ANALYTICAL
BIOCHEMISTRY
An Inexpensive,
96, 90-93
(1979)
Miniature
Potassium-Ion-Selective
Electrode
HARRY ROSENBERG Department
of Biochemistry, Australian National
The John Universiiy,
Curtin School of Medical Research* Canberra, A.C.T. Australia
Received August 22, 1978 A simple method for the construction of a small K+-selective electrode is described. Its performance characteristics are comparable to those of any of the commercial units and it can be made much less expensively. A simple modification enables the electrode to be prepared for sensing other ions.
The measurement of K+ concentration 110 mm long, o.d. 3.5 mm, id. 2.0 mm. The by means of selective electrodes is a well- ends were ground flat and the outer glass established method (6) and is based on the surface at the bottom end was roughened measurement of a potential difference with wet carborundum paper. The tube across a partition which incorporates the was then washed and the roughened end K+-selective depsipeptide, valinomycin, was immersed in chromic oxide solution either in an immobilized liquid film, or (Chromerge, Manostat, New York) overincorporated in a polymer. Electrodes with night. The tube was then thoroughly washed the liquid partition respond rapidly to and dried. changes in K+ concentration with a halfA strip of aluminum foil 12.5 x 90 mm, time below 1 s and the total response time 0.05 mm thick, backed with Scotch adbelow 5 s [Ref. (4), this work]. The solid- hesive transfer tape No. 465 was roiled and polymer-selective membranes give a lengthwise onto the tube, forming a screen somewhat slower response (3). (B) and leaving 20 mm at the bottom end The electrode described below works uncovered. A piece of “shrink-on” insulaessentially on the principle described by tion tubing (C), 90 mm long and 5 mm in diPioda et ul. (5), but dispenses with the ameter, was slipped over the tube, leaving machined electrode assembly. It is robust, 10 mm of screen exposed on the top and inexpensive, and simple to construct, so overlapping 5 mm below the screen on the that it can be considered disposable. When bottom. The tube was heated in the oven used in conjunction with one or two other at 80°C for several minutes to allow the electrodes, it can still be accommodated plastic to shrink tightly over the screen in a small cell (2 ml), which presents a and glass. considerable advantage where limited quanThe bottom end (E) was now coated tities of biological materials are available. with Araldite (CIBA-GEIGY) prepared by mixing the correct proportions of base (AW134) and the corresponding hardener MATERIALS AND METHODS (HY994). This, and the preparation of the The body of the electrode is shown in glass surface, was in accordance with Fig. 1, with relevant parts designated by manufacturer’s instruction (1). An annulus letters in brackets, below. It was con- of mixed Araldite was also applied around structed from borosilicate glass tubing (A), the lower edge of the insulating sleeve (D), 0003-2697/79/09W’O-04$02.00/O Copyright 0 1979 by Academic F’ress, Inc. All rights of reproductmn in any form reserved.
90
MINIATURE
K+-SELECTIVE
FIG. 1. Details of electrode body. Left: lower end: right: upper end. For expIanation of symbols see text.
producing a watertight seal. The resin was cured at 60°C overnight and then slightly abraded, and the surface was cleaned with A.R. grade ethanol and dried. The surface at the bottom was thinly coated with cyanoacrylamide adhesive (I.S. 04E, Loctite, Ltd.), and the tube was pressed vertically onto a piece (about 6 mm*) of Gelman GA8 membrane filter. It was allowed to cure about 30 min at room temperature followed by 15 min at 6OC. The excess membrane was trimmed and a layer of cyanoacrylamide was applied over the join, overlapping the edge of the membrane so as to leave a circle of about 1.5-2 mm in diameter in the center uncovered (F). The resin was allowed to cure at room temperature overnight.
