Anal. Chem. 1990, 62, 2153-2155
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Fabrication Method To Enhance Stability of N,N,N’,N’-Tetracyclohexyl-3-oxapentanediamide Calcium Microelectrodes H. Mack Brown* a n d Sharon K.M a r r o n Department of Physiology, University of Utah, Salt Lake City, Utah 84108 Calcium ion selective microelectrodes (Ca-pISE) have been used extensively for intracellular measurement of Ca2+(1-3) since reasonably sensitive and selective electrodes were developed more than a decade ago (4,5). These early electrodes may be only marginally useful for measurement of Ca2+in cells with high background K+ (200 mM) and Mg2+ (>2 mM) if basal Ca2+levels are below 100 nM. This is the measurement situation encountered in large cells from seawater animals where these electrodes have been used most often. Recently a new ionophore has been developed (ETH 129) that shows promise for punctate analysis of intracellular Ca2+ in cells from seawater animals because of increased selectivity for Ca2+over K+ and Mg2+ (6, 7). However, there was one feature reported that limits the usefulness of electrodes made with this ionophore: sensitivity systematically deteriorates over time. The present communication describes different construction methods aimed at minimizing this deficiency and reports results from solutions with higher background K+ than reported previously. One method described herein significantly improved the stability and longevity of the electrodes.
EXPERIMENTAL SECTION Micropipets. Corning Pyrex tubing (1.2 mm o.d., 0.5 mm i.d.) was pulled in a single step to a tip size of 1.0 pM (Narishige PE2). The pipets (batches of 10) were heated in an oven for 15 min at 200 OC. This was repeated after covering them with a glass beaker containing 30 pM of N,N-dimethyltrimethylsilylamine(Fluka Chemical) to silanize the glass (8). Sensor. The membrane “cocktail” (kindly provided by D. Ammann and T. Buhrer) consisted of 5 wt % ETH 129 (N,N,N’,N’-tetracyclohexyl-3-oxapentanediamide),1 wt % sodium tetraphenylborate, and 94 wt % o-nitrophenyl octyl ether. The cocktail was mixed with 14 wt % PVC (Aldrich, VHMW) and tetrahydrofuran (3 times total weight). Electrode Construction. Three methods were used to make electrodes designated A, B, and C and schematized in Figures 1, 2, and 3. Method A is similar to the method employed by Ammann et al. (6). The pipets were backfilled with an internal reference solution (solution 7, Table I) with a fine needle and stringe. Pressure was then applied to the back of the pipet to fill the void at the tip. The membrane material was introduced into the tip by applying suction to the back of the electrode while the tip was immersed in the mixture of membrane components. Between 100 and 250 pm of material was drawn into the tips; the membrane phase was allowed to ‘shrink” to about 50% of the initial column height before the electrodes were calibrated (30 min). Method B was employed to maintain the effective membrane column height the same from electrode to electrode and to limit exposure of the membrane phase to the aqueous reference solution. The mixture of membrane components was drawn into the tip of a pipet by suction as in A, but the pipet was not prefilled with reference solution. An internal pipet (tip diameter 2-3 pm) filled with aqueous reference solution was introduced into the butt end of the pipet and advanced under microscopic control into the membrane phase. The inner electrode was advanced until the tip to tip distance of the inner and outer pipet was 75 pm. The internal concentric pipet was then sealed to the outer pipet with dental wax. Method C employed air-drying or evaporation of the solvent (THF) from the mixture of membrane components before exposing it to the aqueous reference medium. The tip of the outer pipet was first filled with a column of membrane material to a height of 100-250 pm. The THF in the membrane component was then allowed to evaporate for 24 h at room temperature. 0003-2700/90/0362-2153$02.50/0
Table I. Calibration Solutions pCa 3 4
5 6 7
8 9 m
pH buffer Ca2+liganda CaCl,, KOH, KC1, mM (10mMl (10mM) mM mM MOPS HEPES TAPS MOPS MOPS HEPES HEPES HEPES
1
NTA NTA HEEDTA EGTA EGTA EGTA EGTA
5 5 5 5 5 0.5
6.6 33.0 33.0 37.0 20.0 21.0 15.5 14.2
193.4 167.0 167.0 163.0 180.0 179.0 184.5 185.8
pH 7.30 7.39 8.42 7.70 7.29 7.80 7.80 7.80
