1 - What is SPR?

1.1 TIR – Total Internal Reflection

If a light beam passes a medium (e.g.: glass) with higher refractive index (ռ1) and hits a medium (e.g.: water) with lower refractive index (ռ2) at different angles of incidence, one part of the light will be reflected; another part of the light penetrates the lower refractive medium.

Figure 1-1: Polarized light is directed at varying angles of incidence into a glass prism. The reflected light intensity is detected using an optical detector (e.g. a CCD camera).

At the critical angle, the light will be totally reflected (Figure 1-2). Even at Total Internal Reflection (TIR), the so-called evanescent wave penetrates the lower refractive medium, see Figure 1-1.

Figure 1-2: Relation between reflected light intensity and angle of incidence. At angles ≥ critical angle 100% of the light will be reflected at the interface of both media.

1.2 SPR – Surface Plasmon Resonance

If there is a thin metal layer (e.g.: silver or gold) attached to the medium (e.g.: glass) with higher refractive index (ռ1) as illustrated in Figure 1-3, the relation between the reflected light intensity and the angle of incidence is changing.

Figure 1-3: Polarized light is directed at varying angles of incidence into a glass prism.

At another angle (> critical angle), the so-called SPR-angle, a minimum in the reflected light intensity can be observed, due to resonance of surface plasmons in the metal film and photons of the incident light (Figure 1-4). In a broader sense surface plasmons can be seen as delocalized electron oscillations at the interface of the metal film. Almost the whole energy of the incident light is absorbed by surface plasmons in the metal film, when wave-vector and the electrical field of the surface plasmons equals the wave-vector and the electrical field of the photons of the incident light.

Figure 1-4: Relation between reflected light intensity and angle of incidence. At angles ≥ critical angle 100 % of the light will be reflected at the interface of both media. There is a minimum in the reflected light intensity at the SPR (surface plasmon resonance) angle, due to resonance of photons and surface plasmons in the gold layer.

1.3 SPR Biosensor

In a SPR biosensor a microfluidic channel is attached to the gold surface in order to facilitate sample delivery to and from the gold surface (Figure 1-5).

Figure 1-5: Schematic illustration of a SPR biosensor. The glass / gold / water interface with attached microfluidic channel. Polarized light is directed at varying angles of incidence into the glass prism. The reflected light intensity is detected using a CCD camera.

Furthermore, if the wavelength of the incident light, the temperature and thickness of the metal film are kept constant, the angle at which surface plasmon resonance takes place depends only on refractive index changes of the lower dense medium (e.g.: water). The association and dissociation of biomolecules to or from the sensor surface leads to continuous changes of the refractive index, which result in changes of surface plasmon resonance conditions or changes in SPR angle (Figure 1-6).

Figure 1-6: Relation between reflected light intensity and angle of incidence. At angles ≥ critical angle, 100 % of the light will be reflected at the interface of both media. There is a minimum in the reflected light intensity at the SPR angle, due to resonance of photons and surface plasmons in the metal film. Changes in the refractive index will change the SPR angle.

Most of SPR biosensors report the signal as RU (resonant unit) over time. Thereby 1 RU corresponds to ~10-4 angular shift of the SPR-Dip or 1 RU is approximately ~10-6 refractive index change. The collected data (RU vs. time) is called sensorgram (Figure 1-7).

Figure 1-7: Schematic illustration of a sensorgram. In a SPR biosensor the binding signal in RU is reported vs. time in seconds.

1.4 SPR Language

1.4.1 The SPR sensor or sensor surface or sensor chip

  • Is a gold coated glass slide or prism with a certain surface chemistry.
  • The surface chemistry depends on the application.
  • Is used to attach a reaction partner.
  • Is a consumable.
  • Reusability depends on the application.
Figure 1-8: Schematic illustration of a SPR sensor chip

1.4.2 Surface sample or ligand or target

  • E.g. a protein, peptide, single stranded DNA/RNA, membranes, lipids, small molecules
  • Is the attached reaction partner.
  • Direct: via functional groups of sensor surface chemistry within the immobilization /
    surface creation procedure
  • Indirect: Captured to a secondary molecule on the surface
  • The ability to regenerate depends on the application.
Figure 1-9: Schematic illustration of the surface sample / ligand

1.4.3 Solution sample or analyte or sample

  • E.g. a protein, peptide, single stranded DNA/RNA, membranes, lipids, small molecules
  • Is the reaction partner to be investigated in solution.
  • Via injections over the attached surface sample and reference surfaces
  • Is often tested in multiple concentrations.
  • The ability to regenerate depends on the application.
Figure 1-10: Schematic illustration of the solution sample / analyte

1.4.4 Sensorgram

  • The plotted binding response [RU] of solution sample binding to surface sample.
  • The binding response [RU] is plotted against time [s].
Figure 1-11: Schematic illustration of a SPR sensorgram [from SensorCon-2]. Shown are all individual stages (Baseline, Association, Equilibration, Dissociation, Regeneration) while binding appears.

1.4.5 Baseline

  • SPR signal prior injection of solution sample
  • [solution sample] = 0%

1.4.6 Association

  • [solution sample] = 100%
  • Describes the injection time of solution sample over surface sample, while binding is
    monitored.

1.4.7 Equilibration

  • Part of the association phase
  • Equilibrium of bound solution sample and free solution sample

1.4.8 Dissociation

  • [solution sample] = 0%
  • Describes the time, while the surface is flushed with running buffer.
  • Bound solution sample will dissociate from surface sample.
  • Decay of complex

1.4.9 Regeneration

  • Injection of a solution (e.g. low ph, high ionic strength) to disrupt binding between
    solution sample and surface sample
  • Not always needed

1.4.10 Association rate constant

  • Association rate constant [M-1 s-1 ] or ka
  • Onrate
  • “How fast binds solution sample to surface sample”

1.4.11 Dissociation rate constant

  • Dissociation rate constant [s-1] or kd
  • Offrate
  • „How fast gets the solution sample released from surface sample“

1.4.12 Dissociation constant

  • Dissociation constant [M] or KD
  • Ratio of offrate / onrate
  • „The lower KD, the higher the affinity of an interaction“
Figure 1-12: Schematic illustration of a SPR sensorgram [from SensorCon-2]. Shown are all individual stages (Baseline, Association, Equilibration, Dissociation, Regeneration) as well as the kinetic rates while binding appears.