A big concern in the development of a biosensor is the characterization of the recognition element that will be used to bind the antigen of interest. Having as much information as possible is crucial for the development of a working system. For this purpose, so many techniques can be exploited, and each of them gives a different perspective on the gathered data. Among the instruments that stand out, SPR definitely holds the top spot, as it could allow us to grasp with high accuracy constants like the Kd of the recognition element, its kon, koff and even its thermodynamics. That is why we decided to investigate it, trying to understand the deepest secrets of this technique.

The word SPR by itself looks complicated, and there’s a lot to unpack in the name already. SPR stands for “Surface Plasmon Resonance”, which, if you haven’t studied physics all your life, has no meaning whatsoever. Surface plasmons are electromagnetic waves that propagate on the surface of a metal layer, in a direction parallel to the layer itself. Resonance means that all these waves that are formed oscillate in the same way, with the exact same amplitude and frequency, thereby creating a much larger “interaction wave”, that derives by the sum of all the plasmons.

“So, why do we use it in a laboratory instrument?” Well, before answering this, I first need to explain the behavior of light in different media. When light moves from one media to another, 2 main phenomena compete: reflection and refraction. When the 2 substances are very different, and the second one is much denser and “thicker” than the first, the entirety of the light ray is reflected. However, even in those extreme situations, when the second layer is metallic or conductive, a specific angle of incident light will be able to penetrate it and excite its electrons, creating an electric field, called “evanescent field”. The evanescent field propagates through the second layer, dissipating as it gets further deep in the new media. The excitation of the electrons, however, will force them to start oscillating in resonance, creating exactly some surface plasmons. From a photon perspective, then, there won’t be reflection at that specific incident angle, thereby creating a “negative signal” in the reflected ray. This will both diminish the intensity and cause a slight change in the angle of the whole reflected light. This switch is measured in SPR units, and 1 AU = 0.0001°.

This is the setting of an SPR system. On the right the chip is drawn; that is where the magic happens. Figure drawn using Biorender.com

“Ok, sounds really complicated, but why should all of this help me understand how this machine measures the affinity between two ligands?” Oh that’s easy. The properties of the surface plasmons are influenced by the surface they move through. This means that, if a mass quantity is bound to the surface of the layer, the incident angle causing the evanescent field will change by an infinitesimal of a degree, thus influencing the reflected wave as well. This alteration is dependent on the weight that is bound to the surface. Consequently, if the instrument is tailored to the weight of our recognition element bound to the chip, a binding event will add weight to the chip, modify the surface plasmons and change the reflection angle. Detection of this change will give us all the needed information.

“Is that it, is it that simple?” No, sorry, we are just scratching the surface ☹

References

Bakhtiar R. 2013. Surface Plasmon Resonance Spectroscopy: A Versatile Technique in a Biochemist’s Toolbox. Journal of Chemical Education 90: 203–209.

Drescher DG, Ramakrishnan NA, Drescher MJ. 2009. Surface plasmon resonance (SPR) analysis of binding interactions of proteins in inner-ear sensory epithelia. Methods in Molecular Biology (Clifton, NJ) 493: 323–343.

Roos H, Karlsson R, Nilshans H, Persson A. 1998. Thermodynamic analysis of protein interactions with biosensor technology. Journal of molecular recognition: JMR 11: 204–210.

Sambles JR, Bradbery GW, Yang F. 1991. Optical excitation of surface plasmons: An introduction. Contemporary Physics 32: 173–183.

Saroha D. 2005. Surface plasmon resonance. Applied biochemistry and biotechnology 

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