ANALYST, JUNE 1986, VOL. 111 611 Epoxy-based All-solid-state Poly(Viny1 Chloride) Matrix Membrane Calcium Ion-selective Microelectrodes* Sajedah A. H. Khalil, G. J. Moody and J. D. R. Thomas Department of Applied Chemistry, Redwood Building, UWIST, P.O. Box 73, Cardiff CF7 3XF, UK and Jose L. F. C. Lima Chemistry Department, Faculty of Science, University of Oporto, 4000 Oporto, Portugal Calcium ion-selective microelectrodes have been constructed from PVC matrix membranes containing a calcium bis{di[4-( 1,1,3,3-tetramethylbutyI)phenyl]phosphate} electroactive component with dioctyl phenylphosphonate, tripentyl phosphate or trioctyl phosphate as a plasticising solvent mediator, and from the Philips IS 561/SP membrane. The electrode membranes are backed with a silver-based conductive epoxy implanted with a copper wire for electrical contact.Of the three types of electrodes designed, namely, glass capillaries with a pre-formed membrane (type A) or an applied membrane (type 6) and infrared heat-drawn Perspex capillaries with applied membrane (type C), the most functional are types B and C, which have functional lifetimes of 6 and 10 d, respectively. The electrodes have tip diameters of 1-3 pm for glass and 5-10 pm for Perspex, the bevelled tip in the latter ensuring a sharp edge. Keywords; Ion-selective microelectrodes; calcium ion-selective electrodes; intracellular and intramuscular fluid ions Ion activity measurements with ion-selective microelectrodes (micro-ISEs) are important in many areas of biophysics, physiology and other life sciences.1-4 The availability of liquid ion exchanger and neutral carrier-based liquid membrane types of ion sensors has extended the range of micro-ISEs to include lithium, potassium, calcium and chloride ions. Essen- tially, these are made by placing liquid ion exchangers in a variety of glass micropipettes57 with tip diameters as low as 0.5 vm. Conventional microelectrodes are based on an ion-selective membrane separating the test' solution from the inner refer- ence electrode, which is immersed in an inner reference solution. The small tip geometry offers advantages for the penetration of cell walls not possessed by other microelec- trodes, such as ion-selective field-effect transistors (ISFETs) and coated wires. However, they are fragile and demand patience and care during assembly and measurements.More robust versions with short, or otherwise protected tips, are needed for use in various other situations, i.e., for the measurements of ion activities in muscle fluids. It is with a view to such applications that the microelectrodes described here were developed. The studies are based on PVC mem- brane systems incorporating calcium bis{di[4-( 1,1,3,3- tetramethylbuty1)phenyllphosphate) coupled with an appro- priate plasticising solvent mediator from Orion, dioctyl phenyl- phosphonate (DOPP), tripentyl phosphate (TPP) or trioctyl phosphate (TOP). The sensor membranes heated at the tips of glass or Perspex capillaries are backed with a silver-based conductive epoxy for electrical contact to attain an all-solid- state system.Experimental Reagents and Materials All reagents, except the following, were of the best analytical grade available. Calcium bis{ di[4-( 1,1,3,3-tetramethylbutyl)phenyl]phos- phates and dioctyl phenylphosphonateg were prepared as described previously. * Presented at the 30th IUPAC Congress, Manchester, UK, September, 8-13th, 1985. Glass capillaries (2 mm external and 1.2 mm internal diameter) and Perspex tubes (5 mm external and 3 mm internal diameter) were pulled to tapered tips (of ca. 1 pm diameter) with a commercial micropipette puller at the University Hospital of Wales, Cardiff. These were baked in an oven at 150 "C for 24 h and kept over silica gel. After silanisation with trimethylchlorosilane the tubes were baked at 250 "C for 1 h and stored with the tip upwards in a metal block.Cocktails for the PVC-based sensor membranes consisted of liquid ion exchanger (0.04 g of electroactive component + 0.36 g of plasticising solvent mediator), PVC and tetrahydro- furan in the respective