Making and Using Calcium-Selective Mini- and Microelectrodes - Methods in Cell Biology - page 86

erent
shapes and testing them (ligand filling and calibration) on the same day. Relying on
ME resistance may give misleading results because it is also a

2. The tip diameter is too small. This may entail preparing MEs with di V

V ected by the

geometry of the shank.
3. ME silanization may be insu

Y Y

cient, because of insu cient time of silaniza-

Y

tion (or insu cient beaker seal during exposure to silane vapors), old silane, glass

rehydration (storage problem), and insu Y cient dehydration.

The ME gives bad calibration curves (subNernstian slopes, low detection limit).

1. Make sure that the calibrating solutions are adequate. Check them with
commercial macroelectrodes or the above described ETH 129-based minielec-
trodes, as described in previously published methods (e.g., Bers, 1982 ).
2. The ligand may be too old. This is a common occurrence. Ligand lifetime is
sensitive to exposure to light and air.
3. The ETH 129 is too diluted. This can occur by not letting THF evaporate
enough before ligand filling. In this case, it is better to let all the THF evaporate
and then, to re-add small amounts (few tens of microliter) of THF and stir the
mixture until a syrup-like solution is obtained. It can also happen if after adding
THF to a gelled cocktail, the cocktail is not mixed to homogeneity (by vortexing or
sonication).

IV. Discussion

We here describe the design and preparation of Ca 2 þ -selective mini- and micro-
electrodes, based on the ligand ETH 129 in a PVC matrix. Both mini- and micro-
electrodes have excellent responses during in vitro calibration with a response time
and detection limit superior to that of most commercially available minielectrodes.
These electrodes can have multiple applications. We do note, however, that for
measuring [Ca 2 þ ] i , the ligand ETH 1001 may be preferable.
Other Ca 2 þ -selective ionophores with a higher selectivity for Ca 2 þ against other
ions, such as K23E1 ( Suzuki et al., 1995 ), have been developed after ETH 129 but
their detection limit for calcium is considerably inferior to that of ETH 129-based
electrodes ( Suzuki et al., 1995 ). Thus, K23E1 may be useful for clinical analysis of
calcium in plasma or other biological fluids, but ETH 129-based electrodes remain
superior for calcium measurements in the submicromolar concentration range.
ETH 129-based minielectrodes are economical, easy to prepare, and have suc-
cessfully been used for purposes where the response time of the electrode is appro-
priate. This includes preparation of calibration solutions, determination of the K d
for EGTA, BAPTA, and oxalate in bu er solutions ( Bers, 1982; Harrison and Bers,

1987; Harrison and Bers, 1989; Hove-Madsen and Bers, 1993a ), and calibration of
indo-1 and furaptra signals cell suspensions ( Hove-Madsen and Bers, 1992; Hove-
Madsen et al., 1998; Shannon and Bers, 1997 ). The minielectrodes have also been

V