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Surface Plasmon Resonance (SPR)

Study molecular interactions and reaction kinetics using SPR

SPR at a glance

Surface Plasmon Resonance (SPR) instruments are optical biosensors that allow sensitive detection of molecular interactions in real time. It is a label free technique thus eliminates the risk of label associated interference of the interaction being studied. SPR is typically used for measuring binding constants of molecular interactions (drug-protein, hormone-protein, protein-protein, nucleic acid-protein, carbohydrate-protein and lipid-protein interactions). It is also used in materials characterisation i.e. characterization of polymer layers and abiotic-biotic interactions.

Image 1: Illustration of the Basic Principle of an SPR Measurement

The underlying principle of an SPR measurement is to attach (immobilize) a molecule termed ligand onto a sensor chip surface and measure the change in refractive index of the surface when another molecule (interaction partner called analyte) binds to it. In a standard SPR set up, shown above, a beam of incident polarized monochromatic light is shone through a prism at a thin-layer of gold coating one surface of a glass sensor chip. The prism causes the light to be reflected at the gold-coated surface. At a particular angle of incidence, absorption of some of the light by the electrons in the gold excites charged density waves, called "surface plasmons", which propagate along the metal surface. Plasmon resonance leads to a reduction in the intensity of the reflected light. The angle at which the intensity of refracted light is most significantly reduced (resonance angle) depends not only on the gold layer but also occurs as a function of the refractive index of the medium just above the gold surface i.e. the buffer solution. SPR is thus highly sensitive to changes in the environment close to the gold-aqueous solution interface. A change in the refraction index at the surface of the sensor (due to for example analyte binding or dissociation occurring near the surface) may hence be monitored as a shift in the resonance angle and is recorded as a sensorgram.

Figure courtesy of Marion J. Limo, ISAC, School of Pharmacy, University of Nottingham

Image 2: SPR Kinetic Study of Interaction between Glycopolymers and Lectin

Interaction between carbohydrates and carbohydrate-binding proteins known as lectins has been of interest as a targeting mechanism for drug-delivery because of their known interaction specificity. This interaction is however, weak compared to others in nature but its avidity is ehanced by multivalency. The interaction of two synthesised β-D-galactose bearing polyvalent glycopolymers (ligand 5 and ligand 3) with plant lectin Ricinus communis agglutinin 120 (RCA₁₂₀) was investigated using a Biacore 3000 SPR. Complex polyvalent glycopolymers with defined valency can be synthesised using relatively facile chemistry. The aim of the study was to investigate the avidity of the glycopolymers for lectins towards the development of drug-delivery vehicles. RCA₁₂₀ was immobilised on a CM5 sensor chip and the interaction of the two glycopolymers studied at different concentrations (500 pM to 10 µM) as shown in the sensorgrams. The data was fitted with the BIAcore software using a single sites (1 : 1 Langmuir Binding) model. Fitting with highest concentrations and some low concentrations were removed as binding was either too rapid or too weak to enable accurate fitting. Derived kinetic and thermodynamic parameters are shown in the table. Ligand 3 had a much higher avidity for RCA₁₂₀ binding in comparison to ligand 5, evident from the 5 fold higher k_d of ligand 5.

Image adapted from Sebastian G. Spain and Neil R Cameron. Polym. Chem., 2011, 2, 1552-1560.

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