Biomedical Engineering Reference
In-Depth Information
20.4
Implementation and Illustration of Instrumentation
The Biacore X (Biacore) SPR analytical instrument is a typical device with which this type
of work can be performed. The following is a brief description of the methods involved in
this work.
20.4.1
Immobilization
The IgE-specific aptamer was immobilized to a dextran sensor chip (CM5) obtained
from Biacore (Uppsala, Sweden) via a streptavidin-biotin reaction. The dextran chip
allows for a more “biocompatible” environment, increased receptor activity, increased
receptor density, as well as reduction in nonspecific sample interactions. Once immobi-
lization was complete, binding experiments using IgE could then be performed by run-
ning appropriate sample concentrations and volumes over the sensor surface. After
binding of the target molecule, the sensor surface could be regenerated using a solution
of EtOH/NaOH. A typical result obtained from an SPR experiment, known as a senso-
gram, is shown in Figure 20.9.
20.4.2
Calibration
Using a range of concentrations, it is possible to obtain a calibration curve, which can be
used to extrapolate the concentration of target analyte in solutions of unknown concen-
trations. Such a calibration curve is shown in Figure 20.10.
Although this work has been carried out successfully, there remain a number of areas
where vast improvements can still be made, particularly with respect to the sensitivity of
the measurements. Indeed, in this particular case, the sensitivity of the aptasensor has not
Intensity
(A)
II
I
Angle
(B)
Dissociation
RU
FIGURE 20.9
Sensogram from a typical SPR
experiment. (A) Decrease in inten-
sity of reflected light at resonance
angles. (B) The shift in resonance
angle (from position I to position II)
is due to the binding of the analyte
at the sensor surface. (Modified
from Katsamba PS, Park S and
Laird-Offringa IA. 2002. Kinetic
studies of RNA-protein interac-
tions using surface plasmon reso-
nance.
Methods
26: 95-104.)
Association
Regeneration
Time