91
ELECTRODE
The electrode proper consists of a silver wire 0.9 mm thick, on which AgCl was deposited electrolytically (2). The electrode was fixed into a female LEMO coaxial connector RCOO250 (LEMO S.A., Merges, Switzerland) which was in turn fixed to the tube by a short length (12 mm) of soft silicone tubing, through which was looped a 2-mm-wide band of thin (0.08 mm) bronze foil, to provide electrical contact between the LEMO connector and the screen on the tube (Fig. 2). A male LEMO connector FOO250 was wired to the inner core and screen of a cable leading to a Radiometer pM63 meter, and the electrode was connected through this. The electrode can be used with any standard AgCl reference cell. The one used in this work is a two-compartment concentric glass vessel. The inner compartment contains the reference electrode and connects through a porous silica plug to the outer compartment. Both compartments are filled with 0.1 M KCl. The reference electrode is connected by a single wire to the reference socket on the meter. The one reference electrode may also serve a pH glass electrode which can share the main cell with the K+ electrode. The outer compartment of the reference cell is connected to the main cell by a bridge of 2% agarose in appropriate “support solution,” in this case 70 rnM LiCl, I rnM MgC&, and 20 rnM triethanolamine-HCI buffer, at a pH within the range 6.5-7.5. Both the main cell and the reference electrode cell were jacketed and kept at 37’C by circulating water. RESULTS Operation
The membrane at the end of the electrode compartment is moistened with 10 rnM valinomycin in diphenyl ether (5) and 0.1 M KC1 is introduced into the tube which is “flicked” to displace trapped air. Electrolyte level should be about 4 cm below the top. The electrode proper is inserted and
92
HARRY ROSENBERG
FIG. 2. Construction and assembly details. (1) Glass body, showing screen (A) and abraded lower end (B). (2) Assembled body with shrunk-on insulator (C), showing bare screen on upper end, Araldite seal on lower end of insulation (D), and Araldite coat on lower end of glass (E). (3) Electrode proper fixed in LEMO connector. (4) Complete assembly, showing securing sleeve(F) and screen earthing loop (G).
fastened to the tube by means of the silicon tubing with the brass loop, connected to the meter, and fixed in the cell (Fig. 2). A carefully prepared electrode body can last for several weeks of continuous immersion in support medium and will return to the same potential, about 240-250 mV when washed free of K+.
constants (KM.+) were calculated using the “mixed solution method” (4). These constants were about 10m4 for Na+ and lO-5 for Li+. DISCUSSION
The electrode described here is slim, yet robust, and can be simply and cheaply constructed (six of these, made in a batch, Performance took less than 3 h of total labor). Thus, The electrode shows a fast response to they may be deemed disposable, although changes of potassium concentration and repair is quite simple. The electrode pergood reversibility (Fig. 3). It produces a forms in every way as well as one commernormal Nemstian response (Fig. 4) with a cial instrument in use in this laboratory. slope of 58 mV/log [K+] at 37°C. Selectivity Although the electrode has been spe-
MINIATURE
K+-SELECTIVE
93
ELECTRODE
0.32 7 -
0.34 -
0.36 -
I
1mV
0.42 -
FIG. 4. Electrode response to K+ in the presence of LiCl and NaCl. The voltage was plotted and the curves were extrapolated as described by Moody and Thomas
0.44-
(4). 1
,
1 min FIG. 3. Response time and hysteresis. The electrode was used in a 2.5ml-capacity cell. KC1 was initially present at about 0.32 mM. At A, 3.0 ~1 of 0.1 M KC1 was added, and at B, the cell contents were diluted with 1.0 ml of “support medium” (see text) calculated to bring the K+ concentration back to original.
cifically described here for use as a potassium-sensitive electrode, it can be made selective for other ions by simply modifying the membrane and by the use of appropriate ligand solutions. Its small size should make it quite useful for biological work where sample size is limited. ACKNOWLEDGMENTS I would like to thank Dr. H. A. McKenzie for valuable advice and critical reading of the manu-
script and the Sydney Office of CIBA-GEIGY, Australia, Ltd., which generously provided advice, technical literature, and samples of various Araldite adhesives. Thanks are also due to Ms. Jane Jones for skillful technical assistance and to Ms. Beverley Bernard for preparation of diagrams.
REFERENCES 1. CIBA-GEIGY (1975) Araldite Adhesives, F’ublication 37479le, Basel. 2. Geddes, L. A. (1972) Electrodes and the Measurement of Bioelectrical Events, p. 10, WileyInterscience, New York. 3. Krull, I. H., Mask, C. A., and Cosgrove, R. E. (1970) Anal. Lett. 3, 43-51. 4. Moody, G. J., and Thomas, J. D. R. (1971) Selective Ion-Sensitive Electrodes, p. 14, Met-row, Watford, United Kingdom. 5. Pioda, L. A. R., Stankova, V., and Simon, W. (1969)
Anal.
Lctt.
2, 665-674.
