A Simple Method for Construction of Flexible, Subminiature Ion-Selective Electrodes

Although the latter type of electrode is suitable for intramyocardial and intravascular recording from animals the size of dogs and pigs (Hill et al. 1978), flexible electrodes with smaller diameters and longer shanks are required for recording ion activity in the circulatory system or body cavities of smaller animals such as insects. This paper describes techniques for the simple and rapid construction of flexible pH- and ion-selective electrodes with external diameters as small as 40μm. Double-barrelled electrodes with total outer diameters of 80–100 μm and shanks as long as 50 cm can be inserted into the body cavity of insects such as locusts through syringe needles as fine as 26 gauge.

The electrode body is made from a disposable polyethylene 1ml syringe (Becton-Dickinson & Co., Rutherford, NJ). A 1–2cm region of the barrel is heated over a low flame until it softens and the diameter increases slightly (Fig. 1A). The syringe is then drawn out into a fine tube about 0.1 mm in diameter (Fig. 1B). The procedure to this point is that described previously for producing a disposable syringe with integral plastic needle for use in backfilling glass micropipettes (see Fig. 10 of Thomas, 1978). During or after cooling, the fine tube can be pulled out further by hand to a diameter as small as 40–50 /tm and cut to length with a razor blade. The barrel is then cut to within about 1 cm of the top of the shank. About 0.2ml of an appropriate internal reference solution is injected into the back of the syringe, and forced to the tip of the tubing by the application of pressure either by mouth or by re-inserting the plunger and pressing lightly (Fig. 1C). Under a dissecting microscope, the tip is then inserted into a fine glass capillary tube (Fig. 1D) containing about 1 μl of the appropriate ionophore cocktail (Table 1). The cocktail can be used as supplied if the electrodes are to be used within 1 week. Modifications to provide electrodes with longer lifetimes are described below. After a column of about 1–2 mm (approximately 20 nl) of ionophore cocktail has entered the tip by capillarity, the tubing is retracted from the glass tube. Silanization is not required because the syringe tubing is hydrophobic. Electrodes can be maintained in physiological saline or internal reference solution until use. With practice, the time of fabrication and filling can be reduced to less than 3 min per electrode.

The following ionophore cocktails were obtained from Fluka Chemical Corp., Ronkonkoma, NY: (1) Ca²⁺ ionophore I (ETH 1001), cocktail B; (2) Ca²⁺ ionophore II (ETH 129), cocktail A; (3) H⁺ ionophore I (tridodecyl amine, TDDA), cocktail B; (4) H⁺ ionophore II (ETH 1907), cocktail A; (5) K⁺ ionophore I (valinomycin), cocktail A; (6) Na⁺ ionophore II (ETH 157), cocktail A. For NH₄⁺ electrodes, a cocktail was made by mixing 10 % NH₄⁺ ionophore I (75 % nonactin, 25 % monactin) with 89 % 2-nitrophenyl octyl ether and 1 % sodium tetraphenylborate. Internal reference solutions for each ion (in parentheses) were as follows: (Na⁺), 0.5 mol 1^-1 NaCl; (K⁺), 0.5 mol 1^-1 KC1; (H⁺), 0.1 mol 1^-1 NaCl and 0.1 mol 1^-1 sodium citrate, adjusted to pH 6 (Thomas, 1978); (Ca²⁺), 0.2 mol 1^-1 KC1, 5 mmol 1^-1 CaC12 and 10 mmol 1^-1 EGTA, yielding pCa 7 (Alvarez-Leefmans et al. 1981).

Recording from the subminiature ISE was accomplished by placing silver chloride-coated silver wires into the internal reference solution and connecting the wires to an operational amplifier. Noise due to electrical interference was reduced by filtering the amplifier output through a low-pass RC filter with a time constant of Is. Electrode response times were of the order of 2–4s. The resistance of the subminiature ISEs varied from Ix10⁸-5x10⁸Ω, comparable to or less than the 10⁹Ω typical for solvent polymeric electrodes and the 10⁹-10¹¹ Ω of ISMEs. The potentials generated by the subminiature ISEs, therefore, can accurately be measured by operational amplifiers with input impedances of 10¹¹ Ω or more. These are typically used for recording from standard glass microelectrodes filled with 3 mol 1^-1 KC1, and are cheaper and more readily available than the high-impedance (>10¹⁴Ω) operational amplifiers typically used for ISMEs.

Ionophore cocktail tended to leak out of the tip of the electrode if use involved inadvertent contact between the tip and a hydrophobic surface such as an acrylic or polyvinyl chloride experimental chamber. This problem was eliminated by filling the tip with a mixture of one part of an ionophore cocktail with two parts of 15 % (w/v) polyvinyl chloride in tetrahydrofuran (Fluka). Evaporation of the solvent resulted in a tough gel-like membrane that was not easily displaced or dislodged by contact of the tip with hydrophobic surfaces. Additional advantages were that the lifetime of electrodes stabilized with PVC exceeded 4 weeks, and that hydrostatic pressures within very long (>50cm) electrodes did not force out the sensor, as sometimes occurred in the absence of PVC. Electrode resistance and response times were not noticeably altered by the addition of PVC.

For reference electrodes, plastic syringes were heated and pulled as for ISEs and were backfilled either with 3 mol 1”¹ KC1 (for pH, Na⁺, Ca²⁺) or with NaCl (for K⁺ measurements) in 3% agar. Double-barrelled subminiature ISEs were fabricated by heating two plastic syringes, as described above, then joining the barrels while the plastic was near its melting point. The fused barrels could then be pulled out to the desired total diameter (80–100μm), as for single-barrelled ISEs, and one barrel used for a reference while the other was used for measurement of ion activity.

Flexible subminiature ISEs may be suitable for recording near or on mucosal surfaces, such as gills, bronchial passages or the oesophagus. Levels of pH and NH₄⁺ in the micro-environment near the gills of trout, for example, are different from those in the bulk water (Playle and Wood, 1989), and such local ion gradients might be detectable using flexible subminiature ISEs. An example of measurement of the surface pH of moistened filter paper is shown in Fig. 2A. The flexible double-barrelled electrode was advanced until slight bending of the tubing indicated contact with the surface of the filter paper. This type of ISE may also be useful for the measurement of bathing fluid pH or ion activity in small experimental chambers used for in vitro perfusion, particularly if holes or passageways, which need not be straight, are milled in the chamber. They can also be used for analysis of nanolitre droplets of biological fluids collected in micropipettes and expelled under paraffin oil.

A major advantage of both single- and double-barrelled subminiature ISEs is that they can be inserted into a blood vessel or other tissue compartment through syringe needles as small as 26 gauge (Fig. 2B) or through a fire-polished glass capillary tube (Fig. 2C). The syringe needle or glass capillary can be withdrawn after the subminiature ISE and reference electrode are in place. The flexibility of the subminiature ISE not only prevents damage to the electrode itself but also limits damage to the preparation, for example to the gut wall during peristalsis.

Although suitable for haemolymph pH measurements in insects over short periods (5–10min), longer-term use resulted in blockage of the ISE tip by haemocyte clotting. Electrodes blocked in this way could be restored by simply cutting off about 0.2 mm of the tip with a razor blade, thereby exposing a fresh surface of the ionophore cocktail.

This work was supported by NSERC operating grants. The author is grateful to C. M. Wood for his comments on a draft of the manuscript.