Solid-State Nanopore Sensors for Nucleic Acid Analysis - Nanopores: Sensing and Fundamental Biological Interactions - page 3

species, based on the enhanced mobility of shorter molecules in the gel. This
technique finds application in DNA and protein purification, DNA length separation
assays and in DNA sequencing [ 9 ]. The limitation of this technique however, is that
it involves the simultaneous processing of millions or billions of molecules in order
to provide a macroscale optical readout. This in turn translates to large analyte
volumes, increased preparation time and high cost. In addition, the output is
averaged over a population of molecules and is less sensitive to subtle structural
variations amongst molecules. Single molecule sensing methods could help over-
come these limitations. Single molecule sensing methods employ highly sensitive
optical and electrical technologies to interrogate and analyze individual molecules
thereby reducing required analyte volumes and cost. One such technology that finds
application in single molecule DNA analysis with potential application to next
generation DNA sequencing, is the use of nanopores.
Nanopores are nm sized apertures embedded in biological membranes or
fabricated in solid-state membranes. Though the passage of biomolecules and
ions through nanopores is commonplace in biology, it is only recently that
researchers have been able to successfully drive single biomolecules such as
DNA through proteinaceous and solid-state nanopores in-vitro [ 22 , 43 ]. Solid-
state nanopore platforms are capable of resistively sensing individual biomolecules
including DNA, RNA and small proteins. The concept of resistive particle sensing
in solutions was first pioneered by Coulter in the early 1950s [ 21 ]. This work led to
the development of the Coulter Counter, now a commonly used device for
obtaining complete blood cell counts. The principle governing the operation of
the Coulter counter is relatively simple. An aperture, slightly larger than the analyte
of interest separates two chambers filled with conductive electrolyte. Electrodes
immersed in each chamber are used to apply an electric potential, creating a current
of ions through the aperture. As the analyte of interest passes through the aperture,
the ionic current is partially blocked and this perturbation is sensed electrically
revealing useful information about the particle.
Nanopore based single molecule sensors use this exact same principle, only at the
nano-scale where the size of this aperture is comparable to the 2.2 nm cross sectional
diameter of an individual dsDNA molecule. Briefly, a silicon support containing a
single nanopore of diameter comparable to the diameter of an individual DNA
molecule is fabricated (Fig. 1.1a ) and then inserted into a flow cell containing two
chambers filled with conductive electrolyte (Fig. 1.1b ). An electrode is immersed in
each chamber as shown, and a potential is applied across the nanopore chip, resulting
in an ionic current through the pore corresponding to the open pore current. Target
DNA molecules are next inserted into the cis chamber of the fluidic setup. Two-
terminal electrophoresis is used to drive the negatively charged DNA molecule
through the nanopore (Fig. 1.1c ), resulting in transient current blockades as seen in
Fig. 1.1d . These electrical signatures are then analyzed, revealing useful information
about the translocating molecule [ 89 , 90 ]. This technique has been used to study
various biophysical phenomena at the single molecule level including the label-free