amounts 0.40 g, 0.17 g and 6.00 cm3. Individual Philips IS561/SP calcium ion membranes were dissolved in 1 .O cm3 of tetrahydrofuran to produce the sensor cocktail. Microelectrode Construction Preliminary experiments were made on conventional glass capillary microelectrodes in order to establish the optimum conditions for introducing the PVC-based sensor cocktail into the tapered tip glass capillaries for later back-filling with epoxy to produce all-solid-state electrodes. The vacuum-suction technique (Fig. 1) avoided the air-bubble problem mentioned by Walker,lo and the epoxy-sealed unit gave calcium micro- ISEs with good response characteristics, as illustrated in Fig.2. Three types of all-solid-state calcium micro-ISEs were constructed, namely a glass capillary tip loaded with PVC sensor cocktail followed by back-filling with silver-based conductive epoxy (type A), a glass capillary back-filled with silver-based conductive epoxy followed by application of the PVC sensor cocktail to the tip (type B), and an infrared heat-drawn Perspex capillary back-filled with silver-based conductive epoxy followed by application of the PVC sensor cocktail to the tip (type C); an air-blower heat-drawn Perspex capillary was an unsatisfactory alternative to infrared heat drawing because of threading of the Perspex owing to de-polymerisation. The full constructional details are as described below.612 +40.0 > E E LLi 2 +20.0 0.0 ANALYST, JUNE 1986, VOL.111 ' . Tip diameter -1 pm Reference solution (0.1 M CaCI2) Silanised Pyrex glass ------- - _ - - - _ - ----- ---+ _ _ _ _ _ _ _ _ _ - - - - - - - - ------ Vacuum 4- , Ag - AgCl Epoxy seal Fig. 1. tion of liquid ion-exchanger micro-ISEs Modified procedure to avoid air bubbles during the construc- 1 I I 20 40 60 Time/m in Fig. 2. D namic response of a li uid internal reference calcium micro-ISE Eased on Orion 92-20-02liquid ion exchanger, by spiked additions of 10-1 M calcium chloride to 20 cm3 of calcium chloride solution Type A electrodes The tips of the silanised glass micropipettes were broken under the microscope (Olympus Stereozoom Microscope with Research Instruments Manipulators and Dio Positioners) by gentle rubbing with a piece of tissue paper.The tip was filled by gentle vacuum suction with the appropriate PVC sensor cocktail to a depth of 100-150 pm and left to dry for 48 h. The micropipettes were back-filled with silver-based conductive epoxy using a copper wire (7/0.2 mm strands, RS, low noise cable) to push in the epoxy, the wire being finally left embedded in the epoxy to form an electrical contact. The assembly was then left for 3 d at room temperature to cure the epoxy. The tip diameters of the assembled electrodes (1-3 pm) were measured under the microscope with a calibrated eye-piece graticule. Type B electrodes The silanised glass micropipettes were first back-filled with the silver-based conductive epoxy and the copper wire contacted assembly was oven-cured at 60 "C for 4 h, whilst resting in a horizontal position in order to avoid cracking of the epoxy.The microelectrode tip was polished lightly under the micro- scope with emery paper to give a smooth flat surface, and then lightly coated with the appropriate PVC sensor cocktail. After 30-min intervals, four further PVC sensor cocktail coatings were applied. The resulting micro-ISEs had tip diameters of 1-3 pm. Type C electrodes Perspex tubes (3 mm external diameter) were carefully pulled after heating under an infrared lamp and the constriction cut at an angle with a heated metal blade to give a micropipette with a bevelled tip. The micropipettes were then assembled into micro-ISEs as detailed for type B electrodes.These electrodes had tip diameters of 5-10 pm, but with a sharp edge resulting from the bevelling. Procedures The various electrodes were normally conditioned by immer- sion in 10-1 M calcium chloride solution and calibrated at 25 k 0.1 "C in conjunction with a Corning Model 476002 reference electrode, by spiking aliquots of 10-1 M calcium chloride solution into 20 cm3 of 10-4 M calcium chloride solution. The