Methods and Concepts in the Life Sciences/SPR
Surface Plasmon Resonance
Surface plamon resonance (SPR) is a technique that can be used to study the binding and unbinding of molecules such as proteins. In order to analyze an interaction, one binding partner (the ligand) is immobilized onto the surface of a sensor chip. The analyte, which is dissolved in a sample buffer, is then injected into the flow cell under continuous flow. The binding of analyte to the ligand changes the refractive index of the sensor. This change is measured (as Resonance Units, RU) and plotted as a function of time. The change from running buffer to sample buffer can create a response as well, therefore a second flow cell without immobilized ligand is used as a reference.
When light passes from a medium with a high refractive index into a medium with a low refractive index (e.g. glass to water), some light is reflected from the interface. When the angle of the incident light surpasses a critical value, this reflection is total (total internal reflection). The electric and magnetic fields can, however, not be discontinuous at a boundary. Instead, they propagate into the material as an evanescent wave.
If the surface of the glass is coated with a thin metal film, some energy is lost due to the excitation of mobile electrons at the metal surface by the evanescent wave. The oscillation of free electron density with respect to the fixed positive ions in a metal can be described as plasmons. When the frequency of incident photons matches the frequency of the surface plasmons, resonance occurs, hence the term surface plasmon resonance. The loss of energy is greatest at a specific angle (the surface plasmon resonance angle), consequently the intensity of the reflected light reaches a minimum (or “dip”) at this angle.
Immobilization of the ligand
The sensor consists of a planar metal surface (typically gold or sliver), which is commonly covered with a carboxymethylated dextran matrix. This matrix increases the capacity for ligand immobilization. Ligands can either be immobilized directly or indirectly. Direct immobilization typically makes use of amine, thiol or aldehyde groups, which are covalently coupled to free carboxymethyl groups on the sensor chip. In the case of indirect immobilization, the ligand is captured by a covalently coupled molecule, such as an antibody. In any case, it should be tested whether the immobilized ligand is still active.
A high ligand density creates two potential problems: Firstly, mass transport may become the rate-limiting step, meaning that the analyte binding is faster than the delivery of analyte to the surface. In this case, the measured association rate constant is slower than the actual kon.
Secondly, after dissociation, the analyte may rebind to unoccupied ligand before being washed away. Consequently, the measured dissociation rate constant is slower than the true koff.
- Van Der Merwe, P.A., 2001. Surface plasmon resonance. Protein-Ligand Interactions: Hydrodynamics and Calorimetry