University of Nottingham
Biocore3000 SPR

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.


  • Molecular interaction studies (kinetics, affinity, specificity, strength, concentration etc.)
  • Analyte identification studies (bacteria, viruses, hormones, immunoglobulins etc.)
  • Protein activity and stability analysis
  • Epitope mapping
  • Characterization of mutant proteins
  • Study of layer-by-layer self-assembly

Typical Applications

  • Clinical studies i.e. Identification of binding partners to targets by linking SPR to mass spectrometry (proteomics) which can be carried out without need for purification of complex mixtures such as clinical material
  • Academic research i.e. life sciences, materials science
  • Industry research i.e. pharmaceutical drug discovery (mechanism of action, hit validation and characterisation, lead optimisation), materials characterisation




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

SPR_Image 1


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

SPR_Image 2

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 kd of ligand 5.

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


Our SPR Facilities

Biacore 3000 SPR (GE Healthcare)

The Biacore 3000 instrument integrates SPR technology with a microfluidic system to monitor molecular interactions in real time at concentrations ranging from pM to mM. It is a highly sensitive high throughput instrument (can analyse up to 192 samples per run) that uses label-free technology and can detect an extremely wide range of molecular masses (180 Da to whole cells). Its light source is a Light Emitting Diode (LED) with 760 nm wavelength. It has two liquid delivery pumps, one for maintaining a constant flow of liquid over the sensor chip surface and the other for handling samples in the autosampler. Measurements are carried out over 4 detector flow cells formed by the Integrated µ-Fluidic Cartridge (IFC) pressing against the sensor chip. Measurements can be performed at a temperature range of 4 to 40˚C and its refractive index range is 1.33-1.40.



Pioneer SensiQ SPR (Molecular Devices)

The Pioneer SensiQ is a fully automated high throughput SPR system (can run up to 768 samples in 24 hours in a single experiment) that is used for real-time, label-free measurements of biomolecular interactions. It has 3 channels, its light source is LED, measurements can be performed at a temperature range of 4 to 40˚C and its refractive index range is 1.33-1.40. The SensiQ Pioneer systems extends measurement to include small molecules (Molecular Weight Cutoff < 70 Da) and provides insight into fragment analysis and protein aggregation. It can also carry out OneStep gradient injections, which provides a high-resolution dose response in a single injection eliminating time and error associated with making dilutions.



Interface and Surface Analysis Centre (ISAC)