Biomedical Engineering Reference
In-Depth Information
Fig. 3.2 Single molecule detection with a protein nanopore sensor. The nanopore sensor can be
constructed by forming a lipid bilayer membrane that insulates solutions on both sides. A protein
nanopore embedded in the bilayer constitutes the only path for ionic flow across the membrane.
The voltage applied between the two solutions will drive a pico-Ampere ion current through the
pore, recorded using an amplifier. The protein pore has been engineered with a recognizing probe
in the lumen. Without a bound target, the pore remains open. When a single molecule binds to the
probe, the pore current is blocked, but when the molecule is released, the ionic current resumes.
Repeated cycles of binding and release of individual target molecules to and from the pore can
result in a string of binary (on/off) signature blocks. Different targets in the mixture (represented
by
filled
and
unfilled
balls) may competitively bind to the same pore, but give rise to different
block amplitude and duration, which can be used for target discrimination. The block duration,
t
off
,
is target-specific, and is used to evaluate the dissociation rate constant
k
off
:
k
off
¼
1/
t
off
. The
frequency of block occurrence
f
can be known from the inter-block duration
t
on
:
f ¼
t
on
.
f
is a measure of target quantity because it is proportional to the target concentration ([T]), using
the association rate constant
k
on
as the coefficient,
f ¼ k
on
[T]
1/
conductance (Fig.
3.2
). From this principle, bio-nanopores [
5
] and artificial
nanopores [
42
,
51
,
69
,
71
,
93
,
102
] have been engineered as potential biosensors
for substrates including pharmaceuticals [
33
], secondary cellular messengers [
15
],
metal ions [
11
,
63
], nucleic acids [
47
], and proteins [
82
,
93
,
103
]. Other goals of
nanopore use are the understanding of polymer dynamics and transportation [
7
,
8
],
and the rapid sequencing of DNA [
3
,
21
,
27
,
61
,
62
,
70
,
79
,
81
,
84
,
112
,
113
].
Invariably, these applications take advantage of the potent capabilities of nanopores
in dissecting single-molecule kinetics - a property deduced from the principle that
any change in conformation during a molecular process will affect a molecule's
occupancy within the nanopore, thereby altering the conductance. In addition to
ease-of-use, one of the most outstanding features of nanopores is their ability to
directly measure transition rate constants between states in a kinetic path [
34
-
37
,
46
,
73
]. Furthermore, detection using nanopore requires no molecular labels, such
as fluorescents or microbeads, which could otherwise perturb molecule function.
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