ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
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Neutral Carrier Sodium Ion-Selective Microelectrode for Intracellular Studies R. A. Steiner, M. Oehme, D. Ammann, and W. Simon* Department of Organic Chemistry, Swiss Federal Institute of Technology, Universitatstr. 16, CH-8092 Zurich, Switzerland
junction calomel electrode Philips R11. Ion-Selective Microelectrodes. Glass micropipets were drawn from double-barreled Pyrex glass tubing (W. Krannich KG, D-34 Gottingen, BRD; 80 mol % SiO,, 2 7 ~A1203,13% B203,1% K?O, 3% Ka,O. 1% Fe,O,). They were filled using a modification of the technique described by Lux and Neher (10). The two channels of the microelectrodes contained 0.01 M NaCl and 0.01 M KC1 respectively. The inner wall of the NaCl channel was made hydrophobic by silanization with a solution ( 5 7 ~w / w ) of dichlorodimethylsilane in CC1,. A solution of 10% w/w of the neutral carrier in o-nitrophenyloctyl ether (o-NPOE) (with or without 0.5% wr/w of sodium tetraphenyl borate (NaTPB) in the case of ligand 1) was introduced into the tip of the NaCl channel up to a height of 200 pm. For the characterization of the electrode properties, the reference channel (KC1) of the double barreled electrode was not used (see above); instead an external macro reference electrode was used. E M F Measurements. The ELIF measurements were performed at 20 "C using a digital voltmeter model F 223-A (W-P Instruments Inc., New Haven, Conn.). The operational amplifier had an input impedance of 10'j Q , an input capacitance of 1 pF, and capacity neutralization. It was located directly on top of the electrode. iVith a source resistance of 10" R,the response time ( t o within 1% of final value) is according to the manufacturer's specifications about 0.4 s (evidently response to 90% (11) is faster). The electrode assembly and other high impedance components were located inside a faraday cage. Reagents. All solutions were made from doubly distilled water and salts of the highest purity available. The synthesis of ligand 1 is described in detail in ( 9 ) . Ligand 2 was prepared by a similar procedure. Selectivity Factors, Electrode Response. Selectivity factors were obtained by the separate solution method (SSM, 0.1 M metal-chloride solutions), following the IUPAC recommendations (11). The activity coefficients used are described in detail in (12,
Na+-selective microelectrodes based on new, synthetic, electrically neutral ion carriers are described. The selectivities in respect to K+ and Mg2' are adequate for intracellular measurements of Na' ion activities 1. 10 mM at ionic backgrounds of 100 mM K+, 2 mM Mg2+, and lo-' M Ca2+. Electrodes with tip diameters of around 1 pm have an electrical resistance of about 10" !! and a 9 0 % response time as specified by the IUPAC recommendation of 1 5 s.
Microelectrodes with tip diameters of about 1 pm may be used for the potentiometric measurement of intracellular ion activities (1-3). Although Ka+-selective glasses have been described ( 4 ) which have a sufficiently high selectivity with respect t o K+ for intracellular studies, t h e corresponding microelectrodes have not found wide acceptance in practice ( 3 ) . T h e most attractive glasses of t h e type NAS11-18( 4 ) possess elevated glass transition temperatures and therefore give rise t o vexing technical problems in the preparation of microelectrodes ( 1) . They commonly have a n active membrane of around 10-pm length. In connection with the penetration into cells, this introduces uncertainties which are minimized by using recessed-tip glass microelectrodes (5), which, however, suffer from disadvantages like t h e volume of the recess (5),the possibility of tip blockage (51,and a rather sluggish response (5) which might partly be a consequence of the possible contamination of the glass surface by proteins (see also (6)). Since t h e hydration of the glass seems t o be essential for t h e electrode response. a repeated reactivation of t h e glass is necessary ( 5 ) . Microelectrodes based on liquid ion exchangers sidestep these drawbacks ( 7 , 8 ) . For the measurement of intracellular Na+ activities, an exchanger based on the antibiotic monensin has been suggested (8). Since intracellular ion concentrations are around 200 m M and 10 m M for K+and Na+, respectively, t h e N a + / K + selectivity of 13 claimed for this electrode (8) (KNaKPot = 0.08) is meagre: in the extreme case presented, the interference by K' introduces a n error in t h e Na+ determination of more t h a n 10070. Similarly, the high intracellular Mg2+concentration is a serious source of interference. Recently Xa+-selective neutral carriers have been developed which induce a K+ discrimination in PYC-liquid membrane electrodes ( 9 ) comparable to Ea+-selective glass membrane electrodes. Here we report on the use of this and a similar ligand as ion-selective components in microelectrodes of high Ea+-selectivity.
