Biomedical Engineering Reference
In-Depth Information
tion enzymes that are heavily used in molecular biology as molecular scissors able
to cut DNA molecules at very specific spots. Another example where proteins are
used as “workhorses” is antibodies: When a foreign body or a protein is injected in
an animal, some of its cells produce particular proteins called antibodies that are
highly specific to this “antigen.” This property is the basis of the immunological
response and is often used to produce antibodies that can be isolated, purified, and
tagged. They are then used to specifically localize and quantify the protein of inter-
est for instance in fixed cells or in immunoassays.
We have mentioned earlier the use of DNA microarrays to quantify the level
of gene expression. Measuring directly the level and the activity of proteins with
protein microarrays is the next step in this process. Here, the sequences of DNA
spotted on the surface are replaced by proteins or by molecules with which these
proteins can interact. One then gains access to protein-protein interactions, proteins
interactions with small molecules, and so forth.
This information is richer than that obtained by measuring the levels of RNA
since proteins evolve after their production, they mature, are modified, interact with
other components—all processes that can affect their function and/or activity [9].
The detection of the coupling between the proteins and the molecules immobilized
on the surface is performed in different ways: by fluorescence, radioactivity, surface
plasmon resonance, or mass spectrometry using the strategies illustrated in Chapter
7 and developed here in Section 8.3.
8.1.2 Some Aspects of Cells
Cells are extremely complex arrangements and, of course, living entities (the small-
est there are). Prokaryotic cells do not have nucleus, they include bacteria and ar-
chae and have a simpler organization than eukaryotic cells in which DNA is packed
in a nucleus.
Both cell types have a barrier protecting them from the exterior: a soft phospho-
lipid membrane for eukaryotes and a more rigid wall for bacteria.
8.1.2.1 Eukaryotic Cells
It is often required to sort and characterize particular cells. An interesting example
of this process is the one of circulating cancer cells in the general framework of
early cancer diagnosis. In some cancers such as breast cancer, isolated tumor cells
disseminate in the body of the patients by being conveyed by the circulating blood.
They can also be found in their bone marrow. The challenge becomes the detection
of these cells estimated to 1-10 cells / 1 million nucleated cells. Their detection relies
on specific markers in their cytoplasm or on their membrane and may use fluores-
cence-based techniques such as the ones reviewed later in this chapter. However,
the number of pathologic cells is so low that not only does their detection require
a particularly sensitive and specific technique, but it is also of prime importance to
first enrich the medium with these cells. This results in a two-phase process where
the enrichment does not have to be highly specific but should take all the suspect
cells, and a second step where the true detection needs to be highly specific. An ef-
ficient strategy in this line is to use immunomagnetic enrichment: suspect cells are