e.m.f. measurements were made with a Corning EEL Model 112 digital millivoltmeter pH meter, used in conjunction with a Servoscribe Model RE 4541 potentiometric chart recorder. Measurements of interferences to the calcium ion responses from inorganic ions were based on selectivity coefficients , w:,t,~, measured by the separate solution approach for a 10-3 M metal ion concentration: EB - ECa mt>B = 2.303 RTl2F where Eca and EB are the e.m.f.s of calcium and interferent, respectively.Interferences to the calcium ion response from selected proteins and drugs were studied for the e.m.f. changes for 25 cm3 of 10-2 M calcium chloride solutions spiked with succes- sive 0.05-cm3 aliquots of pH 7.4 Tris buffered 0.05% mlV solutions of interferent. The solutions of proteins were aqueous, while 2.5% methanol was used to solubilise the drug-containing solutions. Results and Discussion The general approach to the study of these micro-ISEs is based on the utility of the electrically conductive epoxy for preparing ordinary all-solid-state ISEs, initially with solid sensor coatings11 and later with PVC matrix membrane trapped liquid ion-exchanger coatings.12 This design was tested in early experiments on prototypes of type A and B micro-ISEs, and the results obtained (Table 1) indicated that functional electrodes could be obtained following pre- conditioning in calcium solution before use. Inadequately pre-conditioned electrodes led to super-Nernstian slopes. (See Table 1 data on serial dilution calibration for electrode B, 2.) Table 2 summarises the characteristics of repeated calibra- tions of further typical electrodes of types A, B and C, based on the calcium bis{di-[4-1,1,3,3-tetramethylbutyl)phenyl]- phosphate} electroactive components and a dioctyl phenyl- phosphonate solvent mediator. These confirm that electrodes of good working characteristics can be obtained for all three constructional procedures.Of the two types of glass micro-ISEs, type B is clearly the more satisfactory, as illustrated by the characteristic responses shownfin Fig. 3. The responses of Type A electrodes are frequently erratic [Fig. 3(a)], the sensing membranes of these electrodes being more susceptible to damage during construc- tion. The membranes of Type B electrodes tend to peel off, which limits their functional lifetime to about 6 d. This can be caused by the hydrophilicity of glass, which causes leakage of the test solution around the PVC - glass interface with consequent peeling. There is greater compatibility between the polymer components and Perspexll,l* so that it is not surprising that type C electrodes have a longer lifetime of up to 10 d (Table 2).However, as mentioned above, these electrodes have larger tip diameters (5-10 pm) , although the bevelling gives a sharp edge, allowing their possible use for the study of ionic activities in intramuscular fluids. As the penetration of muscle flesh is to be the first application objective of these electrodes, the appraisal studies here are based on calcium ion levels in the 10-4-10-2 M range. More detailed studies have been focused on type B electrodes, although as constructional difficulties come to be solved, type C electrodes can be expected to be equally suitable in these application areas, with the advantage of longer functional lifetimes. In all of the studies described below, the electrodesANALYST, JUNE 1986, VOL.111 613 Table 1. Responses of prototype all-solid-state calcium micro-ISEs Range Linear E vs. SCE Electrode tested range at 10-3 M Type No. (-log[Ca2+]) (-log[Ca2+]) Slope/mV [CaZ+]lm\i ( a ) Runs calibrated with serially diluted CaCl, solution: A 1 3.85 - 2.1 3.25 - 2.45 33 - 180 3.85 - 2.1 As tested 28 - 155 4.00 - 2.1 As tested 24 -110 4.00 - 2.1 As tested 27 - 24 4.00 - 2.1 As tested 26 - 15 4.00 - 2.1 As tested 24 - 17 4.00 - 2.1 As tested 23 -18 B 1 4.05 - 1.5 As tested 28 - 10 2 4.05 - 1.5 As tested 42 - 192 4.05 - 1.5 As tested 59 - 183 ( b ) Runs calibrated by spiking 20 cm3 of lop4 M CaCI, with 1 M CuCl, solution: B 2 3.75 - 2.1 As tested 30 - 174 2.75 - 1.3 2.40 - 1.3 30 - 186 3.85 - 2.1 