6. Simon, W., and Morf, W. E. (1973) in Ion-Selective Electrodes (Pungor, E., ed.) pp. 127- 143, Akad. Kiado, Budapest.
BIOCHEMISTRY
An Inexpensive,
96, 90-93
(1979)
Miniature
Potassium-Ion-Selective
Electrode
HARRY ROSENBERG Department
of Biochemistry, Australian National
The John Universiiy,
Curtin School of Medical Research* Canberra, A.C.T. Australia
Received August 22, 1978 A simple method for the construction of a small K+-selective electrode is described. Its performance characteristics are comparable to those of any of the commercial units and it can be made much less expensively. A simple modification enables the electrode to be prepared for sensing other ions.
The measurement of K+ concentration 110 mm long, o.d. 3.5 mm, id. 2.0 mm. The by means of selective electrodes is a well- ends were ground flat and the outer glass established method (6) and is based on the surface at the bottom end was roughened measurement of a potential difference with wet carborundum paper. The tube across a partition which incorporates the was then washed and the roughened end K+-selective depsipeptide, valinomycin, was immersed in chromic oxide solution either in an immobilized liquid film, or (Chromerge, Manostat, New York) overincorporated in a polymer. Electrodes with night. The tube was then thoroughly washed the liquid partition respond rapidly to and dried. changes in K+ concentration with a halfA strip of aluminum foil 12.5 x 90 mm, time below 1 s and the total response time 0.05 mm thick, backed with Scotch adbelow 5 s [Ref. (4), this work]. The solid- hesive transfer tape No. 465 was roiled and polymer-selective membranes give a lengthwise onto the tube, forming a screen somewhat slower response (3). (B) and leaving 20 mm at the bottom end The electrode described below works uncovered. A piece of “shrink-on” insulaessentially on the principle described by tion tubing (C), 90 mm long and 5 mm in diPioda et ul. (5), but dispenses with the ameter, was slipped over the tube, leaving machined electrode assembly. It is robust, 10 mm of screen exposed on the top and inexpensive, and simple to construct, so overlapping 5 mm below the screen on the that it can be considered disposable. When bottom. The tube was heated in the oven used in conjunction with one or two other at 80°C for several minutes to allow the electrodes, it can still be accommodated plastic to shrink tightly over the screen in a small cell (2 ml), which presents a and glass. considerable advantage where limited quanThe bottom end (E) was now coated tities of biological materials are available. with Araldite (CIBA-GEIGY) prepared by mixing the correct proportions of base (AW134) and the corresponding hardener MATERIALS AND METHODS (HY994). This, and the preparation of the The body of the electrode is shown in glass surface, was in accordance with Fig. 1, with relevant parts designated by manufacturer’s instruction (1). An annulus letters in brackets, below. It was con- of mixed Araldite was also applied around structed from borosilicate glass tubing (A), the lower edge of the insulating sleeve (D), 0003-2697/79/09W’O-04$02.00/O Copyright 0 1979 by Academic F’ress, Inc. All rights of reproductmn in any form reserved.
90
MINIATURE
K+-SELECTIVE
FIG. 1. Details of electrode body. Left: lower end: right: upper end. For expIanation of symbols see text.
producing a watertight seal. The resin was cured at 60°C overnight and then slightly abraded, and the surface was cleaned with A.R. grade ethanol and dried. The surface at the bottom was thinly coated with cyanoacrylamide adhesive (I.S. 04E, Loctite, Ltd.), and the tube was pressed vertically onto a piece (about 6 mm*) of Gelman GA8 membrane filter. It was allowed to cure about 30 min at room temperature followed by 15 min at 6OC. The excess membrane was trimmed and a layer of cyanoacrylamide was applied over the join, overlapping the edge of the membrane so as to leave a circle of about 1.5-2 mm in diameter in the center uncovered (F). The resin was allowed to cure at room temperature overnight.
