How Surface Plasmon Resonance WorksSurface Plasmon Resonance Principle
How Does Surface Plasmon Resonance Work?
Surface plasmon resonance can be used to measure binding events because it is very sensitive to changes in refractive index. SPR instrument are made up of an optical measurement system, a fluid handling system, and a sensor chip.
The sensor chip is a gold film coated on a glass substrate that has been chemically modified to make it easier to immobilize one of the binding partners onto the surface of the sensor. The molecule that is immobilized is known as the ligand, and the molecule in solution is known as the analyte. The sensor chip is interface with the fluidic system through the use of a small flow cell, so that the analyte can be injected at different concentrations in a very repeatable manner.
What Happens in an SPR Experiment?
In a typical SPR experiment, the first step is to immobilize the ligand to the sensor surface. There are a number of different types of sensor surfaces and methods to do this, as explained in our blog post. Once the ligand is immobilized the remaining binding sites are blocked, and the surface often conditioned with injections of regeneration buffer. Running buffer is being continuously run over the surface of the sensor chip, and once the surface is stable, the analyte is injected at a specific flow rate for a specific amount of time (usually 2-5 minutes) and at a specific concentration. The time in which the analyte is passing through the flow cell is known as the association phase, and the analyte will accumulate on the surface as it binds to the ligand. The rate of binding will depend on the on-rate of the interaction, as well as the mass transport of the analyte from the bulk to the surface. This why it is important to maintain a high flow rate and use a low ligand density, so that the concentration of analyte at the surface will remain high. Binding of the analyte to the ligand will cause the signal to increase until equilibrium is reached (if it is reached). Once the analyte passes through the flow cell and is replaced with buffer, the analyte will start to come off the surface, and the signal will begin to decrease. This is known as the sensorgram. If the ligand and analyte bind strongly, a regeneration buffer will be injected to break up the ligand-analyte complex, leaving the ligand on the surface. The analyte injection is repeatable multiple times for 3-5 different concentrations, which is needed in order to obtain reliable kinetic constants.