As mentioned in my previous blog, my project is related to iSPR platform and measurement. Commonly, SPR is used for studying interactions between all classes of biomolecules and biochemical mechanisms in real time. In a nutshell, it is an exciting technique to detect changes in refractive index. It can be applied for many purposes from food quality measurement to the study of nanoparticles. Furthermore, the rich information can be obtained from the SPR experiment. It is not only about affinity assays but also acquiring kinetic information. In this blog, I would like to talk about the basic idea of SPR.
First of all, what is a plasmon? Basically, plasmons are collective oscillations of the free electron gas density, often at optical frequencies. The fluctuations of density that appear on the surface of this material are called plasmons or surface plasmons. SPR is an optical phenomenon involving excitation of free oscillating metal electrons. The energy carried by photons of light is transferred to packages of electrons on a metal surface. The optical excitation of plasmons occurs only under proper resonance conditions – under conditions of attenuated total reflection (ATR) when the energy of the photons of light exactly equals the quantum energy level of the plasmons.
In general, there are two kinds of configuration for SPR measurement – the Otto and Kretschmann configurations.
The Otto configuration is shown at the above left-hand side. A light source is used to illuminate a glass block or prism and the light wave is totally internally reflected and a thin metal film (typically a thin gold film) is placed close enough so that the internally reflected light can interact with the plasmons on the surface and excited them. The right-hand side images is the Kretschmann configuration. It is more commonly used and refers to when the thin metal film is evaporated onto the glass block as opposed to being separate. The wave of light passes through the glass block and the metal film, exciting the plasmons on the opposite side of the film.
The above figure shows the most common biosensor setup in SPR instruments (the Kretschmann configuration). The circles and inverted “Y” entities in the flow channel signify analyte and ligand molecules, respectively. In this case, the ligand is a monoclonal antibody immobilized on the gold surface. The circle is a protein antigen that selectively binds to the mAb.