Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 338

electrical potential difference to be applied between the two chambers. Because the
solution is conductor, a majority of this applied potential difference is dropped
across the membrane, leading to a focusing of the electric field in and around
the pore. Articles describing NFS experiments often quote the applied potential
difference, or transmembrane voltage, which gives another experimenter sufficient
information to reproduce the electrostatic force given the same membrane thick-
ness and pore geometry. As shown in Fig. 14.1b , the electric field is sharply focused
at the tightest constriction of the pore, with a magnitude on the order of 10 8 V/m
for a typical nanopore under a transmembrane voltage of 1 V. Analytes in the
surrounding solution of the appropriate charge are drawn to the pore by the electric
field or an electro-osmotic flow. Loading of more than one analyte can be prevented
by tuning the size of the pore to sterically exclude more than one analyte.
There are two common protocols for the application of the transmembrane
voltage in NFS experiments. First, one can use a constant transmembrane voltage,
which applies a constant force assuming a constant pore-analyte conformation, and
measure the distribution of the time interval between the rupture and escape of the
analyte. A second common protocol involves applying a linearly increasing trans-
membrane voltage, or a constant loading rate if one again assumes a constant
pore-analyte conformation.
The answer to question (iii) above is that measurements of the ion current
through the pore, which is driven by the transmembrane voltage, serve as an
indirect means to determine the effect of the force on the analyte. This current,
which typically ranges from a few picoamperes to many nanoamperes, can be
measured by sensitive ammeters as suggested in Fig. 14.1a . Because the opening
of the nanopore is near the size of the analyte, the ion current through it can change
dramatically when an analyte is captured and loaded into the pore. For example, a
drop (or increase) in the ion current can be observed when DNA enters a nanopore.
The exit of the DNA from the nanopore is therefore accompanied by a return of
the current to its original value. Furthermore, the ion current can change in response
to conformational transformations of the analyte. Although protocols for nanopore
assays have been constructed which employ fluorescent markers [ 26 ] or measure-
ments of the potential on electrodes embedded in the walls of the nanopore [ 27 ],
the ion current is invariably a convenient and useful source of information on events
occurring in the pore during NFS experiments.
The unique properties of NFS also lead to some disadvantages with respect to
other single-molecule force probing techniques. In a typical NFS experiment, there
is just one input signal (the transmembrane voltage) and just one output signal (the
measured electrolytic current). In contrast to other force spectroscopy methods, in
pure NFS the force on the analyte cannot be directly measured (although hybrid
experiments including both optical tweezers and nanopores have been performed [ 28 ]
to directly measure the force). In NFS, the experimenter has precise control over the
transmembrane voltage, which only indirectly determines the force on the analyte.
This force depends on the position of the analyte within the pore as well as the pore
geometry, the electrical properties of the membrane and electrolyte, and the
positions of any other molecules that may be associated with the analyte.