Biomedical Engineering Reference
In-Depth Information
it can isolate the electrolyte solutions in the two chamber compartments very well.
Under an applied voltage bias of e.g., 40 mV, the current will be 0 pA even if the
proteins have been added to the
cis
compartment, but have not yet inserted into the
bilayer. In contrast, after one
HL protein channel inserts into the bilayer, the two
chamber compartments (
cis
and
trans
) are connected. Thus, the cations and/or
anions in the electrolyte solutions will be electrophoretically driven toward the
other compartment via the protein nano-channel under the applied potential, and
a sudden current jump could be observed. The single channel currents for the
wild-type and various mutant
a
HL protein pores have been well documented,
usually about 21-32 pA in 1 M NaCl solution under an applied potential of
40 mV depending on the protein pore used [
51
]. If two
a
HL channels insert into
the bilayer, the current will be doubled, around 42-64 pA.
a
a
d
40
0
40
cis
1s
0
e
0.15
trans
b
c
0.1
0.05
0
0
25
50
75
100
[TNT] /
μ
M
→
Fig. 13.5 Stochastic sensing of TNT in an engineered protein pore. (a) Side view of the
Met113Phe7 pore highlighting position 113, where the naturally occurring Met residue has been
substituted with Phe. (b) View into the Met113Phe7 pore from the
cis
side of the lipid bilayer.
(c) View into the Met113Phe7 pore showing a TNT molecule at the same scale. The Phe side
chains are shown in extended conformations and the TNT molecule has been placed with the plane
of the ring perpendicular to the central axis of symmetry in the pore. (d) Current traces in the
absence of TNT (
upper panel
) and in the presence of 50
m TNT (
lower panel
). The TNT was
added to the chamber (1.52 mL) in acetonitrile (17.2 mL). The same volume of acetonitrile was
included in the experiment shown in the upper trace. (e) Plot of 1/
t
on
as a function of TNT
concentration. The calculation of 1/
t
on
(n
¼
3) was based on the long blocking events.
Experiments shown in Figs.
13.5d
and
13.5e
were performed at +50 mV (
cis
at ground) in NaCl
(1 M), Tris-HCl (10 mM, pH 7.5) with the Met113Trp7 pore. Both the protein and TNT were
added to the
cis
chamber. Figure reprinted with permission from [
13
], Copyright 2005 Wiley-VCH
Verlag GmbH & Co. KGaA
m
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