Biomedical Engineering Reference
In-Depth Information
( Dekker 2007 ). The biological nanopore already mentioned in this section, for
example, '-hemolysin, is a protein generated as toxin by Staphylococcus aureus ,
which is inserted in a lipid membrane and has a minimum width of 1.4 nm. This
nanopore was first used for DNA sequencing in the years 90, when nanotechnologies
were in infancy. Today, biological nanopores are not only used to study DNA
sequencing at the single molecule level but are also used to study peptides, nucleic
acids, and polymers ( Ma and Cockroft 2010 ).
Solid-state nanopores are the result of silicon micromachining technology and
the usage of EBL and/or FIB technology to process such small nanopores. There are
two basic methods to obtain nanopores. The first is the ion beam sculpting method,
where a focused ion beam directed to mill a large nanopore in a thin membrane
is combined with exposure to an Ar C beam which, subsequently, activates pore
closure due to a diffusion process; an Ar C ion detection technique placed under the
membrane determines when to stop this diffusion-induced pore shrinking process
( Li et al. 2001 ). The second method is based on micromachining of Si, SiN, or SiO 2
membranes, followed by EBL and etching ( Storm et al. 2003 ). Basically, an electron
beam drills the hole in the Si membrane, and at the same time, the hole is monitored
by TEM, the electron beam reducing the hole dimension in a systematic manner, at
arateof0.3nmmin 1 .
Tunneling junctions are fabricated and embedded in the nanopore for detecting
simultaneously both ionic and tunneling currents, which contain the DNA base
signatures ( Ivanov et al. 2011 ). Short-lived translocation events of type I are
recorded in sub-ms time ranges, and only the ionic current is recorded in this case,
while for type II events, which are assigned for translocation times longer than 1 ms,
both ionic and tunneling currents are recorded.
Solid-state nanopores are used to translocate RNA molecules and to compare the
electronic signature of dsRNA with those of homopolymers poly(A), poly(U), and
poly(C). The blockade ionic current is used for such studies, and the molecular
identification is due to the high applied voltages, of 0.6 V, which stretch these
flexible acids ( Skinner et al. 2009 ). It was observed that while the blockade
conductances of dsRNA and dsDNA are slightly increasing from 1.5 to 2 nS for
an applied voltage range 0.1-0.6 V, the poly(A), poly(C), and poly(U) conductances
decrease in the same voltage interval from 1.5 to 0.5 nS. So, these distinct signatures
could be used for detection of hybridization events and for base identification (see
Fig. 2.22 ).
Solid-state nanopores map also the DNA repair protein RecA attached along the
DNA length, using the distinct electrical signatures of the bare DNA and the RecA-
coated DNA, with a larger diameter of 7 nm. A high speed is achieved to locate and
map the RecA distribution with a spatial resolution of 8 nm, which corresponds to
five RecA proteins binding to 15 DNA base pairs, and single protein resolution is
soon expected ( Kowalczyk et al. 2010 ).
MicroRNAs (miRNAs) are biomolecules with a paramount role in gene reg-
ulation and RNA silencing therapies. MiRNAs play a key role in a variety of
cancers and are associated with RNA-induced silencing complexes, which bind
the messenger RNA and thus stop protein production. Rapid electronic detection
Search WWH ::




Custom Search