HOW DOES SPR WORK?
Surface Plasmon Resonance (SPR) is a label-free detection method that is used to monitor biomolecular interactions on a surface in real time. SPR provides information on protein or small molecule binding kinetics, affinity, specificity and concentration.
SPR platforms consist of a sensor chip, a microfluidic system and an optical measurement system. A thin metal, usually gold, coated glass sensor chip is located in between the prism and the fluidic part. Gold is the preferred surface coating because of its chemical inertness, and it is also suitable for self-assembled-monolayer (SAM) surface chemistry. The gold sensor chip is chemically modified to allow for immobilization of ligands. In an SPR experiment, a binding partner -the ‘ligand’- is attached to the sensor surface, and the interacting partner -the ‘analyte’- is injected in a continuous flow. While the analyte molecules are flowing across the microchannels of the sensor chip, they interact with the previously captured ligand molecules.
The FlexPR instrument employs a prism-based detection scheme based on the Kretschmann configuration. The optical system, comprised of a light source and detector, is responsible for monitoring the change in refractive index. SPR phenomenon occurs when the incident light passes through the prism and is reflected off the backside of the sensor chip to create an electric field called an evanescent wave. The light beam at a certain angle of incidence is absorbed by the electrons in the gold layer of the sensor chip, causing them to resonate. These electrons are surface plasmons that are highly sensitive to the surrounding environment. As molecules bind to the sensor surface, they change the refractive index. This causes a decrease in the intensity of the reflected light that is measured by the detector. The result appears as a dip in the SPR reflection intensity curve and is displayed in a sensogram. The change in SPR angle is directly proportional to the amount of the bound molecules. The software exploits this relationship to quantify the surface-bound molecules, thus enabling you to understand the key bonding properties of the analyte, such as molecular binding, affinity and kinetics.
