Surface Plasmon Resonance (SPR) is an optical technique used for analyzing molecular interactions. It provides a unique, label-free method to observe binding events in real time, offering a dynamic view of how two substances associate and dissociate. This analytical power stems from surface plasmons, which are highly sensitive to minute changes occurring at a metal-solution interface. By converting a molecular binding event into a measurable optical signal, SPR systems allow researchers to precisely quantify the strength and speed of interactions for a wide array of biomolecules.
The Underlying Physics of Plasmons
The core mechanism of Surface Plasmon Resonance relies on the behavior of free electrons at the boundary between a thin metal film and a surrounding dielectric medium, such as water or a buffer solution. A plasmon is the collective oscillation of the free electron cloud occurring at this interface, creating an electromagnetic wave known as a surface plasmon polariton.
For this oscillation to be excited, it must interact with incident light under specific resonance conditions. The momentum of the incoming light must precisely match the momentum of the surface plasmon. This is typically achieved by shining polarized light through a prism at a controlled range of angles. When resonance is met, energy is transferred from the light into the plasmons, resulting in a measurable reduction in the intensity of the reflected light.
This energy transfer generates a strong electromagnetic field, called the evanescent wave, which extends only 100 to 300 nanometers into the medium above the metal surface. Because the plasmons are confined to this interface, the evanescent wave is extremely sensitive to any change in the refractive index within this limited volume. The refractive index is essentially a measure of the speed of light in a medium, and it changes proportionally with the mass concentration of molecules present in that area.
The Sensor Chip and Optical Setup
To harness this phenomenon for molecular analysis, the Kretschmann configuration is commonly employed. The setup uses a high-refractive index prism, often glass, optically coupled to a sensor chip. This chip consists of a glass substrate coated with a thin film of noble metal, typically gold.
The prism directs p-polarized light toward the gold film at an angle greater than the critical angle for total internal reflection. The light is reflected back into the prism, but the resulting evanescent wave penetrates through the gold film into the sample medium. This geometric arrangement enables the light’s momentum to match the plasmon’s momentum, initiating resonance.
The gold film is functionalized with specific chemistry to securely immobilize one binding partner, the ligand. A microfluidic system delivers a continuous flow of buffer and controlled injections of the second binding partner, the analyte, over the immobilized ligand. The optical detection unit continuously monitors the intensity of the reflected light as the angle of incidence is scanned.
Monitoring Molecular Binding
The operational power of SPR lies in translating the physical act of molecular binding into a quantitative signal. The ligand, attached to the gold surface, interacts with the analyte introduced in the flowing solution.
When the analyte binds to the immobilized ligand, it adds mass to the sensor surface within the evanescent wave volume. This mass increase causes a localized change in the refractive index immediately adjacent to the gold film. Because the plasmon resonance condition is highly sensitive to this change, the angle at which minimum light reflection occurs—the resonance angle—shifts.
This shift is measured in real-time and reported in response units (RU), which are directly proportional to the total mass of the bound analyte. The output is a sensorgram, a graph plotting response units over time, which displays the entire interaction profile. The sensorgram shows the association phase, where the analyte binds and the signal rises, and the dissociation phase, where the buffer is flowed over the surface and the signal falls as the bound molecules detach.
Key Applications in Research
The real-time, label-free nature of Surface Plasmon Resonance provides researchers with detailed quantitative data about molecular recognition events. From the sensorgram’s binding profile, researchers calculate the kinetic rate constants: the association rate (\(k_a\)) and the dissociation rate (\(k_d\)).
These constants measure how fast molecules bind together and how fast they fall apart. Dividing the dissociation rate by the association rate yields the equilibrium dissociation constant (\(K_D\)), which is a direct measure of binding affinity. A lower \(K_D\) value signifies a tighter, stronger bond between the two molecules.
SPR is also routinely used for concentration analysis, quantifying the amount of active substance by correlating the maximum binding response to a known standard curve. The technique is widely employed to characterize antibody-antigen interactions, screen small molecule drug candidates against target proteins, and analyze binding between a vast range of biomolecules, including proteins, nucleic acids, and lipids.