13).
RESULTS AND DISCUSSION Among the Na+-selective components suitable for liquid membrane electrodes. the neutral carriers 1 and 2 (Figure 1) are the most attractive ones with respect to selectivity (13). In Table I, selectivities of microelectrodes based on these ligands are compared with those of other Na+-selective electrode systems. Except for Li+, ligand 1 exhibits higher selectivities than ligand 2. Therefore only electrodes with ligand 1 have been studied in greater detail. To reduce t h e electrical resistance of the microelectrode, sodium tetraphenyl borate was added to the ion-selective liquid as described earlier for other systems (15-17). As expected, there is some loss in selectivity with respect t o divalent cations (18). Since t h e pivotal Na+/K+-selectivity is not affected, and simultaneously a reduction in resistance from 2 X 10" 12 to 10" 12 is obtained by adding sodium tetraphenyl borate, this type of electrode is the most attractive for practical applications. A linear regression over t h e response function of such a n electrode M NaCl yields a slope assembly over the range IO-' t o of 53.0 f 2.5 mV (s.d.; N = 3) theoretical: 58.2 mV at 20 "C; see Figure 2). Based on the selectivity factors given in Table I, t h e electrode response (EMF vs. log aNa)presented in Figure 3 was calculated using the Nicolsky formalism (11). Assuming
EXPERIMENTAL Electrode System. Cells of the type Hg; Hg,Cl,, KCl(std.) 1 M CH,COOLilsample solution11 \
/ . 4 -
external macro reference electrode
membraneii0.01 Yi NaCl, AgCl; Ag . . l -
1 on-sel ective
microelectrode
have been used. The external reference electrode was a double 0003-2700/79/0351-0351$01 0010
c
1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
Table I. Selectivity Factors log KNaMPotfor Different Na Microelectrodes +
log pot for M'+ acetylH' choline' electrode based o n Mgz+ C a 2 + Srz+ Ba2 method Li' Cs' K' Rb' --0.7 -1.1 -2.2 -2.7 -1.3 -0.9 ligand 1 (ETH 2 2 7 ) SSMa 0 . 1 M 0.4 - 1 . 7 -2.0 without TPBligand 1 (ETH 2 2 7 ) SSM 0 . 1 M 0.4 - 1 . 7 -1.6 -1.8 -2.4 0.2 -0.2 -0.4 -0.8 -1.8 with TPB- 2.3d ligand 2 (ETH 2 3 7 ) SSM 0.1 M 0.2 -1.1 -1.4 - 1 . 6 -1.9 -0.1 -1.0 -0.9 -0.1 without TPB' monensin ( 8 ) F I M ~1 ~e -1.1 -0.8 -2.2 -1.1 Na-glassc ? -3.0 -3.0 -4.3 -3.0 -4.3 3.0 (NASI , - I (4) a SSM: Separate solution method ( 1 1 ). FIM: Fixed interference method (11 ). Typical values for a Nz+-glass macroelectrode (see (14)). Value adjusted t o the Na' response; background electrolyte considered (see Figure 3 ) . e Experience with various electrode systems ( 2 4 ) shows that the SSM (0.1 M ) consistently gives t o o conservative estimates for the selectivity factors as compared t o the FIM (1 M). +
R)
EMF
T
[m"l
K*
200 0 mM
Mg2* 2 0 mM
50 mV
C2*
O01rnM MONENSIN
CARRIER ETH 227 WITHOUT TPB'
Figure 1. Na+-selective neutral carriers EMF
r
I NEUTRAL CARRIER ETH 227
-5
-4
-3
-2
-4
-3
-2
-1
log a
Na Figure 3. EMF response of microelectrodes to changing Na' activities at constant ionic background. Solid cutves: response computed using selectivity data of Table I and actual slopes of the electrode response (monensin: see (8); neutral carrier 1: see Figure 2). Dashed curve: response computed using selectivity data of Table I and a value KMpd of 5 X found to best fit the experimental results. Dots: experimental values. An arrow indicates the detection limit ( 1 7 ) and a vertical bar represents the physiological Na+ activity
-1
toga
Figure 2. EMF response of the Na+-microelectrode cell assembly (based on ligand 1) to aqueous solutions of the chlorides of Na+ and K'. All measured EMF values were corrected by the Henderson formalism ( 12) before plotting and regression analysis
a representative intracellular fluid containing 200 mM K+, 2.0 m M Mg'+, 0.01 m M Ca2+,varying the amount of Na+ makes obvious t h a t the neutral carrier electrode shows a detection limit (indicated by arrows (11)) which is by about a n order of magnitude lower than the one of the monensin based microelectrode. An analysis of the Nicolsky equation
