Biomedical Engineering Reference
In-Depth Information
with 25% cholesterol; the achieved sealing of about 10 M: cm
2
is
too low for sensitive measurements;
108
the pore distribution is ran-
dom, resulting in merged pores that may affect the bilayer stabil-
ity; the membrane is about 10 nm thick, with a resulting high
as-
pect ratio
(i.e., pore size to membrane thickness); the stability of
coated gold on polymer carbonate may be too low for continuous
use.
Nanoporous alumina membranes with highly ordered pores of
60 and 280 nm in diameter have been frequently used by Steinem
and coworkers to support free-standing lipid bilayers. In their ear-
lier work, a differential capacitance of about 1 PF cm
-2
was ob-
tained for bilayers formed by fusing giant vesicles on nanopores of
30 and 50 nm diameter.
109
In a further development, the nanopo-
rous alumina supports were coated with gold, and 1,2-dipalmitoyl-
sn
-glycero-3-phosphothioethanol was tethered to its surface via its
thiol group.
110
The gold surface so hydrophobized favors the span-
ning of lipid bilayers across the nanopores of the alumina mem-
brane, upon painting its gold-coated side with a DPhyPC solution
in
n
-decane. The membrane was clamped between two Teflon
half-cells. The formation of lipid bilayers in this
nano-bilayer lipid
membrane
(nano-BLM) was followed by measuring the differen-
tial capacitance of the membrane by EIS up to the attainment of its
maximum limiting value. The impedance spectra were fitted to an
RC mesh, simulating the lipid bilayer, with the solution resistance
R
:
in series with it. Upon regarding the resistance of the thiol-
coated gold much higher and its capacitance much lower than
those of the pores, the resistance and capacitance of the whole
membrane were referred exclusively to the active area of the pores,
yielding a specific capacitance of 0.5-0.6 PF cm
-2
and a specific
resistance of up to 0.16 G:cm
2
. As opposed to the membrane
capacitance, the membrane resistance strongly depends on the
quality of the preparation and the age of the nano-BLM. What is
important for recording single channel currents by a low-noise
patch-clamp amplifier is the resistance
R
m
of the whole nano-
BLM, which must be higher than 1 G: to reduce the background
electrical noise to the order of 1-2 pA.
R
m
decreases gradually in
time, due to the progressive rupture of the individual lipid bilayers
spanning the single pores. This gradual decrease in resistance
causes a progressive increase in the current baseline, but the back-
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