Biology Reference
In-Depth Information
II. CaSM Construction
A. Micropipette Fabrication
Ion-selective microelectrodes are based on an ion-selective solvent or liquid
membrane, immobilized in the tip of a glass micropipette with a backfilling
electrolyte. The glass micropipette housings are pulled from 1.5 mm outer diameter
borosilicate (TW150-4 World Precisions Instruments, Sarasota, FL), aluminosili-
cate (A150-100-10 Sutter Instruments, Novato, CA
or quartz glass (Q150-110-10,
Sutter Instruments). Inner filaments, commonly used to load electrolyte solutions
to the tips of micropipettes, are avoided. Although the glass body is fragile, it
provides distinct advantages over other materials including low cost, excellent
resistive properties necessary for use with the high-resistance liquid membranes,
and easy fabrication of small tips. Micropipettes are pulled, silanized, and stored in
bulk,
),
50 per wire rack. The glass is pulled down to a final edge slope of 0.15-0.17
and an inner tip diameter of 2-3
m
m. Borosilicate and aluminosilicate micropip-
ettes are pulled on a horizontal heated filament puller (P-97, Sutter Instruments)
while quartz pipettes are pulled on a horizontal laser puller (P-2000, Sutter Instru-
ments). Latex gloves are worn during handling of the glass before silanization.
B. Silanization
The hydrophilic glass surfaces are coated with a hydrophobic silane to enable
adhesion and high electrical resistance between the glass and the hydrophobic
liquid membrane. While many forms of silanization exist, we prefer vapor deposi-
tion of N,N-dimethyltrimethylsilylamine (cat# 41716 Sigma-Aldrich, St. Louis,
MO) as it enables rapid and uniform coating of numerous micropipettes, simulta-
neously. A wire rack of micropipettes is placed in a small solid wall metal box
(8 cm
10 cm) with a swinging door within the oven so that the silane vapor
can be trapped in a small region around the pipettes. Prior to coating, the glass is
dried for 20 min at 240
C under vacuum (28 in Hg). This shortens drying time and
decreases loss of hydroxyl groups (
Deyhimi and Coles, 1982; Munoz et al., 1983
).
Higher temperatures may dry glass more quickly as well; however, this silane has
ignited two out of four times at
8cm
250
C. After drying, atmospheric pressure is
recovered by purging the oven with Argon. A small volume of silane (20
m
L) is
dropped into a tiny glass beaker in the metal enclosure and the door to the
enclosure is closed before the oven door is closed. The glass is exposed to the silane
vapor for 20 min before removing and placing the micropipettes in a sealed bell jar
with desiccant in the bottom. Functional CaSMs have been produced from micro-
pipettes that have been stored in this manner for up to a month. This method has
reduced variation in the quality of silanization.
