Biomedical Engineering Reference
In-Depth Information
to expose more hydrophobic domains.
13
This sort of effect will clearly be
deleterious if the native tertiary structure of the molecule is altered with respect
to target binding. Although hydrophobic effects can be damaging as indicated,
this sort of effect is by no means restricted to non-polar surfaces. Our
laboratory showed conclusively that the well-known protein, avidin, is
compromised with regard to its structure by hydrophilic interfaces.
Presumably, the reason for this lies in the instigated interactions of surface
polar functional groups with the carbohydrate moieties of
d
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3
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1
the protein
molecule.
14
The last comment leads to a look at the use of intervening adsorbed
molecules as a linker for eventual biomolecule attachment. A system which is
employed extremely widely is avidin/streptavidin/neutravidin chemistry.
15-17
Avidin is a tetrameric protein that contains four binding sites for the ligand,
biotin. Interaction of the protein with this molecule results in a particularly
strong bond (Kd
ΒΌ
10
15
). Accordingly, various biomolecules can be modified
with relative facility by biotin addition in order to link them to surface-attached
avidin, or a sister molecule such as the deglycosylated version, neutravidin.
With respect to device operation, particularly where on-line detection is
involved, it is common practice to introduce the protein into the system where it
is allowed to simply adsorb to the device surface. Although four biotin binding
sites are present, there is no doubt that at least two of these are expected to be
unavailable on steric grounds for the reasons outlined above.
An analogous chemical system is the use of protein A.
18
This is a polypeptide
(molecular weight 42 kD) isolated from Staphylococcus aureus which is capable
of binding specifically to the Fc region of various antibody molecules and has
thus been employed in immunosensor technology. One strategy similar to the
case of avidin outlined above is to introduce this protein (for surface
adsorption) in a system prior to antibody addition.
For both avidin and protein A, it is also feasible to attach the molecules to
the device surface in a chemically bonded fashion which leads to a large
portfolio of methods for biomolecule binding, that of using covalent bonds in
attachment (see section 1.3.3).
d
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.
1.3.2 Entrapment and Encapsulation
In this approach the recognition molecule is trapped within the three-
dimensional structure of a specific chemical matrix. The resulting entity is
placed on the device surface for transduction as described above. The matrix
may simply act as a 'holding' moiety or be modified in some way to take part in
the transduction process. For the former, polymers such as polyacrylamide
have been employed in a number of experimental protocols. Examples are
derivatization of a protein with the monomer followed by polymerization and
direct diffusion of the biomolecule into a preformed polymer gel. It is also
possible to attach the probe to polymer beads to be followed by cross-linking.
There are a number of potential disadvantages to this type of approach for
placing the probe on the device surface including the possibility of biomolecule
