ions of the s i m e valence signI tiic zclcctivity lirincr n fiinction of the , - t r cn~ th of iiitcrnction hcTWW1 the or- g n r i i (, ni I tl i no rpn n ic ions.

.\n ion .qmifir ?l?cTrotl? i; made b y iorminz ;I liquid ioii cschnngrr mem- 1)r:inr i n :I -iiitnhlc lioltlcr PO thn t one iitlc of the mcmhrnnc is in contact n-ith nu :i(iiicoii. rcfcrencc mliition of con-

Table I. Dimensions and Volumes of Some Electrically Excitable Cells Kind of cell Cell shape Diameter Length Volume

Molluscan neuron Spherical 1 mm 0.52 pI

Frog skeletal muscle Cylindrical 0 . 1 mrn 30 mrn 0.25 pl

Frog ventricle Cylindrical 0.01 m m 0.13 rnm 1 X 10-j HI

ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971 8 9 A

Instrumentation

stant composition while the ot’her side can be brought into contact wit>li the solution to be analyzed. Electrical contact is made with tlie internal reference solution by means of an electrode which is reversible to one of the ions in the reference solution. The electrical potential difference across the membrane changes in proportion to the activity in the test solution of the ion(s) for which the liquid ion exchanger is selec- tive.

-4 quantitative theory has been de- vcloped (9. I O ) which describes the electrode potential, but it is cumber- some and contains parameters which ;ire difficult to measure. It is, therefore, more convenient to use the following empirical equation:

E =

E i- tlie electric potential (volts) ; E, is :i constant (volts) : R is the gas constant (5.3 joules deg-lmole-1) ; T is t’he tem- perature (OK) : F is Faraday’s number (96,500 coulomb equivalent-1) ; n is an empirical constant’ (dimensionless) cho- ,sen so tliiit n R T / F is the slope of tlie line n-lien E is plotted as a function of logt, ai when Kija j = 0 ; zi and z j are the valences of the ith and jtli ions, respectively; ai is the activity (arbitrary concentration iiiiits) of the ion tlie electrode i.5 espectecl to measure; the aj’s :ire the activities (same units as ai) of intcrfcring ions whose vrilence sign 1s

that of the principal ion and the K,j’s are the selectivity constants for tlic jtli ions with respect to tlie ith ion. When K , , < 1 the electrode has a higher eclectivity for the ith ion than for the jtli ion. K i j is an empirical number and slioiild not be assigned a strict physicnl interpretation.

The problem, then, is to form a liquid ion eschangcr membrane in a holder smnll enough to insert into a cell so that) one surface of the membrane is in contnct with the intracellular fluid.

, .

Electrode Fabrication

-4 standard technique for making intracellular electric potential measurements has been to piill a glass micropipet with a tip diameter of about 0.5 micron and fill it with 311 KC1. Es- tensive research with this technique has shown that when the tip of such a pipet is inserted into a cell, it does not dis- rupt the normal functioning of the cell (IS) and, therefore, if a liquid ion exchanger membrane could be formed in the tip of such a pipet, the problem would be solved.

The primary difficulty is that a clean glass surface is hydrophilic, so an or-

ganic liquid membrane will not reniaiii in place in the tip of the pipet. Instead, it will lie displaced by one of the nque- 011s phases with wliicli it is in contact. To prevent this, it is necessary to ren- der hydrophobic the portion of the pipet wliicli is to contain tlie organic liqiiid. The metliod used to do this is to apply a n organic silicone compomici to tlic terminal 200 microns of the pipet tip . 1 buccessfiil method for making po-

tassium and chloride microelectrodes involves the folloring steps, Boro,sili- care ghss capillary tubing (1.2 mm od, O..?-mm thick 1v-311) i:: clennecl with hot etlianol vapor and dried. Pipets are piilled from the clenn tubing with a commerci:ill\. nvailable pipet puller niid Iinve n tip diameter of 0.5 to 1.0 micron. Immedintely nfter pulling, the pipet t i p are clipped in :i fresh solution of Silicliad (Clny-.kIams) in 1- cliloroiiaplitlinleiie i i n t il tliere is a col- limn of the solution about ‘200 microns long iiisitie tlie tip. This tnkes npprosi- nintel!. 13 sec. The pipets :ire then p1:iced tip 111) in :I metal lilock and n-lien the de3irctl iiumlm (onc to two dozen) linve I~ecn prepnrcd, the!. :ire placed in :I 250°C oven for one lioiir. *\fter hc- ing removed from the oven anti allonerl t o cool, the pipets ;ire rend!. for filling hut can be held in this condition for :it least one week before being filled.

