Biomedical Engineering Reference
In-Depth Information
Current flow
cis
Recognition site
α
-hemolysin
Lipid bilayer
trans
τ
on
Ope
n
c
h
ann
el current
Amplitude
τ
off
Fig. 13.1 Schematic representation of nanopore stochastic sensing
current which is monitored. In the absence of compounds, the channel is always open
and a constant ionic current (called open-channel current) could be observed. In
contrast, when a target molecule enters the pore, it will physically block the channel,
thus resulting in a decrease in the ionic current flowing through the pore; when the
molecule leaves the channel, the pore re-opens and the ionic current will increase
(back to the open-channel state). In this way, a sequence of individual single-
molecule binding events can be detected as transient modulations in the recorded
current. Although each individual current blockage event is random, the statistical
mean values of the residence time (
t
off
) and amplitude of the events are reproducible
and are also unique for different analytes. Hence,
t
off
and amplitude can serve as
a characteristic current signature to reveal the identity of an analyte (Fig.
13.1
).
Furthermore, the concentration of the analyte can be obtained from the frequency of
occurrence (1/
t
on
) of the binding events. In stochastic sensing, since each analyte
produces a characteristic signature, the sensor element itself need not be highly
selective. Theoretically, this allows several analytes to be quantitated concurrently
using a single sensor element (Fig.
13.2
)[
3
], as long as the sensor itself can provide
enough resolution.
The most often used nanopore stochastic sensor element is a single transmem-
brane protein
HL) channel embedded in a planar lipid bilayer.
a
-Hemolysin is a spontaneous pore-forming toxin secreted by
Staphylococcus
aureus
. The wild-type
a
HL (Fig.
13.3
) forms a mushroom-shaped pore, which
consists of seven identical subunits arranged around a central axis [
4
]. The opening
of the channel on the
cis
side of the bilayer measures 29
˚
in diameter and broadens
into a cavity of ~41
˚
across. The cavity is connected to the trans-membrane
domain, a 14-stranded
a
-hemolysin (
a
-barrel with an average diameter of 20
˚
. The
HL pore
has several properties, which make it unique as a sensor element in stochastic
sensing. First, compared with other protein channels, such as porin [
5
] and leuko-
cidin [
6
], the open
b
a
HL channel is quiet without transient background current
modulation events. Thus, the
a
HL pore is an ideal sensor element for sensitive
detection of trace amounts of analytes. Second, since the three-dimensional struc-
ture of the
a
HL pore is known [
4
], it can be modified with a variety of new
functions, which greatly enhance its potential sensor application. Furthermore,
the transmembrane portion (
a
b
-barrel) of the protein pore is sufficiently stable to
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