A highly-selective chloride microelectrode based on a mercuracarborand anion carrier

Abstract

word count: 238 Word count: 5891 Figures: 7 Keywords: ion-selective electrode, liquid membrane microelectrode, skeletal muscle Abbreviations: 9-ACA: 9-anthracenecarboxylic acid, BMT: bumetanide, ISM: ion selective microelectrode, MC3: [9]mercuracarborand-3, NPOE: 2-nitrophenyl octyl ether, TDMAC: tridodecylmethyl ammonium chloride ABSTRACT The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory GABAA receptors. Measurement of intracellular chloride activity, , using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular HCO3may be up to 4 times more abundant than Cl -(e.g. skeletal muscle) which places severe constraints on the required selectivity of a Clsensing ISM. Previously, a sensitive and highly-selective Cl sensor was developed in a polymeric membrane electrode (Badr et al. 1999) using a trinuclear Hg(II) complex containing carborane-based ligands, [9]mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of (0.1 mM to 100 mM), is highly selective for Cl over other biological anions or inhibitors of Cl transport, and has a response time of less than 5 sec. Importantly, over the physiological range of(1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to HCO3or changes in pH.Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular , and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.

A four-electrode method to study dynamics of ion activity and transport in skeletal muscle fibers

“…Silanized micropipettes must be used for ISM fabrication because the hydrophobic surface is essential for mechanical stability and high electrical resistance at the interface between glass and the liquid membrane at the tip of the electrode. Micropipettes were silanized by evaporation of chlorotrimethylsilane (Sigma-Aldrich) in a 500-ml jar, followed by baking at 250°C for ≥4 h, as previously described ( DiFranco et al, 2019 ). Silanized micropipettes were stored dry in a closed jar (Jar-E215; World Precision Instruments) to avoid dust adhesion and were used for ≤2 wk without noticeable deterioration.…”

“…ISMs were assembled by backfilling through a 34-G microneedle, as previously described ( DiFranco et al, 2019 ). Briefly, an excess of the ionophore-containing cocktail was delivered as closely as possible to the micropipette tip.…”

“…The ionophore cocktails were mixed and stored as stock solutions at room temperature that could be used for up to 1 yr. The composition (wt/wt %) of the ionophore and the backfilling saline solutions were, for the sodium microelectrode (Na-ISM), 10% Na + ionophore IV (#71745; Sigma-Aldrich), 1% Na-tetraphenylborate in 2-nitrophenyl octyl ether (NPOE), backfilled with 100 NaCl and 10 HEPES, pH 7.4 titrated with HCl; for the chloride microelectrode (Cl-ISM), 10% mercuracarborand-3 (custom synthetized; DiFranco et al, 2019 ), 2.5% tridodecylmethyl ammonium chloride in NPOE, backfilled with 100 NaCl and 10 HEPES, pH 7.4 titrated with H 2 SO 4 ; and for proton (pH) microelectrode (H-ISM), 10% H + ionophore-I (#95292; Sigma-Aldrich), 0.7% tetraphenylborate in NPOE, backfilled with 100 NaCl and 100 HEPES, pH 7.0 with HCl.…”

A Four Electrode Method to Study Dynamics of Ion Activity and Transport in Skeletal Muscle Fibers

“…Silanized micro-pipettes must be used for ISM fabrication because the hydrophobic surface is essential for mechanical stability and high electrical resistance at the interface between glass and the liquid membrane at the tip of the electrode. Micropipettes were silanized by evaporation of chlorotrimethylsilane (Sigma) in a 500 ml jar, followed by baking at 250 o C for at least 4 h, as previously described (Difranco, Quinonez et al 2019). Silanized micro-pipettes were stored dry in a closed jar (WPI Jar-E215) to avoid dust adhesion and were used for up to 2 weeks without noticeable deterioration.…”

“…Ion-selective microelectrodes were assembled by backfilling through a 34G microneedle, as previously described (Difranco, Quinonez et al 2019). Briefly, an excess of the ionophore-containing cocktail was delivered as closely as possible to the micro-pipette tip.…”