What is Surface Plasmon Resonance (SPR)?
Surface Plasmon Resonance (SPR) is a proven, label-free biosensor technique for studying interactions between all classes of biomolecules and biochemical mechanisms in real time.
With SPR, you can answer questions including:
- How fast do molecules associate and dissociate?
- How strong is the interaction between molecules (affinity)?
- How specific is the interaction?
- What is the concentration of the interactants?
How SPR WorksTechnically speaking, SPR refers to an optical phenomenon that enables monitoring of changes in refractive index via a quantum mechanical principle.
In a traditional SPR experiment:
- A target is immobilized or captured onto a surface known as a sensor chip
- A pump is used to flow analytes over the sensor chip
- An optical measurement system captures changes occurring on the surface of the sensor chip
- Software plots time-dependent responses in the form of a graph called a sensorgram
Starting with Snell’s LawTo start, it is helpful to think in terms of Snell's law. Snell’s law describes what happens when light is directed through a high refractive index prism (e.g., sapphire, with a refractive index of 1.76) to the surface of a gold-coated chip in contact with a low refractive index medium, such as a physiological buffer (See Figure 1).
Following Snell’s Law:
- Light rays below the critical angle will exit the prism bending toward the prism surface (i.e., refraction)
- Light rays above the critical angle are totally internally reflected back through the prism
- When light rays (specifically, the photons) are totally internally reflected, they create an electric field at the interface
- This electric field extends past the reflecting surface, and oscillates with the usual characteristics of an electromagnetic mode
- The electrical component perpendicular to the interface — the evanescent wave, which is bound to the surface — decays exponentially
Adding a ResonatorIn SPR, a thin metal layer coated on glass — a sensor chip — is added between the prism surface and the aqueous compartment (Figure 2).The metal that is coated on the glass chip is gold because of its chemical inertness, high wavelength surface plasmons and the ability to easily coat the gold with various molecular surfaces using self-assembled monolayer chemistry.
When the gold layer is added, free electrons in this metal layer can then act as a resonator. The energy for the resonance (energy transfer) comes from the evanescent wave produced by the totally internally reflected photons.
With the metal acting as a resonator, coupling/resonance can occur between the plasma oscillations of the free electrons in the metal and the bound electromagnetic field of the totally internally reflected photons. This coupling is the result of the momentum of the incoming light equaling the momentum of the plasma electromagnetic field, and is dependent upon certain conditions, including the wavelength of the incoming light, the illumination angle, and the refractive index of the prism, metal and aqueous layers.
Through coupling, photons are “absorbed” by the gold layer and, as a result, a “shadow” of diminished light intensity occurs in the reflected light since these photons are no longer totally internally reflected from the gold surface. The position or angle of this “shadow” in the reflected light profile is sensitive to changes in refractive index occurring above the gold chip surface.
Reichert SPR systems utilize the Kretschmann configuration, the most proven technology for surface plasmon resonance. The Kretschmann configuration, which utilizes a prism-based system, allows for “a more efficient plasmon generation” according to a leading website dedicated to SPR analysis.
When mass changes occur at the interface between the gold layer and the aqueous compartment, they cause changes in the local refractive index near the gold layer, which then changes the coupling/resonance angle.
Detecting Changes in Mass
Here’s an example of how changes in mass lead to changes in the coupling angle:
- When using a gold sensor chip and a physiological saline solution, the “shadow” (or SPR minimum) occurs at about 66 degrees
- When a protein is injected over a functionalized gold chip, some of the protein molecules couple to the gold chip, and the coupling angle increases. The actual reflectivity data (angle vs. reflectivity) before and after immobilizing a protein to the surface is illustrated in Figure 3.
- SPR instruments are optically configured to illuminate a spot on the surface of the sensor chip over a range of angles (typically 58-85 degrees for Reichert instruments); this range encompasses these shifts in the coupling angle, and is continuously monitored with a linear photodiode array detector to cover a refractive index range of 1.32 to 1.40.
Analyzing and Using the DataThere is typically a linear relationship between how many molecules are coupled to the sensor chip and the refractive index change. During SPR, image analysis determines the angle at which the reflectivity minimum occurs, and this data is continuously sent to the data acquisition computer/software. The software is the center of the user experience, allowing you to study molecular binding, kinetics, affinity and other key binding characteristics.
In SPR, sensorgrams are produced as a visual representation of the molecular interactions. Every sensorgram shows how molecules associate and dissociate throughout the experiment. The curvature of the line also provides key information about the kinetics involved.
By combining sophisticated sensors with powerful software, SPR lets you gain a comprehensive understanding of changes in mass, kinetics, thermodynamics and other key variables involved in biomolecular interactions.
SPR: Versatile, Flexible, Informational
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