Biomedical Engineering Reference
In-Depth Information
most of their time to orient themselves along the field are considerably slowed down
(Figure 10.6). This technique has literally been a breakthrough in the separation of
long molecules (up to 10 Mbp) [13-15]. Extremely efficient, it remains also very
time-consuming.
10.1.4.3 Capillary Electrophoresis and Electrophoresis on Chip
Capillary electrophoresis is a gel-free technique. It is becoming predominant over
gel electrophoresis for challenging separations. In this case, the gel slab is replaced
by a capillary whose typical dimensions are 100
m
m in diameter and 50 cm in
length. Since the capillary geometry is favorable to a good thermal dissipation, high
electric fields can be used (up to 1 kV/cm). This strategy is much easier to automate
and parallelize than the gels and has been massively used in “brute force” sequenc-
ing of various genomes including the human genome. The sieving matrix in this case
is a polymer solution that acts similarly to a gel. The main difficulty in using this ap-
proach is the electro-osmotic flow (EOF) (Section 10.1.2) that superimposes to the
electrophoretic motion and that is quite detrimental to the resolution. This effect is
very dependent on the details of the chemistry of the surfaces and it is necessary to
“hide” them to the electric field, for example by chemically grafting or by adsorbing
neutral polymers on it.
A strong tendency nowadays is to integrate these capillaries within “chips”
where all the dimensions can be reduced and that have on the same chip, the injec-
tion, separation, and detection steps [17, 18]. With smaller devices, the analysis
volumes are reduced and the times are much shorter. Furthermore, heat dissipation
is even better than for standard capillaries enabling higher fields to be used. As it
has been mentioned in the preceding chapters, the fabrication of these channels uses
a technology stemming from the microelectronics industry. Many different materi-
als can be used for the fabrication of the chips (plastics or plastic-silicon hybrids,
for example) although it should be stressed that silicon, the preferred material in
microfabrication, is generally too conductive to be compatible with the high elec-
tric fields required for this particular application. When it arrives at full maturity
(which can be forecast within the next few years) this approach can be expected
to lead to disposable, inexpensive devices. However, there are still hurdles to over-
come: the use of new materials changes the electro-osmosis characteristics as well as
the DNA-surface interactions. Furthermore, there seems to be a limit to the electric
fields that can be used as too high values can have negative consequences such as
the aggregation of long DNA chains by electrokinetic effects [19].
10.1.4.4 New Exploratory Fields
Besides reproducing electrophoresis on a chip, micro- and nanofabrication has en-
abled new ideas and new concepts aiming at the separation of DNA molecules and
more and more of proteins.
Artificial Gel
As early as 1992, there have been seminal experiments where the gel commonly used
as a separation sieving matrix has been replaced by a solid state microfabricated
