Biomedical Engineering Reference
In-Depth Information
the presence of the molecule can be detected by monitoring its effect on the
trans-membrane current, which is measured by a patch-clamp amplifier.
During electrophoretic translocation, molecules typically pass through the nano-
pore at high speed. A 48 kbp double-strand DNA (dsDNA) molecule, for instance,
traverses the pore in only about 1 ms under an applied voltage of 120 mV [
5
]. While
this presents advantages for some applications (high throughput rapid screening to
name one), it poses a challenge for others. For example, accurate detection of sub-
molecular structure like local protein position or even nucleotide sequence - an
especially promising goal in the field - would require very high bandwidth to
achieve. One potential way to address this is to adjust experimental variables like
temperature and viscosity in order to slow down the translocation speed. However,
this places inherent limitations on solvent conditions and thus on what can be
measured. A more elegant solution would be a mechanism by which to “hold on”
to a given molecule and control its position relative to the pore. Such a situation
would offer the ability to slow, halt, or even reverse translocation arbitrarily.
Here, we describe a technique that yields this level of control: solid-state
nanopores with integrated optical tweezers [
6
]. We detail how the experiments
are performed, describe how the resultant measurements are interpreted, and
discuss a model developed along the way that gives insight into the dominant
forces involved in nanopore translocation.
2.2 Experimental Methods
Solid-state nanopores are fabricated using a method that has been described else-
where [
7
]. Briefly, common microfabrication techniques are used to produce a 20-nm
thin, 5 mm wide, free-standing window of silicon nitride supported in a silicon chip.
This chip is then mounted in a transmission electron microscope (TEM) and the
highly-focused electron beam is used to locally ablate the surface, effectively “dril-
ling” a single hole through the membrane (Fig.
2.1a
,inset).Thepropertiesofthe
resultant nanopore (diameter, shape) can be controlled to some degree by adjusting
the beam intensity, beam size, and the exposure time [
8
]. After pore formation, the
entire chip is stored in a solution of 50% ethanol in water. The mixture was chosen
in order to keep the membrane clean and hydrated, but to allow fluid to wet the interior
of the small pore more easily by reducing the surface tension.
Prior to use the chip is cleaned with water, acetone and ethanol, then dried under
nitrogen flow and exposed to an oxygen plasma for 30 s in order to create a hydrophilic
surface. Directly following this treatment, the chip is introduced with measurement
solution (KCl solution with 10 mM tris-HCl at pH 8.0). The sample cell (Fig.
2.1a
)is
composed of a sandwich-type structure with a poly(methyl methacrylate) (PMMA)
flow cell below the chip and a Perspex flow cell above. This allows independent
exchange of solvent to both sides of the membrane while still permitting the optical
path necessary for optical tweezer integration (Fig.
2.1b
). The assembled flow cell
is positioned on a three-dimensional piezo stage above a 60
water immersion
objective, which acts as the focusing lens of both the optical trapping laser (4 W,
l
¼
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