Biology Reference
In-Depth Information
Table II
Preparation of the PVC-based Ca
2þ
-selective ligand MEs
Component
a
Amount
Solution 1
ETH 129 (or ETH 1001)
27.5 mg
NPOE
500
m
l
Na
þ
-tetraphenyl borate
5.53 mg
Solution 2
Solution 1
200
m
l
PVC
36 mg
THF
400
m
l
a
Components can be obtained from Sigma-Aldrich (St. Louis,
MO) and see
Table
I
for abbreviations.
94% (w/w) NPOE, 5% (w/w) ETH 129, and 1% (w/w) sodium TPB) is satisfactory
to start with (
Ammann et al., 1987
), small volumes sometimes provided (0.1 ml) do
not facilitate handling. We prefer to make up our own cocktail, in a larger NPOE
volume, with the same proportions. The composition, for 500
m
l of NPOE is given
in
Table II
.
Because of the small volumes and required stirring, it is preferable to work with
flat-bottomed, small volume glass vials, and miniature stirring bars. ETH 129 and
TPB are dissolved with vigorous stirring in NPOE, in a 2-ml glass vial (Solution 1).
Solution 1 can be kept at room temperature for several months, with a Teflon
screw cap, protected from light. When the final cocktail (Solution 2) is prepared,
PVC is dissolved in THF with stirring. 0.2 ml of Solution 1 is then added, stirred,
and finally sonicated. THF is allowed to partially evaporate to approximately half
of the initial volume and the final cocktail is finally poured in a 0.5-ml conical vial
(Clark Electromedical Instruments).
Before dipping the ME tip, THF is allowed to evaporate until the mixture has
the consistency of a thick syrup. Experience helps to determine the adequate
consistency of the ligand. In fact, if not enough THF has evaporated, the ETH
129 sensor is too diluted and the evaporation in the ME may cause retraction of the
gel, yielding poor responses. On the other hand, in some instances, we have
managed to fill the electrodes even if the ligand appeared to be solidified (as a
rule, the thickest mixture which will fill the tip is best).
Because of the small diameter of the tip and the viscosity of the ligand, negative
pressure is required at the back of the electrode. This is achieved by a
10-ml
syringe connected, via a 3-way stopcock, to a flexible Teflon tubing connected to
the back of the electrode with soft tubing. Vacuum is then applied by pulling the
plunger out and by blocking it with a rod/block or collar placed along the plunger
(care should be taken to regularly check the vacuum of the system). Observation of
the ME and measurements of the ligand column height are performed under
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