Biomedical Engineering Reference
In-Depth Information
oriented and homogeneous attachment obtained, but it is also possible to detach
proteins and make repeated use of the same surface. For example, antigen-antibody
[Figure 9.16(a)] interactions are used to organize monolayers and multilayers of
GOX on glassy carbon electrodes. Alternatively, glassy carbon electrodes are coated
with gelatin and rabbit IgG antibodies are adsorbed onto the gelatin. A conjugate
consisting of GOX linked to the antirabbit IgG antibody is added to interact and
form a complex with the modified glassy carbon electrode surface. Enzyme multi-
layer electrodes are generated by the application of an anti-IgG-GOX conjugate X
and an IgG and anti-GOX antibody Y as linking components. Homogeneous me-
diation is used using ferrocenylmethanol as a freely diffusing mediator. The current
increases with an increase in the enzyme loading on the surface.
A popular technology is based on avidin-biotin affinity interactions. Avidin is a
tetrameric glycoprotein soluble in aqueous solutions and stable over wide pH and
temperature ranges. Biotin (or vitamin H) is a naturally occurring vitamin found in
all living cells. Only a small fragment of biotin is required for the interaction with
avidin. Avidin can bind up to four molecules of biotin via a strong noncovalent
bond (
K
D
10
15
M
−1
). Streptavidin is a closely related tetrameric protein, with a
similar affinity to biotin. Since biotin is a small molecule, its conjugation to mac-
romolecules does not affect conformation, size, or functionality. Both biotin and
avidin/streptavidin are attached to a variety of substrates. A typical biotin/avidin/
biotin multilayer [Figure 9.16(b)] is composed by directly immobilizing biotin and
avidin, creating a secondary layer for binding biotinylated molecules. This approach
is generally preferred due to the higher organization obtained in comparison to that
of the direct immobilization of avidin. Use of biotinylated DNA for complex for-
mation with a surface-confined avidin or strepavidin is also an option. A number of
high affinity recombinant proteins are also produced by genetic engineering, which
are then coupled with affinity tags. Tags are placed at defined positions on proteins,
preferably far away from the active site in order to achieve optimal accessibility of
the ligands. Poly(His) is the most popular tag used in these situations due to the
advantages of small size, compatibility with organic solvents, low immunogenicity,
and effective purification under native and denaturing conditions.
=
9.5.3 Microelectromechanical Systems (MEMS)
Microfabrication technology is borrowed from microelectronic integrated cir-
cuits and is utilized to miniaturize devices and develop portable biosensors.
Biotin
conjugated
sensing element
Antibody 2
Antigen
Avidin
Biotin
Antibody 1
Substrate surface
Substrate surface
(b)
Figure 9.16
Affi nity immobilization of biosensing elements to the surface: (a) antigen-antibody
based immobilization and (b) avidin-biotin based immobilization.
(a)
