Biomedical Engineering Reference
In-Depth Information
symmetrical 1 M/1 M KCl buffer (pH 8.5) conditions, using a nanopore 4 nm in
diameter. Entry of PEG molecules into the pore resulted in brief stochastic block-
ades of the ion current. Figure
10.9
(top panel) displays one second of typical gap-
free recorded ion currents under different applied voltages. When positive voltage
was applied to the
trans
electrode, no ion current blockades were observed.
Reversing the voltage (i.e. when the
trans
chamber was negatively biased), clear
stochastic blockades appeared, the frequency of which grew sharply with the
voltage magnitude. The lower panel of Fig.
10.9
displays the blockade event rate
as a function of voltage applied to the
trans
electrode over a range from
300 to
+300 mV. Clearly, PEG entry to the pore is only observed at negative voltages.
Entry of the uncharged PEG molecules into the nanopore is driven by the EOF of
cations (K
+
) in this system. Therefore entry of the PEG into the nanopore is only
feasible when the
trans
chamber is negatively biased. These measurements suggest
that EOF in the silicon nitride system is produced by
cations
, and is directed
towards the negative electrode.
Given that the EOF in the SiN pores is produced by the cations, we next examine
the effect of salt gradients (which can either enhance or suppress the flow of
cations) on the capture rate of negatively charged DNA. Using one nanopore, the
capture rate
R
c
of 400 bp DNA was measured using three different
trans/cis
salt
concentrations and the same
cis
DNA concentration (3.8 nM), shown in Fig.
10.10a
[
50
]. The top trace corresponds to 1 M/1 M
trans/cis
KCl concentrations, the middle
trace to 1 M/0.2 M, and the lower trace to 0.2 M/1 M. Continuous data streams were
collected for each gradient, and representative 2-s snapshots are shown. Strikingly,
when the
cis
concentration was lowered from 1 to 0.2 M, the capture rate increased
ninefold from ~0.4 to ~3.7 s
1
nM
1
. Reversal of the conditions (to 0.2 M/1 M)
effectively suppressed DNA capture. These results were completely reversible,
i.e. returning to 1 M/1 M yielded the same capture rate as was previously obtained
for those concentrations. Figure
10.10b
shows the enhancement in
R
c
for several
other DNA lengths (400, 2,000, 3,500 and 8,000 bp), over a large range of asym-
metries (
C
trans
/C
cis
from 1 to ~32). Clearly, above a threshold value of roughly
C
trans
/C
cis
~ 1.5, a roughly linear, length-independent increase in the capture rate is
observed. In addition to providing rate enhancement, these asymmetric salt conditions
are particularly advantageous because they allow experiments to be performed where
DNA is kept under physiological conditions (i.e. 125 mMKCl), while still preserving
the signal-to-noise ratio of an experiment performed at high salt concentration.
Under asymmetric salt conditions, the electrical potential near the pore is
proportional, to first approximation, to the ratio of bulk ionic concentrations in
the
cis
and
trans
chambers:
VðrÞ¼V
sym
ðrÞC
trans
C
cis
is the poten-
tial under symmetric conditions. Accordingly, the value of
r*
is also modified to
r
¼ r
sym
C
trans
C
cis
=
, where
V
sym
ðrÞ
[
50
]. The rationale for this is that the voltage drop in the
solution of lower conductance is higher, and therefore a high/low
trans
/
cis
salt
gradient will yield an asymmetric potential profile, with more of the voltage drop
occurring within the
cis
chamber than in the
trans
chamber. Finite-element
Comsol simulations confirm that salt-dependent charge imbalance at the pore
causes the potential to protrude significantly farther into the
cis
chamber for
=
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