Biomedical Engineering Reference
In-Depth Information
Fig. 2.1 The experimental setup. (a) Diagram of the nanopore flow cell, detailing each compo-
nent. Inset: TEM image of a typical nanopore (scale bar 10 nm). (b) Side view of the assembled
flow cell, showing the liquid feedthroughs to both the top (
inner
) and bottom (
outer
) of the
nanopore chip, and the optical path for transmitted light to the objective
which is reflected onto a position-sensitive detector (PSD). The objective is used
additionally as a means for optical feedback using a CCD camera. Electrical contact is
made to measurement solutions with agarose salt bridges and Pt connecting wires
using small, enclosed reservoirs of 1 MKCl containing 100 mM ferro/ferri cyanide as
a redox agent. Voltage application and ionic current measurements are performed
using a patch-clamp amplifier (Axopatch 200B). This experimental setup is described
in greater detail elsewhere [
9
].
A given nanopore is tested for linear I-V characteristics (Fig.
2.2a
) and low
electrical noise (
20 pA RMS) before further use. In the event of unacceptable
properties, the pore can be cleaned briefly (~5 min) with 1 M NaOH and/or rinsed,
plasma treated and remounted for additional testing. Poor qualities are often the
result of bubbles [
10
] and can also be remedied by exchanging the solution with
water and then ethanol before exchanging back to measurement buffer. If all
treatments fail, the pore is abandoned and a new chip is used. Nanopores can
sometimes be unsuitable for measurement even immediately after fabrication due
to unknown reasons. Using the ethanol/water storage conditions, we find that 80%
of pores are fit for measurement.
Following identification of a useful nanopore, its precise location on the mem-
brane is determined. This is done by rastering the high-power trapping laser across
the membrane surface and monitoring the trans-pore current at each step. As the
laser focus comes near the nanopore, it locally heats the solution, increasing its
mobility and thus the measured ionic current. The resulting 2D array displays a
(nominally gaussian) spike [
11
] at the location of the nanopore. The membrane is
then positioned such that the trapping laser is co-local with the nanopore and moved
about 10 mm above the focal plane. Following this, microbeads (2 mm polystyrene)
with target molecules attached to them [
6
] are introduced to the bottom chamber at
very low concentration (0.001% w/v). Once a single bead is trapped in the optical
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