To fill a pipet, the tip is dipped into tlie ion exclinnger (Corning code 477317 potassium exchanger or Cor- ning code 377315 chloride esclinnger) iintil tlie terminal 200 microiis (np- grosimatel!.) of tlie ti11 is filled ivit l i the esclianger (one to tn-o minutes). One-half molar KC1 is then injected as far doivii into tlie top of the pipet as possible using a three-in., 30-gauge needle attached to a syringe. The pipet is placed horizontally on the movable stage of n compoiind microscope and n hand-draIvn, solid glass needle monnted 011 a micromanipulator is advanced down the inside of the pipet while view-

~~ ~~

ing under lO0X magnification. The tip of tlie glnas needle is brought to the cen- ter of the field and tlie microscope stage is carefiill!. moved until the tip of the needle touches the meniscus of the ion eschanger. The KCl solution flows doivii to the liquid ion eschanger, dis- placing the air toward the top of the pipet where it can he fluslied out using tlie 30-gauge needle. -4ir bubbles of 100 microns or less in length may be disre- garded since they will be quickly ab- sorbed. With practice, electrodes can lie dipped and filled in an average of five min. -4 sninll amount of mineral oil i$ then injected into the top of the pipet to prevent water evaporation, nnd the pipet is stored with the tip in a solution of 0.5.11 KC1. Best, results are ob- tained when the filled electrodes nre al- lowed to stnnd for a t least two hr before being used. Figure l shows a sclie- mntic dingrLiin of an electrode ready ior ii.se. and Figure 2 is a pliotograpli of :I potassium niicroelectrode nitli 125 microns of liquid ion esclianger in the tip.

Tn-o tlificulties have been encoun- tered with these electrodes. Sometimes the tips of tlic electrode; will not fill with the ion esclinn vcrj. slo~vly. This : i l ) l ~ . , t o tlic tips being plugged by the Piliclnd. If n pipet doe. not t A e up the ticsireti niiioiiiit of eschnnger within two inin, it is dikcarded. =in nlterna- tive to this is to break the til) slightly, biit this is onl!. useful if n tip diameter of two micron: or lnrger caii be toler- ated. Tlie other difficulty is that the electrodes n-ill sometimes lose the eschanger n-itliin two to three lir after being filled. Tlie cause for this difficulty lins not heen determined. To alleviate tliese problems, other siliconizing pro- cediires wing n variety of organic clilo- rosilnnes in different solvents are being investigated.

.4lthougli both kinds of electrodes csnn be made in the same way, there is another method of siliconiziiig which

Figure 1. Schematic diagram of an ion specific l iquid ion exchanger microelectrode

9 0 A ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971

B x

Figure 2. Photograph of a potassium liquid ion exchanger microelectrode with 125 microns of ion exchanger inside the tip. Magnification, 400X

works better for the potassium electrodes but is not satisfactory for the chloride elcctrodes. After bbing pulled, the pipet tip is dipped in a 5% solution of tri-ii-but~~lclilorosilane in 1- ehloronaplitliale~ie until there is a col- limn about 200 microns long inside tlie tip (about 15 see). The pipet is then :~lloa.ed to air dry for a t least 24 hr before being filled as described above. The tips of pipets siliconized in this m y do not plug up ns they sometimes do vith the Siiiclad solution.

