Biomedical Engineering Reference
In-Depth Information
Figure 10.8
Schematic of a DNA strand translocating the
a
-hemolysin protein embedded in a
lipid bilayer. The drawing is not to scale. The structure of this protein is represented Figure 8.5.
of these experiments, single strand DNA is driven by an electric field through a
single biological pore, the
a
-hemolysin channel, that has been previously isolated
and inserted in a lipid membrane (Figure 10.8) [23]. The structure of this proteic
complex enables the translocation of a single single-strand DNA at a time. The
electric field that drives the chain in the pore is also used for its detection: When the
DNA is inside the pore, it effectively blocks the ionic electrical current increasing
the electrical resistance. By analyzing the details of this time-resolved resistance, one
can get the length of the molecule (by the duration of the pulse) and some informa-
tion on its sequence (by the value of the resistance). It is hoped that, at some point,
this technique will be used for ultra-fast DNA sequencing [24]. To achieve this goal,
it is necessary however to develop a solid-state alternative to the
a
-hemolysin pore.
Intense work is presently devoted to drill nanometer sized holes in thin inorganic
membranes with sophisticated nanotechnology [25]. The next generation of these
devices are expected to be more than simple pinholes. They should carry also in
the same plane some kind of very local detection setup. This can be performed
with the chemical grafting of molecules responding to the passing of some of the
nucleotides (for instance by fluorescence energy transfer) or, maybe more realisti-
cally, in the form of transverse electrodes embedded in the membrane that will
allow to achieve a single base pair resolution through the measurement of a tunneling
current.
Still in the nanoworld, nanochannels are the other field of application of this
“nanoelectrophoresis”. In this case, double strand DNA is forced into nanochannels
or nanoconstrictions with the electric field and observed by fluorescence micros-
copy. There are nowadays several techniques that can be used for this nanofabrica-
tion. The chains can considerably stretch in these structures (up to 60% of their
fully extended length) and it is observed that their extended length is proportional
to the number of base pairs. By a simple averaging of this projected length, an ex-
tremely accurate determination of the molecular weight can be obtained [26]. An-
other application of this confinement is the precise localization of proteins bound to
the chain. If the protein and the DNA are labeled with fluorescent dyes of different
