Chapter 6 – Forces  249

this scenario is the speed of migration: even for the lowest controllable voltage gradient the

translocation speeds are high for unconstrained DNA molecules leading to the unreliability

in experimental measurements of the ion-​flux signature. One method to slow down a DNA

molecule as it translocates through a nanopore is by controllably pulling on the molecule from

the opposite direction to the electrostatic force using optical tweezers (Keyser et al., 2006).

An additional issue with DNA sequencing through a solid-​state nanopore is the finite trans­

location length. The minimum width of a structurally stable silicon nitride sheet is ~20 nm,

equivalent to ~50 nucleotide base pairs of DNA (see Chapter 2) assuming the double-​helical

axis of the molecule is stretched parallel to the central nanopore axis. Attempts to circum­

vent this problem have involved reducing the substrate thickness by using a monolayer of

graphene (Schneider et al., 2010). Graphene is a 2D single atomic layer of carbon atoms

packed into a honeycomb shape with a thickness of only ~0.3 nm but which is structurally

stable. This is comparable to just a single-​nucleotide base pair (Figure 6.10c).

Graphene is not an easy substrate to work with, however, being mechanically quite

brittle, and also graphene is only as strong as its weakest link, such that imperfections in its

manufacture can seed extensive cracks in its structure. Also, graphene nominally has a high

hydrophobicity that can causes problems when working with physiological solutions. An

alternative compromise being developed is to use a molybdenum disulfide three-​atom layer

substrate. This has an inferior larger thickness of ~0.8 nm, but fewer of the problems are

described earlier.

Simulation studies for the translocation of single biopolymers through a nanopore that

incorporate some degree of realistic flexibility of the nanopore wall actually suggest that

allowing the pore, some level of compliant wiggle can increase the speed of biopolymer trans­

location (see Cohen et al., 2011). In this case, nanopores composed of a less stiff material

than graphene, molybdenum disulfide, or silicon nitride might be an advantage, such as those

composed of soft matter, discussed in the following text.

6.6.4  SYNTHETIC SOFT-​MATTER NANOPORES

A number of natural pore-​forming proteins exist, which can self-​assemble within a phospho­

lipid bilayer, and are much more compliant than the synthetic silicon-​based nanopores

discussed earlier. The best characterized of these is a protein called α-​hemolysin. This is a

poison secreted by the Staphylococcus aureus bacterium to kill other species of competing

bacteria (a version of S. aureus that is resistant to certain antibiotics has been much in the

news due to its increasing prevalence in hospitals, called methicillin-​resistant S. aureus).

α-​Hemolysin binds to cell membranes of these nearby competing bacteria and spontaneously

punches a hole in the phospholipid bilayer significantly impairing these cells’ viability by

disrupting the proton motive force across the membrane, which thus allows protons to leak

uncontrollably through the hole and destroy their ability to manufacture ATP from the oxi­

dative phosphorylation process (see Chapter 2).

An α-​hemolysin pore is formed by self-​assembly from seven monomer subunits

(Figure 6.11a). These nanopores can be used in a controlled environment in an artifi­

cial phospholipid bilayer and utilized in a similar manner to solid-​state nanopores to

study the translocation of various biomolecules through the nanopore by measuring the

molecular signature of the ion current as the molecule translocates through the nanopore

(see Bayley, 2009). These naturally derived protein nanopores have advantages over solid-​

state nanopores. First, their size is consistent and not prone to manufacturing artifacts.

Second, they can be engineered to operate both with additional adapter molecules such

as cyclodextrin, which allows greater ion current measuring sensitivity for translocating

molecules such as DNA, and in addition the amino acid residues that make up the inside

surface of the pore can be modified, for example, to alter their electrostatic charge, which

can be used to provide additional selectivity on which biomolecules are permitted to trans­

locate through the pore. This nanopore technology is a prime candidate to first achieve

the goal of reliable, consistent, rapid single-​molecule sequencing of important biopolymers

such as DNA in the near future.