Electrode Resistance and Selectivity

Because of the small size of the tip of the electrodes and the lorn conduc- tivity of tbe liquid ion exchangers, the resist.ance of the electrodes is liigh and a voltage measuring device with an input impedance of a t least 10'3 ohms is required. 4 satisfactory solution has been to use a varact.or bridge operational amplifier (Analog Devices, model 31iK) which has an input impedance of 1014 ohms with an input capacitance of two picofarads. The microelcetrode is connected to the input of the amplifier via a chloridieed silver wire inserted into its top, and the ampiifier output is connected to a digital voltmeter. The resistance of the microelectrodes, as measured from the charging time of the input a i the operational amplifier,is in the range of 109 to io1" ohms.

The response time of the electrodes when the concentration of potassium or chloride is changed has not yet heen measured carefnlly hecause of the tech- nical problems involved. However,

when one of the electrodes is dipped into a solution and the amplifier input is opened as rapidly as possible, the new steady-state potential is reached within five see, which indicates that the time constant of the response is not more than one see. Once the steadystate potential is attained, it remains constant to within -C 1 mV for several hours. I n Table 11, some data from experiments ivitli Aplysia neurons (14) are presented showing the long-term stability of both t.he potassium and chloride electrodes.

The selectivities of the electrodes for interfering ions with respect to the principal ion has been determined for some intcrfering ions commonly found in biological preparations. These have been determined by measuring the clect,rode potentials in mixtures of constant ionic strength and finding the Kij which makes the data fit Equ a t' ion 1. The results of these measurements

are presented in Table 111 for the chloride electrode and for potassium electrodes made by both of the methods described above.

Making Intracellular Measurements

Immediately before making an intracellular measurement with one of the electrodes, the electrode must be calibrated. This is done by measuring its potential with respect to B 3M KCI-filled micropipet in a series of KCI solutions o i known concentrations. The KCI concentrations used should bracket the expected concentration of the unknown solution. The electrode is then moved to the solution bathing the cell in which the measurement is to be made, which normally contains some of the ion to he measured, potassium andlor chloride. The potential of the electrode in that solution should agree with the potential predicted for the electrode from tlie calibration curve, taking into account any interfering ions which may he present in the solution. The tip of the electrode is then inserted into t,lie cell. Since it is not possible to see the t ip of the electrode miter the cell, the criterion used to determine cell entry is an abrupt shift in the potential of the electrode as it is s l o r l y advanced toward the cell. This shift in potential is dne to two fac- tors: (1 ) the difference in activity of the ion being measured between the ex- traeellnlar and intracellular fluids; and ( 2 ) the cell membrane potential. The shift of dlie elcct,ric potential as the electrode tip enters the cell can be written as:

AE =

ai" + Kijaj.

a t + Kija,i where A l 3 (voltq) 1s the difference in electric potential between the inside :md the outside of the cell; E,,$ (volts) is the cell membrane potential; the

Table II. Data from Experiments on Aplysia Neurons with Chloride and

Potential in artificial Potass ium Microelectrodes' (14)

Slope, mV seawater, mV Time in Before' Aftera Before2 After2 cell@), min

58 58 58 58 58 54

58 ~. 58 58 58 58 54

34 31 48 44 35 46

34 30 48 44 36 44

335 486 137 118 347 495

'First five measurements are with potassium electrodes and last five are With chloride eiec- trodes. In most experiments, more than one celi penetration was made with the electrode during the indicated time. Wefore and after intracellular measurement(s1.

ANALYTICAL CHEMISTRY. VOL. 43, NO. 3, MARCH 1971 91 A

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9 2 A ANALYTICAL CHEMISTRY, VOL. 43, NO. 3 , MARCH 1971

Instrumentation

Table 111. Selectivities for Interfering Ions with Respect to Principal Ion for Chloride and Potassium Microelectrodes

Interfering ion O.lM1 1. OM1 K I J ~~~

Bicarbonate2 I set hiona te2 Propionate2 Calciu r n 3

Hydrogen'