3.50 - 2.5 22 - 32 B 3 3.85 - 2.1 3.25 - 2.1 18 - 23 3.85 - 2.1 As tested 31 - 50 3.85 - 2.1 As tested 21 - 17 3.85 - 2.1 As tested 26 - 10 4.00 - 2.1 4.00 - 3.0 33 - 72 Dynamic response time at 10-3 M [Ca,+]/min Stages of electrode treatment and remarks 1 1 1 1 1 1 1 1 1 1 1 - 1 1 1 2 1 1 Dried in an oven for 18 h at 40 "C, left for 2 d in air and calibrated Conditioned for 30 rnin in 0.1 M CaCl, after previous run Conditioned for 1 h in 0.1 M CaC1, after previous run Conditioned for 18 h in 0.1 M CaC1, after previous run Conditioned for 4 h in 0.1 M CaCI, after previous run Conditioned for 45 min in 0.1 M CaCI, after previous run Conditioned for 15 rnin in 0.1 M CaCI, after previous run Air-dried for 15 h.Conditioned in 0.1 M CaCl,. PVC membrane dropped off Air-dried for 15 h. No conditioning Calibrated 15 rnin after previous run Calibrated after conditioning for 3 h in 0.1 M Calibrated immediately after previous run.Calibrated after conditioning for 2 d in 0.1 M Calibrated after air-drying for 2 d Conditioned for 1 h in 0.1 M CaCI, after previous run Conditioned for 18 h in 0.1 M CaC1, after previous run Conditioned for 2 d in 0.1 M CaC1, after previous run Conditioned for 4 d in 0.1 M CaC1, after previous run. Slope falls to near zero at -log aca = 2.1 CaCl, E (10-3 M CaCI,) extrapolated CaCI, +40.0 > +20.0 E E ui 0.0 > 'c: -20.0 10 30 50 10-4 1 10 30 50 10 30 50 Ti me/m i n Fig. 3. Dynamic response to calcium chloride of all-solid-state calcium ISEs calcium bis(di[4-(1,1,3,3-tetramethylbutyI)phenyl]- phosphate} and a DOPP plasticising solvent mediator.Electrodes: ( a ) type A; ( b ) type B; and (c) type C were pre-conditioned by soaking overnight in 10-1 M calcium chloride solution, in which they were subsequently stored. Cation Interferences Fig. 4 summarises the responses to various cations of type B electrodes based on the PVC sensor with DOPP [Fig. 4(a)], TPP [Fig. 4(b)] and TOP [Fig. 4(c)], respectively, as plasticis- ing solvent mediators, and on the sensor based on the Philips IS 561/SP membrane, while k#Yi,B data are summarised in Table 3. The superior selectivity of the Philips membrane with respect to sodium, potassium, magnesium and zinc ions is clearly demonstrated, while the electrodes made from the DOPP-based cocktail [Fig. 4(a)] is at least of equal selectivity with respect to sodium and potassium ions and only marginally less for magnesium ions.However, the DOPP-based elec- trode is far less selective for calcium over zinc ions [Fig. 4(a)] and even less so for the TOP-based electrodes. Zinc interfer- ence has always been a characteristic of the organophosphate- based calcium ISEs,l3 but this does not normally present practical problems in studies on biological fluids. The selectivity coefficient data in Table 3 are calculated forANALYST, JUNE 1986, VOL. 111 614 Table 2. Typical characteristics of calcium micro-ISEs with a DOPP plasticising solvent mediator Slope at 25 "CImV S.d. ( a ) Type A electrode* : 25.3 21.8 25.6 28.4 23.1 22.8 19.4 18.6 ( b ) Type B electrode" 40.9 32.7 28.1 25.4 26.4 26.3 26.3 22.7 22.3 1 .o 0.9 3.5 1.4 0.6 0.6 0.8 0.8 2.4 1.8 2.3 1.1 2.5 1 .o 1.2 0.8 1.1 ( c ) Type C electrode*: 29.1 1.6 31.2 1.4 27.9 1.2 27.6 1.2 27.9 1.4 28.5 0.9 26.4 0.9 28.3 1.2 28.4 1.2 28.7 1.2 30.2 1.3 EdmV +0.8 - 0.2 +8.6 +17.8 +1.4 +30.0 +18.0 +56.9 -25.7 -22.9 -26.2 -32.4 -23.4 -22.4 -1.8 + 10.6 +24.0 108.1 117.1 126.3 125.4 126.2 127.2 120.4 127.9 126.7 126.1 126.0 S.d.3.2 3.2 5.4 4.7 2.0 2.2 2.7 2.6 1.2 3.3 4.5 3.6 4.1 3.5 4.0 2.7 3.6 5.4 4.8 4.0 4.0 4.6 3.1 3.1 4.1 4.0 4.0 4.2 Remarks Conditioned for 24 h in 10-1 M CaCI, solution Conditioned for 1 h in 10-1 M CaCI, solution Conditioned for 2 h in 10-1 M CaCl, solution Conditioned for 3 h in 10-1 M CaCI, solution Conditioned for 4 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Air dried Conditioned for 24 h in 10-1 M CaCl, solution Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 2 h in 10-1 M CaCI, solution Conditioned for 3 h in 10-1 M CaC1, solution Conditioned for 4 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaCl, solution Conditioned