and others can detect binding of the smallest ions to an aptamer. Furthermore, all
available technologies have different prerequisites and requirements with respect to
the sample material. Development of an aptamer for later use in diagnostics or as a
therapeutic agent thus needs careful selection of the right tools and methods at the
right time and for the right application.
2.1 Biophysical Principles and Readouts of Selected
Techniques
Typically, a complex of two interaction partners differs from the respective individual molecules in many molecular parameters, such as size, structure, shape, energetic
state, charge, or hydration shell. Biophysical technologies either directly read out
these changes in order to obtain basic binding parameters or they indirectly monitor
effects correlated to these changes. Table 1 summarizes the readout principles of the
biophysical methods described below, as well as their key information content.
Furthermore, the key advantage of each respective technique is indicated.
2.1.1 Surface Plasmon Resonance (SPR) [1–5]
SPR appears when a polarized light beam hits a metal layer (commonly a gold film)
at the interface of two media with different refractive indices. Monitoring changes in
refractive index upon binding of an interaction partner (analyte) to an immobilized
partner (ligand) on the metal layer enables to calculate kinetic parameters (k on and
k off ) and steady-state affinity (K D ). Furthermore, thermodynamic parameters can be
estimated from experimental repeats at different constant temperatures [19].
In a typical SPR experiment, one of the interaction partners is immobilized on the
surface of an SPR sensor chip, whereas the other interaction partner is supplied in
different concentrations via a microfluidic system. Numerous different immobilization strategies and coupling chemistries are available for both nucleic acids, proteins,
peptides, and larger particles so that aptamers can be used both as the immobilized
binding partner and the partner being free in solution.
2.1.2 Biolayer Interferometry (BLI) [6, 7]
BLI analyzes interference patterns of white light that is reflected from two optical
layers of a sensor tip. One internal reference layer is located inside the tip and one
layer at the interface between the tip and the surrounding liquid phase. Each
reflection generates constructive and destructive interferences that vary with the
wavelength of the incident light. Any change at the outer layer of the tip
(a biocompatible surface with one interaction partner immobilized on it), for
4
M. Plach and T. Schubert
available technologies have different prerequisites and requirements with respect to
the sample material. Development of an aptamer for later use in diagnostics or as a
therapeutic agent thus needs careful selection of the right tools and methods at the
right time and for the right application.
2.1 Biophysical Principles and Readouts of Selected
Techniques
Typically, a complex of two interaction partners differs from the respective individual molecules in many molecular parameters, such as size, structure, shape, energetic
state, charge, or hydration shell. Biophysical technologies either directly read out
these changes in order to obtain basic binding parameters or they indirectly monitor
effects correlated to these changes. Table 1 summarizes the readout principles of the
biophysical methods described below, as well as their key information content.
Furthermore, the key advantage of each respective technique is indicated.
2.1.1 Surface Plasmon Resonance (SPR) [1–5]
SPR appears when a polarized light beam hits a metal layer (commonly a gold film)
at the interface of two media with different refractive indices. Monitoring changes in
refractive index upon binding of an interaction partner (analyte) to an immobilized
partner (ligand) on the metal layer enables to calculate kinetic parameters (k on and
k off ) and steady-state affinity (K D ). Furthermore, thermodynamic parameters can be
estimated from experimental repeats at different constant temperatures [19].
In a typical SPR experiment, one of the interaction partners is immobilized on the
surface of an SPR sensor chip, whereas the other interaction partner is supplied in
different concentrations via a microfluidic system. Numerous different immobilization strategies and coupling chemistries are available for both nucleic acids, proteins,
peptides, and larger particles so that aptamers can be used both as the immobilized
binding partner and the partner being free in solution.
2.1.2 Biolayer Interferometry (BLI) [6, 7]
BLI analyzes interference patterns of white light that is reflected from two optical
layers of a sensor tip. One internal reference layer is located inside the tip and one
layer at the interface between the tip and the surrounding liquid phase. Each
reflection generates constructive and destructive interferences that vary with the
wavelength of the incident light. Any change at the outer layer of the tip
(a biocompatible surface with one interaction partner immobilized on it), for
4
M. Plach and T. Schubert