Surface Plasmon Resonance
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Authored by: Paul Taylor, Boehringer Ingleheim Inc.
Molecular labels have served a key role in providing detection capability of most biological assay formats. An important question that persists is whether labeling itself perturbs the binding characteristics of the molecule in question, thereby leading to erroneous conclusions. Label-free technologies have seen use over the last ten years as a way to more directly measure biological binding events and (for molecular interactions) commonly incorporate an assay component tethered to a sensor surface. Surface plasmon resonance (SPR) has been a key component of this technology. When a second assay component is added to the tethered molecule, binding activity results in a change of refractive index of the buffer near the sensor surface. A key advantage is that, unlike separation format assays, the binding event takes place in the presence of unbound reagent. However, the tethering event itself needs to be validated as providing a molecular component that is biologically relevant. Typically, there is an association phase wherein the binding event approaches equilibrium and this is then followed by a washing step which allows the binding interaction to dissociate over time. Data that results allows the determination of on and off-rates which can then be used to calculate binding constants for the interaction. In the context of biomolecular interactions, the detection format has the capability of determining very low affinity interactions. However, being that the biological activity is a binding event only, the methodology is commonly used in secondary screening to determine primarily if a compound hit is real and how fast and stable the binding event is.
The technology has also seen expansion into cell-based formats such as cytotoxicity, receptor-ligand functional assays, cell proliferation, endothelial barrier function, adhesion/spreading assays and monitoring of cell migration and invasion. When cells are stimulated, as with a ligand, cellular events take place that lead to changes in cell adherence, interaction, shape and volume and these changes affect the flow of current across the cells leading to changes in the measured impedance. Two types of sensors are commonly used: resonant waveguide grating (RWG) and electric. RWG sensors consist of a grating that is embedded in conjunction with a waveguide on a glass substrate. Upon stimulation, any dynamic mass redistribution (DMR) that takes place on the cell surface is optically detected by the physical properties of light reflected off the bottom of the sensor/cell interface. Alternatively, electric biosensors monitor the ionic environment of the sensor/cell interface and cells are cultured on gold electrode/substrate surfaces.
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