Biomedical Engineering Reference
In-Depth Information
DNA chips are another example requiring surface modification. DNA oligo-
mers are spotted onto a surface (usually glass) and need to be permanently an-
chored on it. Glass is negatively charged, so only a few of these molecules would
naturally remain stuck on it once it is in contact with a water-based buffer. On the
other hand, if the surface is coated with amine groups by silanization, the surface
becomes positively charged and these oligomers then stick irreversibly to it.
In the case of proteins, a covalent reaction is even better. Very often, a group
able to react on amines is chosen because the exterior surface of proteins is rich
in these chemical groups. The aldehyde group present in glutaraldehyde or the
N-hydroxy-succidimide group (NHS) are common choices for this purpose. Other
groups such as vinyl sulfone can react on thiols also often available on the proteins
[21]. This strategy however has several drawbacks: it needs a high enough density
of amine groups on the protein surface; it can interfere with the function of the
protein if it reacts precisely on the functional site and, of course, even if it reacts on
some other random place of the protein, the orientation information is lost.
To overcome these difficulties, strategies that involve “molecular glues” by spe-
cific and sturdy interactions such as the one of streptavidin with biotin or the hexa-
histidine sequence (His) 6 with Ni-NTA (nitrilo-tri acetic acid) are preferred [22].
Antigen-antibody interactions can also be used to the same end. These strategies are
particularly seducing as groups such as biotin or (His) 6 can be genetically included
in the protein during its synthesis by cells and are actually often used to purify them
after cells' lysis. The position of these groups on the protein is thus well known and
chosen to interfere as little as possible with their function. If the linker of the strep-
tavidin or the NTA to the surface is sufficiently rigid, the orientation of the protein
is preserved. On the other hand, with long spacers, the proteins can have all the
possible orientations and their interaction with the surface is reduced.
8.3  Experimental Methods of Characterization
8.3.1  Microscopies
Within the last years, optical imaging techniques have seen extraordinary develop-
ments. The advances in computing techniques and the widespread use of lasers
have made possible to image processes that were thought to be only indirectly ac-
cessible.
Although they are all called microscopies and are all imaging techniques, there
is little in common between optical microscopy, electron microcopy, and atomic
force microscopy.
8.3.1.1 “Classical” Optical Microscopy
The first microscopy technique that comes to mind to characterize particles is opti-
cal microscopy. Optical microscopes, although all based on the same basic design,
constantly improve, adding new potentialities that the use of lasers as light sources
and the computer analysis of images have contributed to enhance. They are an in-
valuable compromise between ease of use, versatility, and performance.
 
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