Biomedical Engineering Reference
In-Depth Information
(100 µg/L) of the aptamer with respect to the antibody but with an extended linear detec-
tion range to tenfold higher concentrations of IgE. In addition to this, the study demon-
strated for the first time that an aptamer-based biosensor could specifically detect an analyte
in a complex protein matrix. Lastly, regeneration of the aptamer receptor layer was shown
to be possible.
The work used to illustrate this chapter is based on SPR aptasensors, and as such the
theory of SPR and aptamers is expanded on in the Sections 20.2 and 20.3, respectively.
20.2
Surface Plasmon Resonance
SPR, as applied to biosensors, is a relatively new technology which has been used in a
number of fields to determine a desired quantity by measuring the change in refractive
index occurring due to the interaction between, for example, a receptor molecule and its
ligand (32). The development of SPR-sensing configurations and applications has been
described for the measurement of physical, chemical, and biological quantities. It is, how-
ever, in the field of affinity biosensors that SPR has shown the greatest potential, allowing
real-time (i.e., under continuous flow conditions) analysis of biospecific interactions with-
out the use of labeled molecules (33).
Much of the success of SPR is because of the fact that it is a fast, reliable, and sensitive
method that can be used to answer a number of fundamental questions about the interac-
tions of the molecules being investigated. The information that can be gained includes the
following (34):
Concentration determination of the interacting molecules.
●
Determination of the association and dissociation rate constants and affinity.
●
Determination of active binding regions and relative binding patterns
(e.g., epitope mapping).
●
Specificity of the interaction (for a particular molecule or a class of molecules).
●
The potential of SPR for the characterization of thin molecular films and monitoring
processes at metal interfaces was recognized in the late seventies (35; 36). Currently, sev-
eral companies have commercialized SPR sensor technology, which has become a leading
method for the direct real-time observation of biomolecular interactions (37).
20.2.1
Theory
A surface plasmon (SP) is a charge-density oscillation (wave) that may exist at the inter-
face of two media with dielectric constants of opposite signs, such as a metal and a dielec-
tric (e.g., water) (32). The wave is strongly localized and propagates along the interface
between the metal and the ambient medium (34).
The basic principle behind using SPR as a biospecific-interaction analysis (BIA) tech-
nique is as follows. Incident light is used to excite an SP. At the SPR angle, where the
energy and momentum of the incident light coincide with that of the charge density wave,
the photon energy is transferred to the charge density wave (38). This phenomenon is
observed by a sharp dip in intensity of the reflected incident light. The resulting wave
propagates along the interface between the metal and the ambient medium and is
extremely sensitive to changes in refractive index near the metal surface, for example due
to receptor-ligand interactions (38). Changes in the SPR angle, for small angular shifts, are
