Biomedical Engineering Reference
In-Depth Information
Practically, one of these two components is immobilized on the gold surface us-
ing a coupling strategy based on the ones described in Section 8.2.3. Therefore, the
results obtained by this technique describe a particular situation where the ligand
is anchored to, and thus influenced by, a solid surface. Even though this aspect can
be minimized for instance by the use of long polymeric linkers or of biological gels,
it is sometimes a severe limitation. In some other studies, the orientation is favored
and an NTA-based approach is preferred.
The analyte is then injected and the kinetics of association is followed by quanti-
fying the amount of material on the surface. The shape of the evolution of this surface
excess can be modeled by classical kinetic equations (for details, see Chapter 7):
d LA
[
]
(8.16)
=
k
×
[
L A k
] [
×
]
-
×
[
LA
]
on
off
dt
“L” represents the ligand and “A” the analyte. k
on
is the association constant of the
complex, k
off
is the dissociation constant.
After a certain time, the system reaches a steady state described by equilibrium
constants K
a
and K
d
that are given by :
K
=
k
/ k
and K
=
k
/ k
(8.17)
a
on
off
d
off
on
After this steady state, analyte-free buffer is flown over the surface. As there
is no more analyte in solution the mass action law imposes a desorption of the
ligands. This step is described by kinetic equations very similar to the ones describ-
ing the association. Finally, a dissociating agent is injected to remove the remaining
ligands and to regenerate the surface (Figure 8.19).
Figure 8.19
Typical sensorgram obtained by SPR illustrating the three steps of association, dissocia-
tion and regeneration.