Key: NTA, nitrilotriacetic acid; HEEDTA, N-(Hydroxyethy1)ethylenediaminetriacetic acid; EGTA, ethylene glycol bis(@-amino-
ethyl ether)-N,”-tetraacetic acid. Another pipet (tip diameter 2-3 pm) filled with reference solution was advanced under microscopic control into the well formed at the back of the shrunken membrane. The internal pipet was sealed to the outer one by dental wax. Reference solution was then gently ejected onto the back of the membrane by applying pressure to the internal pipet. Calibrating Solutions. The composition of the calibrating solutions is shown in Table I. They are essentially the same as those employed for calibrating an earlier ionophore Ca-MISE (9) with the exception that they contained a K+ concentration of 0.2 M instead of 0.125 M. This more closely approximates the intracellular K+ concentration of cells from seawater animals,which is the emphasis of this communication. Electrode Calibration. The electrochemicalcell for electrode measurements was Ag/AgC1110-’ M CaClzllmembranelICa2+sample13 M KClIAg/AgCl. The salt bridge to the sample was through a glass pipet with tip diameter 20-30 pm. The indicator half cell was led off to a varactor bridge amplifier (input impedance 10“ Q ) . The potential difference between indicator and reference electrode was displayed on a digital voltmeter (Data-Tech) and chart recorder (Hewlett-Packard 7128A). The electrodes were sequentially immersed into sealable cups containing 100 mL of the calibrating solution and allowed to attain a steady-state value (1-4 rnin). Selectivity Coefficient. The selectivity of the electrodes for K+ with respect to Ca2+(kC& was calculated at Ca2+concentrations where the electrode response deviated significantly from linearity. The selectivity coefficientK w was obtained from the Nikolskii (10) equation E = Eo + S loglo (aA+ kmF’’’t(aB)Z@B) (1) where A and B represent the primary (Ca2+)and interferent (K+) ions, respectively.
RESULTS AND DISCUSSION It has been stressed that microelectrodes made with ETH 129 must contain PVC or they develop nonideal electrical behavior in certain Ca2+ranges, and even those containing PVC tend to lose sensitivity to Ca2+ within 8 h after construction (6). We describe here that the internal reference junction also has a critical bearing on the performance of ETH 129 electrodes. In the present study, reliable measurements were difficuIt to obtain from electrodes fabricated by method A for 30-60 min after they were made. The erratic behavior observed was correlated with “shrinkage” of the membrane component mixture after it was drawn into the tip of the electrode. The major constituent of this mixture is T H F which is miscible with the aqueous reference solution. One effect of this mis0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 19, OCTOBER 1, 1990 Method A
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potential variation measured with the five electrodes. Inset: Membrane material (hash marks) drawn into micropipet previously filled with reference solution (stippled). Method B
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Figure 1. Response characterlstics of five electrodes made according to method A. Data points are the average of the potential difference between pCa 3 and the other test solutions. Bars represent range of
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Method C
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Flgure 2. Response characteristics of 10 electrodes made according to method B. Data points are the average of the potential difference between pCa 3 and the other test solutions. Bars represent standard deviations of the potential differences. Inset: Concentric electrodes. Internal pipet containing reference solution inserted into membrane phase.
cibility is that the reference boundary is continuously changing after the electrodes are fabricated. For example, a 2 0 0 - ~ m column shrinks to about 100 Fm over the first hour, and an additional 20% over the next 24 h. The performance of a group of five electrodes made according to method A and
Figure 3. Response characteristics of five electrodes made according to method C. Data points are the average of the potential difference between pCa 3 and the other test solutions. Bars represent range of potential variation measured with the five electrodes. Inset: Concentric electrode. Airdried membrane backfilled with internal pipet.
