Surface plasmon resonance

What is surface plasmon resonance?

Surface plasmon resonance (SPR) allows real-time, label-free detection of biomolecular interactions.

SPR occurs when polarized light strikes an electrically conducting surface at the interface between two media. This generates electron charge density waves called plasmons, reducing the intensity of reflected light at a specific angle known as the resonance angle, in proportion to the mass on a sensor surface.

Biacore SPR systems are used primarily in pharmaceutical development, quality control, and basic life science research.

Follow label-free interactions in real-time

In Biacore assays, target molecules, most frequently proteins are immobilized on a prepared gold sensor surface and a sample containing a potential interacting partner in solution is injected over the surface through a series of flow cells.

During the course of the interaction, polarized light is directed toward the sensor surface and the angle of minimum intensity reflected light is detected. This angle changes as molecules bind and dissociate and the interaction profile is thus recorded in real time in a sensorgram.

What kind of interactions can be studied?

Biacore systems can be used to study interactions involving (in principle) any kind of molecule, from organic compounds to proteins, nucleic acids, glycoproteins, and even viruses and whole cells. Since the response is a measure of the change in mass concentration, the response per molar unit of interactant is proportional to the molecular weight (smaller molecules give lower molar responses).

There is no lower limit for organic molecules with today's instrumentation.

What samples can be used?

The SPR detection principle does not require any of the interactants to be labeled and measurements can be performed on complex mixtures, such as cell culture supernatants or cell extracts, as well as purified interactants. The identity of the interactant monitored in a complex sample matrix is determined by the interaction specificity of the partner attached to the surface. The SPR detection principle is non-invasive and works equally well on clear and colored or opaque samples.

How is surface plasmon resonance data displayed?

Biacore systems monitor the interaction between two molecules, of which one is attached to the sensor surface and the other is free in solution. A sensorgram is a plot of response against time, showing the progress of the interaction. This curve is displayed directly on the computer screen during the course of an analysis.

What is shown in the sensorgram?

During sample injection, a positive response can be viewed in the sensorgram, as analyte (the interacting partner in solution in Biacore-based assays) binds to the ligand (the interaction partner that is attached to the sensor surface in Biacore-based assays). The response decreases during dissociation. After an analysis cycle is completed, regeneration solution is passed over the sensor chip, removing bound analyte, preparing for the next analysis cycle.

The schematic illustration below shows a sensorgram. The bars below the curve indicate the solutions that pass over the sensor surface.

Schematic_illustration_of_a_sensorgram

What data can be obtained from an interaction?

The data from an interaction is presented in a sensorgram, providing quantitative data on:

  • Binding
    Does the interacting partner bind to the target molecule?
  • Specificity
    To what extent does an interacting partner cross-react with other molecules?
  • Concentration
    How much of a given molecule is present and active?
  • Kinetics
    What are the rates of association and dissociation?
  • Affinity
    How strong is the binding?

How do I interpret a sensorgram?

The shape of the sensorgram gives information about the interaction. A few examples of how to interpret sensorgram shapes are shown below.

Binding of target molecule

Binding of target molecule

No binding of target molecule

No binding of target molecule

Rapid association and dissociation (curve slope)

Rapid association and dissociation curve slope

Slow association and dissociation

Slow association and dissociation

Strong interaction

Strong interaction

Weak interaction

Weak interaction

Multiple binding, changes in response

Multiple binding changes in response

Multiple binding, no response

Multiple binding no response

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