Biomedical Engineering Reference
In-Depth Information
For instance, it is possible with these instruments to sort cells by their protein
content provided the protein of choice is fluorescently labeled using immunofluores-
cence. In the same line, assays based on GFP fluorescence (Section 8.2.1.2) can be
used to analyze protein expression of certain cells. In these situations, the cell can
be kept alive and cultured after this sorting.
Different aspects of cell phenotype or genotype can be tested with assays based
on DNA fluorescence. Live/dead assays for instance are routinely performed. They
are based on the membrane permeability: when cells die, their membrane is dis-
rupted and dyes can freely penetrate them. Dyes that fluoresce only in the presence
of nucleic acids are used for this purpose. They can penetrate dead cells membranes
to bind to the nucleus DNA so dead cells become fluorescent while live cells, whose
membranes are intact, are not.
Drugs can drastically modify the biochemistry of cells and their influence can
thus be monitored by FACS. Some of these drugs modify the cell cycle, a measure-
ment of the DNA content of each cell of a population then gives a “signature” of
this particular treatment. These measurements of DNA content are useful in other
situations such as the expression of a particular gene. To perform these measure-
ments, cells are first permeabilized to allow the entry of the dye to the nucleus and
thus the measurement of the DNA content.
Similar architectures of cell sorting devices have been implemented in micro-
systems with good results, although these lab-on-chip systems are still much slower
than the traditional version.
8.4 Molecular Micromanipulation
8.4.1 Force Measurements
One of the main micromanipulation techniques at the molecular scale uses the AFM
described earlier. With this instrument, forces between individual objects such as
proteins can be measured. The principle is quite simple: one of the interacting pro-
teins is immobilized on the tip of the instrument and the other one on a facing solid
surface. The tip and the surface are first bought into contact and then separated.
The force necessary to separate them is measured by the deflection of the cantilever
(Figure 8.21). The small radius of the tip ensures that only events involving single
molecules interactions are measured. Using this technique, antigen-antibodies in-
teractions have been measured and the stretching of several biomolecules including
DNA and proteins have also been characterized. Probing such interaction ener-
gies (close to k
B
T) necessitates a particular theoretical treatment. In particular, the
lifetime of a bond under a force is affected by this force [38]. This model has been
adapted to the problem of the measurement of rupture forces between single pro-
teins and it has been shown that this force F varies logarithmically with the velocity
of separation [39]:
F (k T / x ) Ln[r x /(k k T)]
=
×
(8.18)
B
β
f
β
off B
Where r
f
is the so-called loading rate (product of the cantilever spring constant by
the velocity of separation) and x
β
a characteristic length of the bond.
