09 Mar About SPR
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For SPR analysis, the ligand is immobilized on the surface of the biosensor chip, while the analyte is injected to flow through the surface. When the interaction occurs between the two molecules the refractive index at the surface changes and it can be monitorized as SPR signal over time resulting in a sensorgram.
Fitting the sensogram data at different concentrations of analite allows calculation of affinity constant of the complex, KD.
Single cycle kinetics is suitable to obtain rate constants kon and koff. Different kind of kinetic models are available such as 1:1; 2:1 interaction, heterogeneous ligand or analyte, and two state reaction.
SPR is based on a complex optical phenomenon that occurs in thin conducting films at the interface between media of different refractive index. When a polarized beam reaches the interface at a specific angle range one part reflects out but other interacts with the free electron constellations in the gold surface. The incident light photons are absorbed and the energy is transferred to the electrons, which convert into surface plasmons. The plasmons create a field that extends into the medium of lower refraction index. This field is called the evanescent wave because the amplitude of the wave decreases exponentially with increasing distance from the interface surface. The depth of the evanescent wave which is useful for measurements is within ~300 nm of the sensor surface.
Because the evanescent wave field penetrates the solution, conditions for this resonance effect are very sensitive to the refractive index of the solution composition. Changes in the solute concentration at the surface of the sensor chip cause changes in SPR. When the detecting molecule is attached to the sensor chip or when analyte binds to the detecting molecule, the solute concentration at the sensor chip surface increases, leading to a change in the SPR signal. The response measured is related to the mass of analyte bound, and for biological molecules is largely independent of the nature of the analyte.