In Surface Plasmon Resonance biosensors, the relative position of a sharp decrease or 'dip' in the intensity of light which is reflected at a thin metal surface is detected. The position of this dip depends not only on the quantity of bound biomolecules, but also on other factors such as the wavelength of the incident light.
Most frequently, a monochromatic light source is used and the angular shift of the SPR minimum detected (middle diagram). Another, more primitive setup, measures the intensity of the reflected light at a fixed angle, which changes when the SPR curves shifts (left diagram). Finally, one can exploit the wavelength dependency of the SPR phenomenon, irradiate the chip surface with white light at a fixed angle and detect the wavelength at which the resonance occurs (right diagram). In this case, the shift occurs not in the resonance angle, but in the emitted wavelength upon a binding event at the sensor chip surface.
Three SPR detection approaches: Intensity, resonance angle, resonance wavelength (left to right)
In order to generate surface plasmons, the incident light has to be coupled into the substrate of the sensorchip, and the reflected light needs to be coupled out to a suitable optoelectronic detector. Again, this can be achieved in three ways:
In this most widely used SPR sensor geometry, light is coupled into the sensor chip via a prism below the chip substrate. Chip and prism are optically coupled by matching fluid, usually immersion oil or an elastomer with suitable refractive index.
The detection of the SPR minimum, in this so called Kretschmann configuration, can be done by analyzing the reflectance of a whole angle range with a high resolution photodiode array. This setup can be made in a compact and robust design and is very flexible, too.
Kretschmann geometry with photodiode array detector One disadvantage is that the fixed angular range cannot usually be adjusted by the user, so a wide dynamic range covering at least 10 degrees is important.
An alternative is the detection of the minimum angle by means of scanning optics which constantly scans over an adjustable angle range. The advantage is somewhat lower manufacturing (and/or end-user) cost because simple photodiodes instead of the more expensive CCD or CMOS arrays can be used.
Kretschmann geometry with scanning mirror optics The downside of this approach is an about a ten-fold higher noise level compared to advanced setups using photodiode array detectors and use of moving parts (the scanning mirror) which require regular service.
Instead, via a prism, the light can be coupled through a grating which is embossed into the sensor chip substrate. The incident optical wave is diffracted, forming a series of beams directed away from the surface at a variety of angles. An important difference is that the light impinges through the sample on the top of the sensor chip, so the analyte and the flow cell need to be optically transparent.
SPR grating coupler – here with spectral detection
As it is not necessary to optically couple the chip to a prism, chip handling is relatively uncomplicated. Also, accurate control of the thickness of the plasmon-active metal layer is not required. On the other hand the necessity of a grating complicates the chip manufacturing process. The third option for coupling light into an SPR transducer is through a waveguide or an optical fiber. Though this approach holds potential for extreme miniaturization and allows sensing in inaccessible locations, fiber optic setups are sensitive to mechanical disturbances and due to the cylindrical shape of the sensing area, fabrication of homogeneous metal coatings and economic surface functionalization is difficult.
Optical fiber / waveguide SPR with spectral detection
Sensitivities The above described three detection modes can be combined with the three optical geometries (exception: optical fibers do not allow for angular detection) resulting in eight possible combinations. Aside from the individual advantages and drawbacks, the sensitivity of all these approaches is significantly different as shown in the diagram below.
Sensitivity of different SPR setups (in pg/mm²) With a lower detection limit of around 0,1 pg/mm² the prism coupler with angular detection is clearly the most sensitive approach which explains its widespread use in today’s SPR biosensors.
Literature Homola, J., Yee, S. and Gauglitz, G., Surface plasmon resonance sensors: review, Sensors and Actuators B 54, (1999) 3 - 15