for 24 h in 10-1 M CaC1, solution Air dried Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 24 h in 10-1 M CaC12 solution Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 2 h in 10-1 M CaC1, solution Conditioned for 3 h in 10-1 M CaC1, solution Conditioned for 4 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Conditioned for 24 h in 10-1 M CaCI, solution Conditioned for 48 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCl, solution * The lifetimes of the electrodes were 5 , 6 and 10 d for types A, B and C, respectively. Table 3.Calcium micro-ISEs (type B) selectivity coefficients keitB (separate solution method at a cation concentration of 10-3 M) Interferent DOPP TPP TOP Philips Na+ . . . . 0.15 0.18 0.22 0.089 K+ . . . . . . 0.14 0.16 0.18 0.087 Mg2+ . . . . 0.21 0.24 0.39 0.104 Zn2+ . . . . 0.24 0.28 0.87 0.14 (B) membrane membrane membrane membrane Table 4. E.m.f. changes for type B calcium micro-ISEs for proteins added (0.001%) to calcium chloride test solution M) in Tris buffer at pH 7.40 (n = 3) AElmV DOPP electrodes TPP electrodes TOP electrodes Protein added AE S.d.Humanalbumin . . +2.1 0.07 Bovinealbumin . . -1.7 0.10 a-Globulin . . . . -1.9 0.23 fl-Globulin . . . . -0.4 0.10 y-Globulin . . . . -0.5 0.07 A E S.d. +0.2 0.20 +1.8 0.07 +0.1 0.12 -0.2 0.09 -0.9 0.11 A E S.d. -0.4 0.05 -0.6 0.17 -0.2 0.12 -0.3 0.05 -0.3 0.10 a cation concentration of 10-3 M. It is stressed that the actual w;,, data will be different at different cation concentrations, and will become smaller in magnitude for higher cation concentrations. Table 5. E.m.f. changes for type B calcium micro-ISEs for drugs added (0.001%) to calcium chloride test solution (lo-, M) in Tris buffer at pH 7.40 ( n = 3) AElmV DOPP electrodes TPP electrodes TOP electrodes Drug added AE Aminoglutethimide . . +0.7 Glutethimide . . . . -1.9 Dapsone . . . . . . - 1.7 Hydrocortisone .. -0.7 Insulin . . . . , . -0.3 Nitrofurantone. . . . -0.5 Prilocaine hydrochloride . . -3.3 Methanol . . . . . . -1.2 S.d. 0.10 0.07 0.14 0.06 0.20 0.01 0.30 0.01 AE S.d. +1.8 0.21 +5.7 0.14 +1.3 0.08 +1.0 0.13 +1.4 0.22 -2.7 0.32 -0.4 0.10 -0.9 0.07 AE S.d. +1.4 0.10 -1.7 0.08 -0.3 0.14 +1.2 0.21 -3.5 0.07 +0.2 0.17 +1.3 0.20 +0.9 0.10 Protein Interferences The TOP-based electrodes show more tolerance towards the presence of protein than either of the other type B electrodes (Fig. 5 and Table 4). This trend parallels the observations made in another study14 on the effect of biochemical components on conventional calcium ISEs. In this study the added biochemical components presented minimum interfer- ence to electrodes with an organophosphate electroactive component with a TOP solvent mediator.ANALYST, JUNE 1986, VOL. 113 61 5 50.0 c /I / Fig.4. Res onse of all-solid-state calcium micro-ISEs to various metal cations by spiking M metal chloride solutions with 10-l M metal chloriie solution. (a), ( b ) and (c) type B electrodes based on calcium bis{di 4-(1,1,3,3-tetramethylbutyl)phenyl]phosphate} and (a) DOPP, (b) TPP and (c) TOP plasticising solvent mediators; and ( d ) type B electrode fi ased on Philips IS 561/SP membrane Human albumin and bovine albumin are exceptional in giving an increase in the ISE response for the DOPP-based electrodes and the DOPP- and TPP-based electrodes [Fig. whilst impaled will be in continuous contact with the fluid under study. I . " 5(a) and (b)], respectively.Such interferences are always undesirable and can be of even greater significance in Drug Interferences intracellular fluids where protein levcls are relitively higher than in extracellular fluids. The protein interference manifests itself as a coating on the electrode tips. This can be minimised by having samples in flowing streams, but this facility is not available for micro-ISEs in intracellular and intramuscular studies. Nevertheless, towards this ideal, a single electrode Contrary to studies on the effects of other added biochemical components, in which the TOP-based electrodes showed the minimum of interference, it is the DOPP-based electrodes that generally show the least interference