Sodiu rn

0.05 0.2 0.5 0.002 0.002

a' b

0.05 0.2 0.7 0.03 0.03

0.02 a 0.03 0.025 b 0.016 0.02 a 0.02 0.02 b 0.014

1 Ionic strength. .' Ions interfer ing wi th chloride. I Ions interfer ing w i th potassium. ' a and b refer t o Sil iclad and tr i -n-butylchlorosi lane potassium microelectrodes, respectively.

superscripts on tlie activities, o and i, refer to outside and inside of the cell: xiid j t . tic~tcrmined from the calibratioii curve for each electrode, is 1.0 for the

iiiiii electrodes :inti in the range of 0.!10 to 0.97 for the chloride electrodes. Ts'liilc ) i varies from one cliloritlc electrode t o another, it is constuiit for any one electrode and cmst : i i i t over tlic range of :it least 1.0 x 10 :{JI KC1 to 1.0.11 KC1 for liotli pot:i-siuni antl eliloridc clectrodes. I;,,, i i nic:isureti by impaling tlie cell n-ith :I 3.11 KCl-filled micropipet,

Klieii Equation 2 is >oIved for tlie dr~ioniiiiator of the 1og:irithmic term, it t:tkc- tlic form of Equation 3.

all + f i , , u l l =

It is ion. :I biinple matter, knowing all the fnctors 011 tlie right side of Equa- tion 3, to cdciiltite :i numericnl value for the left d e . The only remaining prolileiii is to determine the 1-due of tlie t m n , Ai,cr,l. If microelectrodes , - p i f i r for the interfering ions are :ivnilable, tlie q i r s cni i be niewured di- rectly. I7nfortumite1yj this is not 11s- 1i:iil:. po,Gsilile and, therefore, it is necessary to use e4mntcs arrived at by other nicaiii. TYlieii whole t imie annly- TS h v e lxen done, the values of the coiiccntratioiis can lie iised by assuming values for tlic intrncellular activity co- rflicieiits ( 2 ) . 1ntr;icellulnr sodium ne- tivity c n n lie estimated from the over- s h o t of t l i p nction yotmtial (?,? 1, and in the c a w of ion.? which appear to be

i d y distributed across the cell mpmhranc, tlic intracellular activity can he calculated by iisinp the extracellular activity of t ha t ion a n d assliming a Doiiiiaii distribution (-31.

TVlien the cells are large, a,= in tlie ( m e of .4pl,vsin neurons or frog skeletal mmsclc, the IiC1-filled micropipet c a n be srcn i n tlic sninr cell as tlie ioii sgecific electrode, hiit with srn:illPr cell9 t1ii.s

i,s iiot po.ssible. Khen the cells are too small to see if both electrode.5 are i n the sxne cell, tlie membrane potential and ion specific electrode measurements can be made separately in ,m-cr:il cells and the average vnlues of tlie iiien$uremeiits used to calciilate in- trace1lul;ir :ictivity. -4 better solution to the problem is to make double-b;irrcl electrodes where one barrel is tlie ion sliecific electrode and tlie otlier bnrrel i-. tlie reference electrode. -4ttempt.s to hliricate fiicli electrodes are currently lieiiig nixle . -I> .slion-n in Table 11, both tlie

iiot:iAum :iiid chloride microelectrode. :ire stable over a period of severd lioiirs even n-hen several cell penetrn- tion+ are m n t l e with the same electrode. Wien an electrode has slionii n DC drift, it has usually been traced to a cliniige in the potentinl of the .lg- ;IgC1 electrode mnking contnct with the interiinl reference solution of the elec- trodr. Most important is the fact that t h c slopes of the electrodes do not c1i:iiige during tlie coiirse of tlie es- perimeiits. Sirice tlie intracellulnr iiiea.iirenienta ;ire mndc with respect to the estrncellulnr solurion, rvliicli is of know11 composition, a .slow DC drift is of little consequence as long ns the slope of tlie respoiise ci:rve remniii,. c0lldt:lllt.

is the case wit11 any new tcch- iiiqiic, it iq necessary T O c.tnblisli tliat the dntn obtniiied with the technique are vdid . In measuring iiitracclliilar :le-

tiTiTir.5 with ion specific electrodes, this is not ency t o do because of the kick of ;icciir:itr information concerning in- tracc1liil:ir nctil-itirs of ions, both or- gnnir nnd inorganic, n-liicli ma:. inter- fere n i th tlic meawrcment. This is erpecially true of tlie rhloride electrode lxxiii,=r of the presence of organic anion:: in the intrncelhilnr fluid. Some rsperimeiitril re.sii1ts bcnring o n this point linvc l>eeii presented :inti di.=cnssetl liy Cornn-all e t a l . ( 1 6 ) .

