Third Generation DNA Sequencing with a Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 292

to produce the most sensitive electronic device - the single-electron transistor
[ 23 ] - and now the same techniques are being leveraged to measure the charge
distribution in a single DNA molecule.
So far, the focus of attention has been on sequencing strategies that rely on
electric signals such as the blockade current that develops when DNA translocates
through a pore in a membrane. Alternatively, using electrodes which are placed
extremely close to the DNA molecule on either side of a nanopore orthogonal to the
DNA backbone, each nucleotide might be probed directly as it translocates passed
through the pore. Theoretical studies have shown that with such electrodes, the
electron tunneling current should be significantly different between the different
nucleotides. There is an overwhelming advantage to this approach - e.g., the
nucleotide is directly probed, rather than indirectly measured using the the blockage
of ionic current. Lagerqvist et al. suggests that 10 7 current measurements per second
should be sufficient to identify individual bases - or 10MHz - which is possible with
proper electrode design [ 24 ]. However, differences in tunneling current have not yet
been experimentally shown to differentiate individual bases, or base pairs, from each
other using typical STM probes [ 25 , 26 ]. Moreover, the tunneling current is exqui-
sitely sensitive to position, which can be problematic especially considering thermal
fluctuations that occur in an oversized constriction [ 27 ].
Another method of potentially detecting the nucleotide present in the nanopore
is through capacitive detection. By placing electrodes in the nanopore, the electro-
static potential in the pore can be measured. If DNA is cycled back and forth
through the pore (with an amplitude of oscillation of ~1 nm), an effect due in part to
the dipole moment of the base present in the pore constriction and in part to the
velocity difference for different bases is measured. The combination of the dipole
moment and the velocity are characteristic of the base in the pore, allowing for
identification of the DNA sequence [ 28 ].
All of these implementations suffer from the same limitations: i.e. lack of control
over the translocation kinetics and the molecular configuration. Prior work [ 21 , 29 ,
30 ] has indicated that the velocity of DNA through a solid-state nanopore can be
large, exceeding 1 bp/10 ns even at low voltage (200 mV). On the one hand, the
high velocity promises high throughput. On the other hand, it also makes it difficult
to resolve the electrical transients associated with a single nucleobase, especially if
the bandwidth is narrow enough to minimize noise. Sequencing with single base
resolution also demands sub-nanometer control of the molecular configuration in
the pore because the configuration affects the signal derived from the ion current.
This stringent condition follows because B-form double-stranded DNA ( dsDNA )
has a twisting, propeller-like, helical structure ~2 nm in diameter with an axial rise
of 0.34 nm per base-pair. Repeated measurements made using either multiple pores
with multiple copies of DNA [ 31 ], or multiple passes with a single molecule [ 32 ]
might address throughput, but they don't offer control of the molecular configura-
tion in the pore.