What is SPR?

SPR is an optical technique that measures the refractive index within ~300nm of a sensor surface in real time.

How does it work?

The resonance of delocalized electrons (plasmon) in a thin gold layer at the boundary between glass (the sensor chip) and fluid (the flow cell), allows some absorption of polarized light when it is reflected from the boundary. The angle at which absorption is maximal is directly proportional to the refractive index within ~300nm of the surface. The instrument measures this angle with a very high degree of accuracy and precision in real time using an optical detector with a shift in the angle of 0.0001 degrees coresponding to 1 response unit or RU which in turn represnts the binding of ~1pg of protein per square mm of the sensor surface. The Sensor surface forms one part of a tiny flow cell (volume 60nL) through which various solutions are passed .

Typically one molecule (the ligand) is immobilized to the sensor surface either directly (e.g chemically bound) or indirectly (e.g. capture antibody) then a solution containing the second molecule (the analyte) is injected through the flow cell and if the analyte binds to the ligand there is an increase in the concentration of protein at the surface which results in a proportionate increase in the refractive index and by measuring refractive index in real time SPR allows the measurement of the equilibrium (affinity) and kinetic (on-rates) properties of the interaction.

What is SPR used for?

SPR biosensor are for studying interactions between molecules. They employ an optical technique to measure the binding of an injected molecule (analyte) to a second molecule (ligand) that has previously been immobilized in the instrument. 

SPR is typically used to determine

1. whether two molecules interact directly,

2. the activity of the interacting molecules, and

3. the kinetic and thermodynamic properties of the interaction.

SPR can also be used to

4. look for the presence of a molecule in a crude mixture,

5. or measure the concentration of a molecule in a crude mixture. 

In both case a purified ligand must be available.

Animatied examples of data traces can be found at www.biacore.com or by following the links below

1. A trace you might expect to see in a Specificity assay

2. A trace you might expect to see in a Kinetics assay

3. A trace you might expect to see in a Affinity assay