SPR Technology

Figure 8. Refraction of light at an incident angle at the interface of two materials with refractive indices n1 and n2 (adapted from [B26])

Before discussing SPR technology, it may be appropriate to visualize the evanescent wave, which is the center of SPR sensing, a little more. This is conveniently done by contemplating the phenomenon of total internal reflection.

One has to watch the behavior of light at the interface of two separate media with differing refractive indices to understand this phenomenon. Light is refracted at the interface (Figure 8) after Snellius´ Law:

After supposition of refractive index n1>n2, total reflection is observed initiating from a fixed angle αc:

However, light intensity is not decreasing rapidly to zero at the interface, it is decreasing exponentially with distance. The field in this perpendicular direction, the evanescent field, is reflecting the bound, non-radiative nature of surface plasmons. The exponentially intensity decay of the evanescent field with increasing distance is presented in Figure 9.

Figure 9. Exponentially intensity decay of the evanescent field with increasing distance from the metal layer (adapted from [B26])

If the surface of a glass substrate is coated with a thin metal film, a part of the incident light can refract into the metallic film. Typical coatings consist of noble metals such as silver, gold, copper, titanium, and chromium. In this assembly, a second critical angle exists that is greater than the angle of total reflection. At this angle, the surface plasmon resonance angle, a loss of light appears and the intensity of reflected light reaches a minimum. This results from the interaction of the incident light with oscillation modes of mobile electrons at the

26

surface of the metal film. These oscillating plasma waves are called the surface plasmons. If this metal surface is coated by a thin layer of affinity ligands, the binding of biomolecules, e.g. proteins, causes a change of the refractive index. This is detected by a shift in the resonance angle.

Figure 10. Scheme of the Surface Plasmon Resonance (left) and Kretschmann configuration for SPR sensors (right) [adapted from [B28] and [B29])

The frequently used Kretschmann configuration (Figure 10) is based on a metal film which is evaporated on one face of a glass prism. The light is coupled into the prism above the critical angle of total reflection, and the resulting evanescent wave penetrates the metal film. The plasmons are excited at the outside of the film. The angle of resonance is dependent on the refractive index of the surface. SPR reflectivity measurements can be used for the detection of specific molecular interactions of bound receptor molecules on the metal surface with their corresponding targets (e.g. DNA or proteins) [B30, B31]. The greatest attraction of SPR measurements is due to direct, label-free and real-time measurement of the refractive index at the surface. These sensors offer the measurement of low levels of biological and chemical compounds near the sensor surface. The sensor recognition of a biomolecular binding event happens when these molecules accumulate at the sensor surface and change the refractive index by replacing the background electrolyte. Water molecules have a lower refractive index than protein molecules [B26].

27 Assay Process: from Buffer to Analyte

A binding cycle observed with an optical biosensor is presented in Figure 11. Prior to the experiment, receptor molecules are immobilized on the surface via adequate coupling chemistry. At t=0 s, the cell containing the receptor is floated with running buffer to have a reliable baseline before capturing starts. At this point, active receptors are on the surface, ready for analyte binding. An analyte solution in running buffer is passed over the receptor at t=100 s. The refractive index of the medium adjacent to the surface is increasing after binding of analyte to the surface. This is monitored by increasing resonance signal. When analyzing this step of the binding curve, the observed association rate kobs is received.

Furthermore, the association rate constant kass is determinable if the analyte concentration is noted. At the equilibrium, the amount of analyte that interacts with the receptor by association and dissociation is equal. The response level at this point is related to the active analyte concentration in the sample. At t=320 s, the analyte solution is replaced by buffer, the receptor-analyte complex dissociates. The dissociation rate constant kdiss can be obtained here. At t=420 s, a pulse of regeneration solution (high salt or low pH) is used to disrupt binding and regenerate the free receptor. The binding cycle is repeated several times with varying analyte concentration to receive a data set for global fitting to an adequate binding algorithm [B26, B28].

Figure 11. Binding cycle observed with an optical biosensor (adapted from [B28])

28 Kinetics

Interaction affinity can be calculated from the ratio of dissociation and association constant (KD = 1/KA = kdiss/kass) or by linear or nonlinear fitting of the response at the equilibrium of varying analyte concentrations. Again, buffer is injected to condition the surface for the next analysis circle. If regeneration is not complete, remaining mass causes an increased baseline level. Typical values for KA are within the range of 10⁵-10¹² L/mol, the values for KD within 10

-5-10^-12 mol/L. The dimensions for both rates are different and vary with stochiometry of the complex. Typical ranges show large variations and depend most on temperature. When starting, no product is present at the surface. At this point, the association rate is highest and dissociation rate is lowest. More and more of complex is produced and enhances the rate of dissociation during the process. Paralelly, the association rate might decrease.

Equilibrium is reached when both rates are equal [B26, B28].