91
ELECTRODE
The electrode proper consists of a silver wire 0.9 mm thick, on which AgCl was deposited electrolytically (2). The electrode was fixed into a female LEMO coaxial connector RCOO250 (LEMO S.A., Merges, Switzerland) which was in turn fixed to the tube by a short length (12 mm) of soft silicone tubing, through which was looped a 2-mm-wide band of thin (0.08 mm) bronze foil, to provide electrical contact between the LEMO connector and the screen on the tube (Fig. 2). A male LEMO connector FOO250 was wired to the inner core and screen of a cable leading to a Radiometer pM63 meter, and the electrode was connected through this. The electrode can be used with any standard AgCl reference cell. The one used in this work is a two-compartment concentric glass vessel. The inner compartment contains the reference electrode and connects through a porous silica plug to the outer compartment. Both compartments are filled with 0.1 M KCl. The reference electrode is connected by a single wire to the reference socket on the meter. The one reference electrode may also serve a pH glass electrode which can share the main cell with the K+ electrode. The outer compartment of the reference cell is connected to the main cell by a bridge of 2% agarose in appropriate “support solution,” in this case 70 rnM LiCl, I rnM MgC&, and 20 rnM triethanolamine-HCI buffer, at a pH within the range 6.5-7.5. Both the main cell and the reference electrode cell were jacketed and kept at 37’C by circulating water. RESULTS Operation
The membrane at the end of the electrode compartment is moistened with 10 rnM valinomycin in diphenyl ether (5) and 0.1 M KC1 is introduced into the tube which is “flicked” to displace trapped air. Electrolyte level should be about 4 cm below the top. The electrode proper is inserted and
92
HARRY ROSENBERG
FIG. 2. Construction and assembly details. (1) Glass body, showing screen (A) and abraded lower end (B). (2) Assembled body with shrunk-on insulator (C), showing bare screen on upper end, Araldite seal on lower end of insulation (D), and Araldite coat on lower end of glass (E). (3) Electrode proper fixed in LEMO connector. (4) Complete assembly, showing securing sleeve(F) and screen earthing loop (G).
fastened to the tube by means of the silicon tubing with the brass loop, connected to the meter, and fixed in the cell (Fig. 2). A carefully prepared electrode body can last for several weeks of continuous immersion in support medium and will return to the same potential, about 240-250 mV when washed free of K+.
constants (KM.+) were calculated using the “mixed solution method” (4). These constants were about 10m4 for Na+ and lO-5 for Li+. DISCUSSION
The electrode described here is slim, yet robust, and can be simply and cheaply constructed (six of these, made in a batch, Performance took less than 3 h of total labor). Thus, The electrode shows a fast response to they may be deemed disposable, although changes of potassium concentration and repair is quite simple. The electrode pergood reversibility (Fig. 3). It produces a forms in every way as well as one commernormal Nemstian response (Fig. 4) with a cial instrument in use in this laboratory. slope of 58 mV/log [K+] at 37°C. Selectivity Although the electrode has been spe-
MINIATURE
K+-SELECTIVE
93
ELECTRODE
0.32 7 -
0.34 -
0.36 -
I
1mV
0.42 -
FIG. 4. Electrode response to K+ in the presence of LiCl and NaCl. The voltage was plotted and the curves were extrapolated as described by Moody and Thomas
0.44-
(4). 1
,
1 min FIG. 3. Response time and hysteresis. The electrode was used in a 2.5ml-capacity cell. KC1 was initially present at about 0.32 mM. At A, 3.0 ~1 of 0.1 M KC1 was added, and at B, the cell contents were diluted with 1.0 ml of “support medium” (see text) calculated to bring the K+ concentration back to original.
cifically described here for use as a potassium-sensitive electrode, it can be made selective for other ions by simply modifying the membrane and by the use of appropriate ligand solutions. Its small size should make it quite useful for biological work where sample size is limited. ACKNOWLEDGMENTS I would like to thank Dr. H. A. McKenzie for valuable advice and critical reading of the manu-
script and the Sydney Office of CIBA-GEIGY, Australia, Ltd., which generously provided advice, technical literature, and samples of various Araldite adhesives. Thanks are also due to Ms. Jane Jones for skillful technical assistance and to Ms. Beverley Bernard for preparation of diagrams.
REFERENCES 1. CIBA-GEIGY (1975) Araldite Adhesives, F’ublication 37479le, Basel. 2. Geddes, L. A. (1972) Electrodes and the Measurement of Bioelectrical Events, p. 10, WileyInterscience, New York. 3. Krull, I. H., Mask, C. A., and Cosgrove, R. E. (1970) Anal. Lett. 3, 43-51. 4. Moody, G. J., and Thomas, J. D. R. (1971) Selective Ion-Sensitive Electrodes, p. 14, Met-row, Watford, United Kingdom. 5. Pioda, L. A. R., Stankova, V., and Simon, W. (1969)
Anal.
Lctt.
2, 665-674.
6. Simon, W., and Morf, W. E. (1973) in Ion-Selective Electrodes (Pungor, E., ed.) pp. 127- 143, Akad. Kiado, Budapest.
