Chloride ion-selective microelectrodes

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Bioelectrochemistry and Bioenergetics, 22 (1989) 411-415 A section of J Electroanal Chem, and constituting Vol 276 (1989) Elsevier Sequoia S A, Lausanne - Punted in The Netherlands

Short communication

Chloride ion-selective microelectrodes Takayuki Abe, Takeyuki Itabashi, Tomokazu Matsue and Isamu Uchida Department of Molecular Chemistry and Engineering, Faculty of Engineering, Tohoku University, Sendai 980 (Japan) (Received 12 September 1989)

INTRODUCTION

Microelectrodes have attracted considerable interest in electrochemistry [1,2] One of the unique characteristics of the microelectrode is its applicability to in vivo electrochemical measurements without damaging the cell So far, many amperometnc measurements have been carried out using microelectrodes in vivo [3], especially in brain tissue, to detect neurotransmitters However, these amperometnc microelectrodes cannot be used for important measurements such as those of intracellular ion activities in living cells For such purposes, glass capillary microelectrodes provided with an internal filling solution have been used in the field of modern electrobiology [4] The internal resistance of their tips is usually quite large, so noise is frequently a serious problem in the measurement of weak responses Furthermore, although several recent papers report the use of polymer-modified electrodes as ion-selective sensors [5], such electrodes are not suitable for in vivo electrochemical measurements because of their large size In this paper, we report a convenient and straightforward method for the fabrication of a Cl - -selective microelectrode, the Ag/AgC1 microelectrode sealed in a thin glass sheath, which can be used as a micropotentiometnc electrode, and we describe its behavior in a microcapsule and a single cell EXPERIMENTAL

The fabrication of the Ag/AgCl microelectrodes was conducted as follows An Ag microelectrode was fabricated by drawing a heated quartz tube containing an Ag wire by means of a micro-capillary puller (Nanshige Model PD-5) Figure 1 shows a typical scanning electron micrograph of an Ag microelectrode cracked with a knife It can be seen that the diameter of the Ag wire is about 1 µm, the thickness of the quartz glass is from 2 to 3 µm, and the diameter of the electrode is about 6 im We also fabricated even smaller electrodes 0302-4598/89/$03 50

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Fig 1 Scanning electron nucrograph of an Ag nucroelectrode The scale bar has a length of 5

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The Ag microelectrodes were immersed in 3 M HNO 3 for ca 10 s to increase the surface roughness of the electrode The deposition of AgCI was conducted by electrooxidation in a 0 1 mol/l HCl solution at 1 2 mA/cm 2 for 90 mm The current density and electrolysis time greatly affected the stability of the electrodes The above conditions seemed to be optimal for the present study The microelectrode thus fabricated was rinsed with distilled water Microcapsules were prepared by dropping a sodium alginate solution (0 2 g sodium alginate dissolved in 20 ml distilled water) into a 0 15 mol/l Ca(N03)2 aqueous solution This procedure creates insoluble calcium alginate at the interfacial region of the droplet, resulting in inicrocapsule formation The diameter of the microcapsule was controlled using a microsynnge The microelectrode was inserted into the microcapsule using a three-dimensional micromampulator system (Nanshige MO-188) under a microscope equipped with a TV monitor (Nikon, diaphot-TMD) Potentiometnc measurements were carried out using a high impedance electrometer (Keithley Model 610C) A saturated calomel electrode (SCE) was used as the reference electrode



413 RESULTS AND DISCUSSION

Figure 2 (A) shows the relationship between the steady-state values of potential and C1 - activities measured with the Ag/AgCI microelectrode in a beaker Figure 2 (a) shows the potential response after injection of a KCI solution into the beaker while stirring the solution The response was very quick and the potential was stabilized in a few seconds The experimental plots accords well with the theoretical line (slope, 59 mV) over a wide range of Cl - activities This result indicates that the miniaturized Ag/AgCI electrode shows a quite normal potential response Therefore, the Cl - activity can be determined from the potential response according to the theory Figure 2 (o) shows the potential vs Cl - activity relationship measured in a microcapsule as a model system for a single cell The Cl - activity in the microcapsule was altered by changing the Cl - activity of the outer solution Since the wall of a calcium alginate microcapsule was no special ion selectivity, we put the reference electrode in the outer solution of 0 15 mol/l Ca(N0 3 ) 2 and measured the potential difference between the microelectrode and the SCE Figure 2 (b) shows the potential response after injection of a KCl solution in the outer solution The potential decay indicates Cl - permeation through the microcapsule wall from the outer to the inner solution The potential values measured in this case also agreed with the theoretical line The above results indicate that the Cl - activity in a small volume such as that of a single cell can be determined by potential measurement with Ag/AgCl microelectrodes In addition to artificial microcapsules, we used salted salmon eggs to test the Ag/AgCI microelectrodes Figure 3 shows a photograph of the cell configuration In

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Fig 2 Relationship between the steady-state values of potential and Cl - activities measured with the Ag/AgC1 nucroelectrode, and typical potential responses measured m a beaker (o, a), and in a microcapsule (o, b) ( ) Theoretical line

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Fig 3 Schematic representation of the cell configuration used to measure the Cl - activity in a salted salmon egg using the Ag/AgC1 microelectrode, and photograph of the egg with electrodes

this case, the SCE was inserted into the salmon egg using a glass capilary filled with 2 M KCl solution to eliminate the influence of the membrane potential The concentration of KCl in the glass capilary was chosen to be near that of Cl - in the salmon egg to avoid contamination The value of the Cl - concentration was estimated roughly, in advance by using a capillary filled with saturated KCl solution Figure 4 shows the time dependence of the measured potential The steady-state potential was obtained within 2 min after inserting the microelectrode into the salmon egg The Cl - activity was estimated from this potential to be ca 2 3 mol/l In conclusion, we have demonstrated a suitable method of fabrication of Ag/AgCl microelectrodes with diameters of a few micrometer, and proved that the Cl activity inside a single cell can be measured with such microelectrodes No serious limitation due to the miniaturization of Ag/AgCI electrodes has been found We are now trying to fabricate Ag/AgCI microelectrodes of sub-micrometer diameter, including the glass seal, and to measure Cl - concentration in a much smaller cell of about ten micrometer diameter The Ag/AgCI microelectrode is superior to the



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t/min Fig 4 Time dependence of the potential measured as shown in Fig 3

ordinary glass capilary microelectrodes in terms of improved signal-to-noise ratio, and can be used as a microreference electrode for in vivo microvoltammetric measurements ACKNOWLEDGEMENT

This work was supported by Grant-in-Aid for Developmental Scientific Research (01850175) of the Ministry of Education, Science and Culture REFERENCES 1 R M Wightman, Anal Chem, 53 (1981) 1125A 2 S Pons and M Fleischmann, Anal Chem, 59 (1987) 1391A 3 R N Adams and J B Joseph, Jr, Voltammetry in the Neurosciences, Principles, Methods, and Applications, The Humann Press Inc, Clifton, NJ, 1987 4 M Lavallee, 0 F Schanne and N C Hebert, Glass Microelectrodes, Wiley, New York, London, Sydney, Toronto, 1969 5 N Oyama, T Hirokawa, S Yamasguchi, N Ushizawa, and T Shimomura, Anal Chem, 59 (1987) 258