Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit

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Abstract
In intracellular electrolyte solutions a Ca2+-selective microelectrode based on the synthetic electrically neutral carrier N,N,N′,N′-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca2+ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa=9.2 in Ca2+ buffers containing an intracellular background of K+ (125 mM). Selectivity studies in mixed solutions show a preference of Ca2+ over Na+ of 6·105, over K+ of 1.6·106, and over Mg2+ of 5·106. The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca2+ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa=7 and pCa=8 are reproducible within 24.7±0.4 mV (SD,n=8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca2+ electrode response is not disturbed by the contact of a cytosolic milieu.