Issue link: https://resources.nanotempertech.com/i/1050614
5 Surface plasmon resonance (SPR) is a common biosensor-based technique for measuring biomolecular interactions. How it works SPR is a surface-based biosensor reporting technology. It requires the immobilization of one binding partner, the ligand. The other binding partner, the analyte, is injected into a flow cell and comes into contact with the ligand on the surface at a constant flow rate. The interaction of ligand and analyte leads to an increase in mass bound to the surface, which results in a signal change. The signal is plotted in resonance units (RU) (also referred to as response units). The resulting sensograms display an association phase which depends on the association rate constant (k on ), followed by a dissociation phase (only buffer is pumped through the flow cell) which depends on the dissociation rate constant (k off ). By testing different analyte concentrations presented to a constant amount of immobilized ligand, the k on , k off and K d of the interaction can be determined. Strengths SPR determines binding kinetics and constants using label-free detection, which eliminates the need for dyes and tags and allows for higher-sensitivity measurements. This method also conserves lab resources by consuming low volumes of sample. Weaknesses Immobilization of the ligand is not always straightforward. Although diverse chip- surfaces, coupling chemistries and even specific chips are available, some ligands cannot be immobilized in a functional way. This is especially important in small molecule research, where capture antibodies cannot be used (to avoid unspecific binding to the antibody). Many "difficult" targets, such as membrane proteins, are fragile and do not tolerate the acidic surface of the SPR assay. Also, for interactions where the analyte does not wash off easily, scientists must identify regeneration conditions that can break the ligand-analyte interaction to facilitate dissociation but leave the ligand intact for another binding experiment. Additionally, SPR measures changes in refractive index. Any buffer component that influences the refractive index of a sample (e.g. DMSO, a common solvent for small molecules) can cause artifacts in SPR measurements. Usually, control experiments are necessary when working with DMSO. Finally, because SPR depends on mass changes on the chip surface, this technology is not an optimal solution for small molecule research. Conclusion SPR is a highly sensitive and precise tool that provides kinetic and affinity data. Considered a gold standard for both academic and industry research applications, it has many established standard protocols. However, it can be costly and a bit complicated to operate, and is not suited as a walk-up instrument in a dynamic research setting. Surface Plasmon Resonance