In the absence of the availability of structural information at an appropriate temporal resolution, several optical techniques have been utilised to provide information on the function of proteins. The most common optical method deployed has been SPR. The phenomenon of plasmon resonance was first observed in 1968 (Kretschmann and Raether 1968). The first commercial SPR based biosensors were released in 1990 and have proved useful in the study of equilibrium and kinetic aspects of the interactions of biological macromolecules (Shuck 1997). The basic optical configuration comprises a prism coated with a thin gold (or silver) layer (Fig. 11).
Light that is coupled into the prism is totally internally reflected at the metal-coated surface. When the angle of the incident light meets the resonant condition, surface plasmons, oscillating electromagnetic waves on the surface of the metal layer, are excited causing a reduction in the (energy) intensity of the reflected light. The surface plasmons have an associated electromagnetic field (evanescent field) that decays exponentially normal to the layer surface, which extends a few hundred nanometres from the metal surface. Changes in the refractive index (dielectric constant) within this region leads to changes in the observed resonance angle. Therefore, when macromolecules are adsorbed or desorbed in the immediate vicinity of the metallic sensor surface, the refractive index changes, leading to a commensurate change in the measured resonant angle. Commercially
Fig. 11. Typical surface plasmon resonance (SPR) experiment
9 = resonance angle (an intensity minimum with respect to reflected light due to excitation of surface plasmons).
Fig. 11. Typical surface plasmon resonance (SPR) experiment available instruments measure only the effective refractive index and therefore it is not possible to obtain analytical analyses of the structure of surface layers directly. Structural data cannot be obtained directly from SPR-based techniques, the information being confined to temporal information on the rate at which molecular associations and disassociations occur.
There are two key aspects that need to be addressed to produce instrumentation of value to researchers in the field of drug discovery and protein science. The first is detailed information on complex three-dimensional molecular systems (proteins), the second is the exquisite sensitivity necessary to determine the binding of small molecules (drug molecules typically have a molecular mass of 200-500 Da) to large molecules (protein molecules span the range between 10,000 and 2,000,000 Da). Before we discuss the nature of DPI in detail, it is necessary to understand the basics of the propagation of light within a waveguide. A schematic is shown in
It can be seen that whilst the majority of the light is confined to the core of the waveguide (depicted in grey), there is a residual, or evanescent, field that extends beyond the boundary of the core. The evanescent field can be seen to decay exponentially with distance from the waveguide surface and, as a result, only changes occurring within the first 100 nm or so of the waveguide surface will influence this field. Material changes in the refractive index in this region will affect the propagation speed within the waveguide as a consequence of changing the effective optical path. By allowing molecules of interest (e.g. proteins) to be attached to the sensing surface, it is possible to use this effect as a means to measure molecular interactions.
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