SPR basics

What is a biosensor?

Biosensors are devices which detect interactions of biomolecules on the spot by integrating a sensing element of biological origin into a physical signal transducer. The signal is created by the interaction between the biological sensing element - an immobilized biorecognition molecule - and the target analyte.
Chemically similar molecules can be detected by their biospecificity for an immobilized molecule. These biomolecular interactions can be directly monitored using so-called evanescent field sensors. The most frequently used technique is Surface Plasmon Resonance (SPR). SPR signals change proportionally to the refractive index close to the sensor surface and are therefore related to the quantity of bound macromolecules. Conventionally, biomolecular interactions are studied using techniques as immunoassays (ELISA or RIA), equilibrium dialysis, affinity chromatography and spectroscopy. SPR gives two main advantages over these techniques: The binding events are monitored in real-time, and it is not necessary to label the interacting biomolecules.

Surface Plasmon Resonance

To obtain SPR it is necessary to generate an evanescent field, that is, an exponential-decaying wave, at the sensor surface. An evanescent field is generated when total internal reflection of incident light occurs at the interface of two different substances: one with a high-refractive index and another with a low-refractive index - for example a glass-air interface.

The incident light is completely reflected, even though an electromagnetic field component of the light penetrates the substance with a low-refractive index. Surface Plasmon Resonance l-4 occurs under certain conditions when a thin film of gold is placed inside the evanescent wave. When the incoming light is monochromatic and p-polarized (i.e. the electric vector component is parallel to the plane of incidence), the free electrons of the metal will oscillate and absorb energy at a certain angle of incident light. The angle of incident light when SPR occurs is called the SPR angle.
SPR is detected by measurement of the intensity of the reflected light. At the SPR angle a sharp decrease or 'dip' of intensity is measured. The position of the SPR angle depends on the mean refractive index within the first few hundred nm above the sensor chip surface, which changes upon adsorption of macromolecules to the surface. As a result, the SPR angle will change proportionally to the amount of adsorbate. There is a linear relationship between the amount of bound material and the shift of the SPR angle.

SPR biosensors measure the SPR angle shift in (milli)degrees as a response unit to quantify the refractive index change caused by the binding of macromolecules to the sensor surface. The magnitude of the angular shift vs. the refractive index change depends on the wavelength of the light source plus the refractive index of the sensor chip substrate. . Therefore, the more universal unit is the overall refractive index change in µRIU, as it allows data comparison obtained with different optical setups and detection principles. Interestingly, the most frequently employed unit RU is the one that is most apparent, as it has been derived from CCD pixel counts of a particular optical detector.

 A consequence of the detection via refractive index changes is that the signal intensity is proportional to the analyte molecular weight. If the molecular weight of the compound is below 200 Dalton, then the change in refractive index during analyte binding becomes very small and direct detection increasingly difficult. Sensorchips with the capability to immobilize very high ligand densities, such as C200d or HC1000m, are required in such cases to obtain a sufficient signal.