from added drug components (Fig. 6 and Table 5). However, the TOP electrodes showed less interference from added dapsone and616 ANALYST, JUNE 1986, VOL.111 +2.0 0.0 -2.0 +2.0 > E $ 0.0 -2.0 +2.0 I I I (b) -2.0 1 I I 1 2 6 10 [ I n t e rfe ren t 1/10 -4 O/O Fig. 5. Effect of added protein components on type B elec- trodes. Calcium bis{di[4-(1,1,3,3-tetramethylbutyl)phenyl]phos- phate} based electrodes with ( a ) DOPP, (b) TPP and (c) TOP as plasticising solvent mediators. Samples: 0, human albumin; 0, bovine albumin; A, cu-globulin; A , P-globulin; and W, y-globulin glutethimide, and differences in sign of some of the deviations. In many instances the drugs give rise to positive deviations of e.m.f., which can contribute to reducing the negative devi- ations observed for most of the protein component studies above and also for biochemical components.14 Conclusion Clearly, all-solid-state micro-ISEs based on PVC matrix membrane sensors for calcium ions and backed by a silver- based conductive epoxy are functional systems.Their selectiv- ity features resemble conventional calcium micro-ISEs. The tip diameters of 1 pm, characteristic of the electrodes studied here, were sufficiently large to permit e.m.f. measurements with a conventional millivoltmeter. This gives encouragement to applications of the devices for studies on intramuscular fluids, the choice of electrode being between types B and C. Within these types, the effect of interferences must balance selection between those based on dioctyl phenylphosphonate 0. -5A O.( > E a -5.a 0.0 -5.0 e ‘C) 2 6 10 [Interferent]/10-4°/~ Fig.6. Effect of added drug type components on type B electrodes. Calcium bis{ di[4-( 1,1,3,3-tetramethylbutyl)phenyl]phosphate} based electrodes with (a) DOPP, ( b ) TPP and ( c ) TOP as plasticising solvent mediators. Samples: 0, aminoglutethirnide; 0, dapsone; A, glutethi- rnide; A , hydrocortisone; W , insulin; V, methanol; V, nitrofuran- tone; and trioctyl phosphate plasticising solvent mediators with a calcium bis{ di-[4-( 1,1,3,3-tetramethylbutyl)phenyl]phos- phate} electroactive component and those of the neutral carrier system of the Philips IS 561/SP membrane. The authors thank the Foundation of Technical Institutes, Baghdad, Iraq for paid leave of absence and a studentship (granted to S. A. H. K.) and the North Atlantic Treaty Organisation for a travel grant (069/84).Professor A. A. S. C. Machado of the University of Oporto is thanked for helpful discussions and suggestions. References 1. LavallCe, M., Schanne, 0. F., and Herbert, N. C., Editors, “Glass Microelectrodes,” Wiley, New York, 1969. 2. Walker, J. L., and Brown, H. M., Physiol. Rev., 1977,57,729. 3 . Thomas, R. C., “Ion-selective Intracellular Microelectrodes,” Academic Press, New York, 1978. 4. Brown, H. M., and Owen, J . D., Ion-Sel. Electrode Rev., 1979, 1, 145. 5. Brown, H. M., Pemberton, J. P., and Owen, J. D., Anal. Chim. Acta, 1976, 85, 261.ANALYST, JUNE 1986, VOL. 111 617 6. Ammann, D., Morf, W. E., Anker, P., Meier, P. C., Pretsch, E., and Simon, W., Ion-Sel. Electrode Rev., 1983,5, 3. 7. Ujec, E., Keller, E. E. O., Kriz, N., Pavlik, V., and Machek, J . , Bioelectrochem. Bioenergetics, 1980, 7 , 363. 8. Craggs, A., Delduca, P. G., Keil, L., Key, B. J., Moody, G. J., and Thomas, J. D. R., J. Inorg. Nucl. Chem., 1978,40, 1483. 9. Craggs, A., Delduca, P. G., Keil, L., Moody, G. J., and Thomas, J. D. R., J. Inorg. Nucl. Chem., 1978,40, 1943. 10. Walker, J. L., Anal. Chem., 1971, 43, 89A. 11. Lima, J. L. C . , and Machado, A. A. S. C., in Albaiges, J., Editor, “Analytical Techniques in Environmental Chemistry, 2. Proceedings of the Second International Congress, Bar- celona, Spain, November, 1981 ,” Pergarnon Press, Oxford, p. 419. 12. Alegret, S., Alonso, J., Bartroli, J., Panlis, J. M., Lima, J. L. F. C., and Machado, A. A. S. C.,Anul. Chim. Actu, 1984, 164, 147. Moody, G. J., Oke, R. B., and Thomas, J. D. R., Analyst, 1970, 95, 910. Khalil, S. A. H., Moody, G. J., and Thomas, J. D. R., Analyst, 1985, 110,353. 13. 14. Paper A51359 Received October 9th, 1985 Accepted November 27th, 1985