Measurement of Na+ and K+ in nanoliter droplets by ion-specific microelectrodes

The measurement of electrolyte concentration in nanoliter quantities of fluid is a fundamental step in the utilization of micropuncture and isolated tubule microperfusion techniques for studying ion transport. A number of methodologies have been developed for these analyses, including helium glow photometry [1], electron probe microanalysis [2], and atomic absorption spectrophotometry [3]. Because of the significant cost of the equipment for performing atomic absorption spectrophotometry or electron probe microanalysis, and the lack of commercial availability of helium glow photometers, we decided to utilize ion-specific electrodes for the measurement of sodium and potassium in samples from isolated tubule microperfusion experiments. The application of ion-specific micro-electrodes to the measurement of intracellular ion activity and macroanalysis is well established [4], but the technique has not been widely applied to the analysis of electrolyte concentration in nanoliter droplets, although Garvin has recently developed a flow-through ion-selective electrode apparatus for measurement of potassium concentrations [5]. Since aqueous droplets must be analyzed under oil to prevent evaporation, one problem we had to overcome was the deterioration in the sensitivity of liquid ion exchanger microelectrodes on contact with oil. Other investigators have utilized ion-selective microelectrodes to study intracellular ion activities or ion activities in tubular fluid in situ, circumstances in which the tissue is bathed in an aqueous electrolyte solution rather than mineral oil [6–8]. In the case where measurements have been made in aqueous droplets under oil, an ion-selective glass rather than a liquid ion exchanger microelectrode was used [9]. With liquid ion exchangers, we found that the sensitivity of the electrode decreased markedly on contact with mineral oil, probably due to the lipophilicity of the ionophore. Although it is possible to utilize very small tips and holders with sidearms to express contaminated ionophore (S. Agulian, personal communication), we found this technique cumbersome and have adapted the methods described by Vogel et al [10], which utilizes a desilanization step to allow a droplet of aqueous solution to protect the ionophore from oil contamination. We have applied electrodes constructed using this technique to the measurement of electrolyte concentrations in aqueous nanoliter droplets under oil. The equipment for this analysis is relatively inexpensive, available in many microperfusion laboratories, and can be applied to a variety of ions, and therefore the technique may be valuable to a wide spectrum of investigators