calibrated from 30 to 60 min after construction is shown in Figure 1. The data were normalized to the potential in the pCa 3 solution; the bars represent the range of variability in measurement among the five electrodes. Their characteristic is linear between pCa 3 and about pCa 6 with a rate of 26.7 mV/lO-fold change in Ca2+concentration. The response is shallower between pCa 6 and 8 and there is no potential difference as a group between pCa 8 and the Ca2+-freesolution (a). The log kca,Kat p c a 7 and 8 was -5.14 and -5.48, respectively. These selectivity coefficients are very similar to those that can be obtained from Figure 4 of Amman et al. (6) for “aged” electrodes but are greater than kCa,K reported in Table 3 for their freshly made electrodes ( k c e , K = -7.2). It is not readily apparent what contributes to this difference in “fresh” electrodes made by the same method. However, a comparison of Figures 1and 3 in the present study indicates that our electrodes displayed the “aging” effect as soon as 1 h after they were constructed. Extremely variable results were obtained from method B. Ten electrodes so constructed were evaluated. Two of them had response characteristics that were linear between pCa 3 and 8, but the slope was depressed (about 20 mV/decade). The majority of them had supertheoretical (>30 mV/decade Ca2+concentration) from pCa 3 to 5 and no sensitivity to Ca2+ at concentrations below pCa 5. The variability in performance of this group of electrodes is portrayed graphically in Figure 2. Data from the 10 electrodes were normalized at pCa 3. The average of the potential difference from pCa 3 is represented by the solid data points. Bars represent the standard deviation of measurement in this case, rather than the range of measurement shown in Figures l and 3. The variability in measurement indicates that there is no significant difference among any of the points between pCa 5 and pCa m. In several cases (6),observation of the electrodes following calibration indicated that the reference side of the membrane had been altered due to the reference solution exuding into the membrane phase. In these cases, electrode failure could be attributed to an unstable reference boundary. In the other cases,
Anal. Chem. 1990, 62, 2155-2158
there were no obvious visible changes associated with the poor electrode performance. For the reasons noted above, this procedure is not a recommended method of construction. Electrodes with the most desirable performance were obtained from method C, i.e. by "air-drying" the membrane component mixture for 24 h before introducing the reference solution via an internal pipet. Figure 3 shows results from five representative electrodes with this type of fabrication technique. The average of the potential changes from pCa 3 to the other solutions is represented by (0);bars represent the range of potential variation among the electrodes. The rate of potential change from pCa 3 to 7 was 30 mVll0-fold change in Ca2+concentration. Even though the sensitivity rolls off below pCa 7 , a 20-mV change between pCa 8 and 9 remained. The calculated log kCa,K a t pCa 8 and 9 is -6.84 and -7.27, respectively. Repeated calibration of these electrodes showed that all of them could be reliably calibrated for 6 h after they were made and two of the five were still reliable 48 h later. After hanging dry for 24 h following calibration, the response characteristic of the three that The rate of potential change/lO-fold changed is shown by (0). change in Ca2+concentration was 25 mV (line) instead of 30 mV and the response began to "roll off" at pCa 6. In addition to the change in sensitivity, the selectivity was reduced; calculated values of log kCa,K at pCa 8 and 9 were -6.4 and -6.84, respectively.
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A comparison of data presented in Figure 3 with those in Figure 1 indicates that a "hardened" ETH-129 membrane retards deterioration upon exposure to aqueous solutions. However, even a "hardened" membrane eventually deteriorates even though not exposed to aqueous solution. Five electrodes that were allowed to air-dry for 3 weeks and then filled with reference solution showed no sensitivity to Ca*+. ACKNOWLEDGMENT We wish to thank T. Buhrer for providing us with the ETH-129 cocktail. Registry No. ETH 129, 74267-27-9; Ca, 7440-70-2.
LITERATURE CITED Brown, H. Mack; Owen, J. D. Ion-Sel. Electrode Rev. 1979, 1 . 145. Levy, S.; Tillotson, D. Can. J. Physlol. Pharmacol. 1987, 65,904. Ammann, D. Ion -Selective Microelectrodes : Springer-Veriag: Berlin, 1966. Brown, H. Mack; Pemberton, J. P.; Owen, J. D. Anal. Chim. Acta 1976, 85, 261 Ammann. D.; Guggi, M.; Pretsch, E.; Simon, W. Anal. Lett. 1975, 8 , 709. Ammann, D.; Buhrer, T.; Schefer. U.; Mulier, M.; Simon, W. Pfluegefs Arch. 1987, 409, 223. Buhrer, T.; Gehreg, P.; Simon, W. Anal. Sci. 1988, 4 , 547. Erne, D.; Ammann, D.; Simon, W. Chlmis 1979, 33, 88. Tsien, R.; Rink, T. Blochim. Blophys. Acta 1980, 623. Nikolskii, B. P. Zh. Fiziol. Khim. 1937, 10, 495.