.It tlie present time, oiilj- tlie po- irim niid chloride ioii eschanger

microelectrodes are of practical use. Attempts to make a calcium microelectrode have not been comiiletely ,-iicce.d'iil, altliougli come progress has heen made. They can be made to es- liibit n Sern>t slope in solutioiis of C:iCl, from 1.0 x 10-iJI to 1.0.11 with :I time coiistant aiici stability compa- rlible to tlie potassium and chloride microelectrodes. Ilowever, when po- tas>iuni> i n concentrations upproxi- mating those found inside cella, is added to tlie CnC1, solutions, it reduces the calcium re;ponse dra-ticall:.. K o r k is continuing on the calcium niicroelectrode and as liquid ion escliniigers for otlier ions of intere-t to biologists be- come available, the!- ni l1 lie u e d to m i k e ion specific microelectrodes or' tlie type described above.

References

(1 ) ,J. -4, ,Jolinson, Anlei.. J . Physioi.

( 2 1 E. .J. Conivny, Piiysioi. Rrc. 37,

( 3 ) E. Page, J . Gen. Physiol. 46, 201 ( 1962) .

i 4 i ,J. -1. 11. Hinke in ,'GI elcctrotlcs," Laxilee e t Kilt!., S e n - Tork, S. I-. 1969.

( 5 ) S. \I-, Carter, F. C. Rector, Jr., D. E. C,impion, and D. W. Seldin, J . C' l i i i , Zi iz i rs t . 46, 020 (1967 I ,

( 6 ) G. -1. Jierkiit antl R . S T . l l eec l i , LiJC s c i . 5, 453 (19GBi.

(7 ' ) I?. Brntner, A m p i , . J . Physiol. 31, 34:3 ( l 9 l S I .

(Si G. Eisenmnn, ,J, P. Saiidhlom, and .J. L. TT'nlker, ,Jr,, Scie i i cc 155, 965 (I9G7).

9 i ,T. P. Sandblom, G. Eisenman, nnd .J. L. JYalker, Jr., J . P h y s . Chem. 71, 3S62 (1967).

10) ,T. P. Sandblom, G. Eisenman, 2nd , J , L. Ynlker, Jr . , J . P / y s . Cheni . 71, *>\I 1 ( 1 9 6 7 .

c i e ) i c e 156, 1;iTF (10071. 12) I:. liunin a n d -4. G. ITiiiger,

d?igcic$. Chem.. Z i t t . Ed. Eugi. 1, 149 (1962).

(1:; I IT. I,. S;ihtuk n r i d -1. L. Hocigkiii. J . C'cli . C ' o v ) p . Piqsiol . 35, 39 (19501,

( 1 4 ) D. L. Kunzc nnd A. 11. Rroivn, wipiihlislicd rewlts! 1970.

(131 ;1. L. Hodgkin and R. I i n t z , J . P / y s i o / . 108, 37 (1949).

(16) 11. C . Cornrr-all. D. F. Petereoil, n. L. Kunze, ,J. L. Kn lke r , ,Ti-,, Lind -1, 11, Brown, Riniu Rcs. 23, 133 (1970).

181, 263 (19%).

b-1 (1957 1 ,

r , --

. \ ~ a & a t i n n Grant #70 887. Yt,nh Heart .\sincia- tion Giant 1T. and rile Tnitecl Sta tes Public H m l t l i %r\-ire Grnnr G l I 14328.

ANALYTICAL CHEMISTRY, VOL. 43, NO. 3, MARCH 1971 9 3 A

ion specific liquid ion exchanger microelectrodes

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