No posts published in this language yet
Stay tuned...
Surface Plasmon Resonance
SPR is a phenomenon in which the reflected light disappears at a specific angle of incidence when light is incident on the side of the prism on which the gold thin film is placed. When biomolecules bind on the sensor chip, the surface refractive index changes and the angle of the reflected light shifts. Molecular interaction is monitored by acquiring sensorgrams that record this angle change in real time.
SPR measures the binding of various molecules such as antigen-antibody, protein, nucleic acid, carbohydrate, fat, cell, and virus.
In addition, label-free real-time analysis enables kinetic analysis of intermolecular association affinity (affinity) as well as
association rate constant and dissociation rate constant.
Applications of SPR in Life Science
Yes/No: Checks whether or not there is a connection. The presence or absence of reaction can be checked by simply flowing the analyte (B)
to the sensor chip on which the ligand (A) is immobilized.
Screening: Multiple analytes can be run sequentially on the same ligand to evaluate which analyte binds best.
Concentration: After acquiring a sensorgram for each concentration of an analyte, a standard quantitation curve can be created through the
concentration-signal graph.
Affinity: Evaluate the equilibrium constant after creating a Langmuir isothermal curve by acquiring the association section of
the sensorgram for each concentration up to the equilibrium stage.
Kinetics: Evaluate the rate constants ka and kd through curve fitting by acquiring the association/dissociation section of the sensorgram
for each concentration.
Ligand Immobilization
The ligand immobilization step is the step of immobilizing the ligand on the surface of the sensor chip.
Substances used as ligands such as antibodies, proteins, nucleic acids, and peptides are very diverse.
Depending on the properties of the ligand material, the surface functional groups (COOH, Biotin, NTA) of the sensor chip and the immobilization method can be determined.
Immobilization with Amine coupling
Analyte binding and regeneration
Analyte binding is an association step in which an analyte solution is injected to bind to a ligand, and the graph rises in real time.
By injecting only running buffer, it is divided into a dissociation phase in which some analytes attached to the ligand fall off.
After that, if the remaining analyte attached to the ligand is removed using a regeneration solution, the experiment can be continued with different concentrations or different types of analytes on the same sensor chip. The regeneration solution can be selected from a variety of solutions such as a low pH buffer, a high pH buffer, and a solution with high ionic strength depending on the properties of the ligand-analyte.
Kinetic Evaluation
Kinetic parameters can be calculated through a sensorgram that observes association and dissociation sections by flowing different concentrations of analyte.
The association rate constant (ka) and dissociation rate constant (kd) are obtained through a 1:1 binding model.The equilibrium dissociation constant (KD) obtained by dividing the dissociation rate constant by the association rate constant can be used as a parameter to evaluate the bonding force between two molecules.
