Biomedical Engineering Reference
In-Depth Information
Fig. 13.4 Diagram of the
nanopore stochastic sensing
chamber device
Holes for holding two
electrodes
Polymeric nanopore
membrane/Teflon film
cis compartment
trans compartment
an aperture of ~120
m. As mentioned in the introduction section, in stochastic
sensing, the protein pore is usually supported by a phospholipid bilayer. Thus far,
there are a number of methods for the formation of planar lipid bilayer, including
painting, tip dipping, apposition, and microfluidics. Among them, the apposition
approach (also called the Montal-Mueller or monolayer folding method [
49
]) is the
most popular one because the method is easy to use and also the bilayer formed in this
way is very stable, which usually can last for at least several hours. To form a lipid
bilayer by using theMontal-Mueller method, the aperture in the film is first pretreated
with an organic solvent, e.g., hexadecane. Then, the two chamber compartments
are filled with an appropriate volume of an electrolyte (e.g., 1 M NaCl/KCl) solution
to make sure that the solution levels are below the aperture. Next, one or two drops of
phospholipid solution such as 1,2-diphytanoylphosphatidylcholine in
n
-pentane are
added to the surface of electrolyte solutions in both the compartments. After waiting
for ~1-2 min to allow the pentane to evaporate and leave the lipid monolayers on the
surface of the electrolyte solutions, certain volume of the same electrolyte solution
is added to both the chamber compartments to raise the solution levels past the
aperture. In such a way, a bilayer of phospholipid is formed on the aperture. The
formation of the lipid bilayers can be monitored by using a function generator.
In general, the bilayer recording experiments are performed under symmetrical buffer
conditions with each compartment containing a ~1.0 mL solution of 1 M NaCl/KCl
and 10 mMTris-HCl (pH 7.4). Proteins (e.g.,
m
HL) are added to the
cis
compartment,
which is connected to “ground.” In such a way, after insertion of a single
a
HL
channel, its mushroom cap is located in the
cis
compartment, while the
b
-barrel
of the
a
HL inserts into the lipid bilayer and connect with the
trans
of the chamber
device. An appropriate transmembrane potential is applied to the chamber device
using two Ag/AgCl electrodes with 3%agarose bridges containing 3MKCl. Currents
are recorded with a patch clamp amplifier. To shield against ambient electrical
noise, a metal box is used to serve as a Faraday cage, inside which the bilayer
recording amplifying headstage, stirring system, chamber, and chamber holder are
enclosed.
It should be noted that, whether the observed ionic current is attributed to the
insertion of a single
a
HL channel into the lipid bilayer or not can be conveniently
judged from the planar bilayer recording experiment. Since the bilayer lipid
membrane typically has a sealing resistance on the order of 100-200 G
a
O
[
50
],
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