Single Molecule Analysis Using Single Nanopores - Nanofluidics: Nanoscience and Nanotechnology - page 114

attempts have been made to adapt such nanopores for use in high-speed DNA sequencing. 2
Nanopore-based analysis methods are typically premised on the concept of passing a
molecule, e.g. single-stranded DNA (ssDNA), through a nanoscopic opening while
monitoring a signal. 2-6 Typically, the nanopore is designed to have a size that allows the
ssDNA to pass in a sequential, single file order. As the ssDNA passes through the
nanopore, differences in the chemical and physical properties of the nucleotides that
compose the ssDNA are translated into characteristic electrical signals. The signal typically
detected is a modulation of an ionic current, created by an applied voltage across the
nanopore-bearing membrane or film, by the passage of the DNA through the nanopore.
Because of structural differences between different nucleotides, each type of nucleotide
interrupts the current in a different way, producing a type-specific modulation in the
current as it translocates. 7,8
The majority of the work performed in this area pertains to the use of a protein
channel in a lipid bilayer. 3 It is known that proteinaceous nanopores, such as those formed
by the toxin protein -hemolysin secreted by the bacterium Staphylococcus aureus, possess
a well-defined shape. Each pore is 1.5 nm at its narrowest point and consists of seven
identical -hemolysin molecules. The attractive features of protein nanopores include the
well-established procedure for their synthesis. 2-6 The -hemolysin protein nanopore is the
archetype for rapid characterization and sequencing of nucleic acid molecules using high
resolution analysis based on local light source and electrical read-out. 2,4,7 However,
because it is not possible to tune the diameter of protein nanopores in both directions,
investigating the structure, dynamics, and interactions of DNA/RNA molecules
electrophoretically translocating through these ion channels has some limitations. It should
also be noted that the -hemolysin nanopore has a limiting aperture approximately 1.5 nm
in diameter. 2 As a result, the pore is large enough to allow the passage of single stranded
DNA, but too small to accommodate double stranded DNA.
An alternative to protein channels is the use of solid-state nanopores. These offer
several advantages over phospholipid-embedded protein channels. The solid-state
nanopores can be tuned in size with nanometer precision and also display an improved
mechanical, chemical and electrical stability. However, the fabrication of these nanometer-
sized pores on solid state materials represents a significant challenge, especially in the
control and reproducibility of both the size and shape of the nanopores. Much research has
focused on the fabrication of nanopores in solid-state thin films 9-14 and typically involves
sophisticated instruments and complicated procedures. Furthermore, the mechanisms for
solid-state nanopore formation are still not well understood, and the geometry and surface
chemistry of such nanopores are not well characterized or controlled. The aims of this
chapter are to outline some of the options that are available when choosing single
nanopores for macromolecular characterization, and how they have been or can be
manufactured using NEMS technologies.

6.2 FABRICATION OF SINGLE NANOPORES

6.2.1 Formation of -Hemolysin Pores on Lipid Bilayers

Protein ion channels are nanometer-scale pores that span cell membranes.
Depending on their size and function, they conduct the traffic of ions and/or
macromolecules into and out of cells and organelles. The most well-known channels play