Solid-State Nanopores: Methods of Fabrication and Integration, and Feasibility Issues in DNA Sequencing - Nanopores: Sensing and Fundamental Biological Interactions - page 195

It was found (Peng and Ling, [ 27 ]) that, after a DNA is captured, it is possible to
trap indefinitely a single DNA by reducing the applied voltage quickly to prevent
multiple DNAs being captured into the nanopore, as well as the breakage of the
streptavidin-biotin bond. At 50 mV bias, we found that a DNA can be trapped inside
the pore for a long period of time (hours), as also observed by Keyser et al. [ 28 ].
In contrast to standard DNA translocation experiment, the driving force is
localized at the pore region, extending only over a few nanometers. While in reverse
translocation, the driving force is tension which is extended over the entire DNA
length between the pore and the bead. The entropic spring effects (Marko and Siggia,
[ 21 ]) of the DNA can be suppressed. In addition, the center-of-mass of the DNA
moves with the bead, thus acquiring a smaller diffusion constant. As discussed in
Sect. 4.2 above, suppressing the diffusive motion of the DNA inside the nanopore
will be crucial to positional measurements on the nanoscale features on a DNA.
To pull the DNA out of the nanopore mechanically, a pair of magnetic tweezers
was used. For fine-tuning the magnetic force on the bead, the magnets with
calibrated field profile are mounted on a Burleigh Inchworm nano-positional
stage. The gradual increase of magnetic force is achieved by moving the magnets
slowly towards the nanopore chip. In the distance range over which the DNA is
pulled out, the force increase rate is about 0.2 fN per step on the Burleigh stage.
(The Burleigh stage can move 20 nm per step, and it can traverse at a speed from
0.01 to 2,062 m/s.) Once the magnetic force exceeds the electric force (at 50 mV),

the magnetic bead moves away from the pore region, as indicated from the real-
time video images. Coincidentally, the nanopore ionic conductance drops.
This technique is compatible with simultaneous ionic-current measurements and
is suitable for multiple nanopores, paving the way for large scale applications. Since
the DNA is held under tension during the translocation, the center-of-mass diffu-
sion constant of the DNA is controlled by the bead, and is thus suppressed.
The current increase when a DNA is captured by the nanopore was due to the low
ionic strength of the buffer used. This effect was first discovered by Chang et al. [ 29 ].
Here it is used as a reliable method for identifying DNA capture.

m

8.5.2 Nanopore DNA Translocation Experiment
for Hybridization Detection

To investigate the feasibility of electrical detection of hybridization probes on
a DNA using nanopores, a 12 mer hybridization experiment was carried out
(Balagurusamy et al. [ 31 ]). The choice of 12 mer hybridization is based on the
stability consideration. Shorter probes are less stable at room temperature. In future
experiments on shorter probes, a cooling device for the buffer will have to be used.
For HANS (hybridization-assisted nanopore sequencing) studies, the solid-state
nanopores are preferred over a -hemolysin pores because the latter do not allow
the passage of double-stranded DNA.
Using the Zucker mfold software ( http://mfold.bioinfo.rpi.edu/ ), available at the
Rensselaer bioinformatics web server, a model DNA molecule was designed for the
experiment. The details of the molecule are shown in Fig. 8.13 . For this experiment,