indicates that the detection limit of the monensin based sensor is given mainly by the interference of K+ and Mg'+ whereas the neutral carrier electrode suffers almost exclusively from K+ interference. In contrast to the monensin based electrode, where the detection limit is slightly above the physiological Na+ activity (lo-*M, vertical bar in Figure 3), the neutral carrier electrode does make intracellular Na+ activities accessible. This is all the more true if the intracellular Kt concentration becomes smaller, as is observed in several living cells (19-21). \%h' ereas the classical Na+ selective glass electrodes exhibit a preference of H30+over Na+ by a factor of about lo3 (Table I), the neutral carrier systems discriminate H30+with respect to Ka+. Therefore p H changes of the sample solution in the range of pH 2 to p H 10 do not heavily influence the EMF if the concentration of Na+ is kept constant either at 10-' M (fl mV (2 < p H < 10)) or M (h5 mV ( 2 < p H < 10)). Therefore pH changes over the physiological range will not
ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
interfere with the Na' determination. T h e 90% rise time of the electrode obtained by adding 1 m L of 1 M solution of NaCl to 50 m L of a M NaCl is about 5 s, significantly longer than the rise time of the electronic equipment with a source resistance of 10" Q. T h e double barreled electrodes (tip diameter 2 pm) described here have a resistance of about 10'O R. Since the speed of mixing in the sample solution after the stepwise addition of NaCl solution is rather critical, the actual response time of the sensor will be considerably smaller than indicated. Indeed, 90% response times of 0.2 s were reported for the same electrode (22). T h e lifetime of the microelectrode in contact with aqueous electrolytes is several weeks and the drift is 51 mV/day. T h e microelectrode described here is designed for intracellular use. A first series of intracellular measurements has been successfully conducted (22); in analogy to observations on other microelectrodes based on electrically neutral carriers (13), no interference due to the presence of proteins was detected. Since the extracellular ion activities differ sharply from the intracellular ones, extracellular measurements with this electrode are prone to interference due to the rather poor Naf/Ca2+-selectivity. It is a fair assumption that a useful Na+ microelectrode on the basis of electrically neutral ligands can be developed for extracellular work, inasmuch as correhave sponding macroelectrodes with KNaCaPot as low as been described (9,23). Because of their small selectivity with = 5 X lo-' (9,23))!their application respect to A? (KNaKPot will be limited to extracellular environments. The same might hold for the monensin-based electrode (see Table I (8)).
LITERATURE CITED (1) M. Lavallk, 0. F. Schanne, and N. C. Hebert, Ed., ''Glass microelectrodes'', Wiley & Sons, Inc., New York, London, Sydney, Toronto, 1969.
353
H. J. Berman and N. C. Hebert, Ed., "Ion Selective Microelectrodes", Plenum Press, New York, London, 1974. M. Kessler, L. C. Clark, Jr., D. W. Lubbers, I . A. Silver, and W. Simon, Ed., "Ion and Enzyme Electrodes in Biology and Medicine", Urban and Schwarzenberg, Munich, Berlin, Vienna, 1976. G. Eisenman, Ed., "Glass Electrodes for Hydrogen and Other Cations", M. Dekker Inc., New York. 1967. R . C. Thomas, in ref. 3, p 141. R. N. Khuri, in ref. 3, p 123. J. L. Walker, Jr.. Anal. Chem., 43 (3), 89A (1971). R. P. Craig and C. Nicholson, Science, 194, 725 (1976). M. Guggi, M. Oehme, E. Pretsch, and W. Simon, Helv. Chim. Acta, 58, 2417 (1976). H. D. Lux and E. Neher, Exp. Brain Res., 17, 190 (1973). G. G. Guilbauit, R. A. Durst, M. S. Frant, H. Freiser, E. H. Hansen, T. S. Light, E. Pungor, G. Rechnitz, N. M. Rice, T. J. Rohm, W. Simon, and J. D. R. Thomas, "IUPAC Information Bulletin, No. l " , 1978, p 70. P. C. Meier. D. Ammann, H. F. Osswald, and W. Simon, Med. Progr. Techno/., 5 , 1 (1977). P. C. Meier, D. Ammann, W. E. Morf, and W. Simon, in "Medical and Biological Applications of Electrochemical Devices", J. Koryta, Ed., John Wiley & Sons, Ltd., Chichester, 