RECEIVED for review March 26,1990. Accepted June 26,1990.
Voltammetric Determination of the Ion-Exchange Behavior of Poly(ester sulfonic acid) Anionomers in Acetonitrile Thomas Gennett*J and William C. Purdy Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, Canada H3A 2K6 INTRODUCTION Over the past few years, electrodes modified with ionomeric polymers have been the focus of a significant amount of research (1-11). Recently, reports have described the ion-exchange properties of a series of Eastman Kodak AQ polymers in aqueous solutions (9-11). These poly(ester sulfonic acid) anionomers exhibited transport properties and ion-exchange selectivity comparable to those reported for Nafion (1,6,10, 11). In aqueous solutions the AQ membranes preferentially bind large hydrophobic cations and exclude negatively charged species from entering the membrane. The complete structure of the three different AQ polymers is not known; however, the proposed backbones of the AQ55, AQ38, and AQ29 polymers are illustrated in Figure 1 (12). The equivalent weights of the three polymers, as determined from the percent sulfonation, are 1500,2500, and 2500, respectively (12).
The use of ionomer-modified electrodes in nonaqueous electrochemistry has been limited because of several inherent problems with ionomeric membranes in nonaqueous solvents: swelling, solubility, lack of structural integrity, etc. However, we have found the AQ polymers to be stable in several nonaqueous electrochemical solutions. We report the ion-exchange and electrochemical behavior of platinum disk electrodes coated with an AQ55 polymer film.The investigations, conducted in acetonitrile, found a strong correlation of ionexchange properties and electrochemical response to the size Current address: Chemistry Department, Rochester Institute of Technology, Rochester, NY 14623. 0003-2700/90/0362-2155$02.50/0
of the electrolyte cation. Also, the nonaqueous ion-exchange selectivity was markedly different from that observed in aqueous systems.
EXPERIMENTAL SECTION An EG&G PAR Model 273 potentiostat/galvanostat and EG&G PAR Model 175 universal programmer in conjunction with a Hewlett-Packard 7015B x-y recorder were used for voltammetric analysis. An EG&G PAR Model 174A Polarographic Analyzer equipped with a Houston Instruments Model REO089 x-y recorder was used for differential pulse voltammetry. An IBM Instruments electrochemicalcell was used for all electrochemical experiments. The typical three-electrode cell consisted of a 1.6-mm platinum disk electrode, a saturated Ag/AgCl reference electrode, and a platinum wire auxiliary electrode. All electrolyte salts, NaClO, (Aldrich), KPF, (Aldrich), [(CHB),N]PF6(Aldrich), [(CH3CH2),N]C1O4(Eastman Kodak), and [ (C4H9),N]PF6(Aldrich Co.), were recrystallized twice and dried in vacuo before use. All electrochemical experiments were conducted in 0.01 M supporting electrolyte unless stated otherwise. The solvents, acetonitrile (Aldrich, HPLC grade) and methylene chloride (Aldrich,anhydrous spectro-grade),were used as received. Ru(bpy),C12 was purchased from Sigma and used as received. All electrochemical solutions were degassed for 20 min with nitrogen prior to data acquisition. Electrochemical experiments were performed under an inert gas atmosphere and at ambient temperature. The AQ55D polymer was purchased as a 28% dispersion from Eastman Kodak and diluted to a 1.0% aqueous dispersion with distilled water from a Millipore Milli-Q system. Prior to membrane coating, the platinum disk electrodes were polished with 1.0-, 0.3-, and 0.05-pm alumina on a Buehler Ecomet I1 polisher. After being polished, the electrodes were sonicated 0 1990 American Chemical Society
