Biomedical Engineering Reference
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Fig. 9.4 Binding isotherm obtained by current recordings through nanopores of particles deco-
rated with monoclonal anti-streptavidin antibodies that bound to streptavidin on the surface of
these particles [
38
]. Equation
9.6
was used to fit the data in order to calculate
K
a
and
K
d
. Adopted
from Uram and Mayer with permission [
47
]
9.4 Determining On- and Off-Rates of Binding with Nanopores
Methods for characterizing the kinetics of protein-ligand binding using nanopores
employed the biological pore
-hemolysin and solid-state nanopores fabricated in
quartz pipettes [
12
,
21
,
24
,
33
]. These methods share the capability to detect the
association or dissociation of individual protein-ligand complexes. Applying (
9.5
),
the value of
K
d
can be calculated when the values of
k
off
and
k
on
are determined.
To detect these individual binding events, Bayley and coworkers used a ligand
attached to the interior wall of
a
-hemolysin by a polymer chain [
22
,
33
]. In these
experiments, the polymer was long enough to protrude from either side of the pore.
Consequently, in the absence of binding, the polymer chain freely moved between the
cis
and
trans
chambers. This flexibility resulted in rapid, transient current decreases
when the polymer extended through the pore (Fig.
9.5
). Figure
9.5
illustrates that the
binding of a protein to the ligand prevented the movement of the polymer chain
between the
cis
and
trans
chambers. This signal permitted the detection of association
and dissociation of individual protein-ligand complexes. Movileanu et al
.
used a poly
(ethylene glycol), PEG, polymer with a biotin moiety at the free end and a mutant
form of streptavidin (W120A), which has a lower affinity for biotin than the wild
type [
33
]. Figure
9.5
illustrates that binding of W120A to the biotin-PEG on the
cis
side of the pore prevented the polymer from entering the lumen of
a
-hemolysin. This
binding resulted in a stable, large current value. Hence, the return to rapid current
blockages signified the dissociation of the W120A-biotin complex, and the average
duration,
t
off
(s), of the large, steady-state current value was related to
k
off
by:
1
t
off
ΒΌ k
off
:
a
(9.7)
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