1978 (in press). K. Cammann, "Das Arbeiten mit ionenseiektiven Eiektroden", Springer Verlag, Berlin, Heidelberg, New York, 1977. M. Oehme and W. Simon, Anal. Chim. Acta, 86, 21 (1976). M. Oehme, M. Kessler, and W. Simon, Cbimia, 30, 204 (1976). R . C. Thomas, W. Simon, and M. Oehme, Nature (London), 258, 754 (1975). W. E. Morf and W. Simon, in "Ion-Selective Electrodes in Analytical Chemistry", H. Freiser, Ed., Plenum Publishing Co., New York, 1978 (in press). T. Zeuthen, J . Mernbr Biol., 39, 185 (1978). J. A. Coles and M. Tsacopoulos, J . Physiol., 270, 12P (1977). S. W. de Laat, W. Wouters, M. M. Marques da Silva Pimenta Guarda, and M. A. de Silva Guarda, Exp. Cell Res., 91, 15 (1975). S. Levy. Experimental Ophthalmology Laboratory. Geneva, Switzerland, private communication, 1978. D. Ammann, R. Bissig, Z.Cimerman, U. Fiedler, M. Guggi, W. E. Morf, M. Oehme, H. Osswald, E. Pretsch, and W. Simon, in ref. 3, p 22. W. Simon, D. Ammann, M. Oehme, and W. E. Morf, Ann. N. Y . Acad. Sci., 307, 52 (1978).
RECEIVED
for review August 28, 1978. Accepted November
9,1978.
Reticulated Vitreous Carbon Flow-Through Electrodes A. N. Strohl and D. J. Curran" Department of Chemistry, GRC Tower I, University of Massachusetts, Amherst, Massachusetts 0 1003
Flow-through electrodes have been fabricated from Reticulated Vitreous Carbon (RVC). This material has low electrical resistance, large surface area, and a physically continuous structure. The resistance to solution flow is low and flow rates up to 25 mL/min were easily attained. Solutions in the concentration range 1 to 1000 pM produced currents from about 1 to 1000 PA, respectively, using flow rates from 0.5 to 25 mL/min. Current-voltage curves were generated using these electrodes. At constant potential, the analyte solution was pumped through the electrode at a constant rate, thereby generating a steady-state current. The linearity and slope of calibration curves (steady-state current vs. concentration) were found to depend on flow rate. Higher sensitivities were obtained at higher flow rates and coulometric conversions occurred at the lowest flow rates. Ferricyanide ion and ascorbic acid were used as test systems, and ascorbic acid in vitamin C tablets was determined with results that were in excellent agreement with a titrimetric check method.
Electrodes based on various forms of carbon have received increasing attention in recent years for use in flowing streams. In general, three configurations have been used which are described by the position of the electrode in relation to the 0003-2700/79/035 1-0353$01 O O / O
flowing stream: flow-by, where the electrode surface is parallel to the direction of flow; flow-onto. where the surface is normal to the direction of flow; and flow-through. Pungor and coworkers have developed a silicone rubber based graphite flow-by electrode (1-3) and have summarized earlier work. The same electrode has been used for turbulent hydrodynamic voltammetry in a flow-onto mode (4-6). Carbon paste flow-by electrodes have been used by Kissinger and others in electrochemical cells for liquid chromatography detectors ( 7 ) . Curran and co-workers ( 8 ) have used a pair of carbon-wax electrodes for the same purpose. Two types of flow-through electrodes can be distinguished, depending upon whether the electrode is simply a hollow cylinder or is a porous matrix of some sort through which the solution flows. SVith respect t o the former, the work of Blaedel with tubular electrodes should be mentioned (9). An excellent discussion of the latter is found in the paper by Johnson and Larochelle where a packed electrode is used as a coulometric detector for liquid chromatography ( I O ) . Various forms of porous matrix flow-through carbon electrodes have been used previously. A review of electrolytic chromatography by Fujinaga and Kihara ( 1 1 ) describes the work of these authors with columns containing glassy carbon grains, which served as the electrode material. Crushed graphite was used in a short column electrode by Blaedel and Strohl (12). Finely divided 1979 American Chemical Society
