Surface Plasmon Resonance Spectroscopy

INTRODUCTION

Surface plasmon resonance (SPR) spectroscopy is a powerful, label-free technique to monitor noncovalent molecular interactions in real time and in a noninvasive fashion. As a label-free assay, SPR does not require tags, dyes, or specialized reagents (e.g., enzymes–substrate complexes) to elicit a visible or a fluorescence signal. During the last two decades, SPR has been broadly applied to study of noncovalent interactions of protein–DNA, protein–cell, RNA–DNA, DNA–DNA, protein–protein, protein–carbohydrate, small molecule–macromolecule (e.g., receptor–inhibitor complex), protein–peptide, and self-assembled monolayers. In addition, SPR has been successfully applied to drug discovery ligand-fishing and clinical immunogenicity studies (i.e., to monitor an immune response against a therapeutic agent). SPR spectroscopy can address questions such as specificity of an interaction, kinetics, affinity, and concentrations of selected molecules present in a sample of interest.

PRINCIPLE

When incident light propagates in a medium of relatively higher refractive index to a medium of lower refractive index, the ray of light tends to reflect as opposed to refract. In refraction, the light ray changes direction and bends as it passes through two media. In reflection, the light beam rebounds after impinging on a surface with angles of incident and reflection being the same. When the light beam seizes to cross the boundary and is entirely reflected, a total internal reflection (TIR) occurs. A common example of TIR is in professionally cut diamonds where it renders their maximum sparkle. TIR is also critical in the operation of fiber optics where light travels along the optical fiber, reflecting off its walls, within the core of the cable with minimal loss.

During the occurrence of TIR at the interface between the two nonabsorbing media, the fully reflected light beam leaks some electrical field intensity into the medium that has the lower refractive index. The leaked electrical field is referred to as the evanescent field. The evanescent field wave’s amplitude decays with distance from the interface in an exponential fashion. In SPR, the evanescent wave excites electrons within the metal layer of a metal−dielectri interface, yielding surface plasmons. Surface plasmons or surface plasmon polaritrons are electromagnetic surface waves that propagate parallel to the interface region. As the plasmon waves penetrate into the medium with the lower refractive index, any time-dependent shift in the intensity of the reflected “angle” of polarized light is recorded. The reflected light intensity is calculated as a function of the incident light angle. A shift in the SPR angle by 0.0001 degree corresponds to one unit shift in SPR signal.

Figure 1. A schematic of the conventional Kretschmann optical configuration for SPR biosensing and the associated angle shift and sensorgram plot of resonance signal change with time

The application of SPR transduction technologies to small molecule immunoassays directed to different classes of small molecule antigens, including the steroid hormones, toxins, drugs and explosives residues. SPR transduction has been used to detect small molecules such as steroid hormones, chloramphenicol, and ochratoxin A. SPR provides information about binding and recycling, as well as signal enhancement through the use of methods such as secondary resistance or gold nanoparticles to enhance binding mass and cooperative plasmon activation. These methods have been standardized and provide a common platform for small molecule detection in immunobiosensors, opening up the possibility of applications to a wide variety of small molecules.

Figure 2. A sensorgram of the primary monoclonal antibody (mAb) binding response to a progesterone-immobilized SPR sensor surface and enhancement of binding signal with secondary antibody followed by regeneration.

The SPR sensor records the changing values of the monoclonal antibody binding response during the sencodary antibody and regeneration stages, thereby monitoring and measuring the interactions between small molecules such as steroid hormones and antibodies. When these molecules bind or leave the sensor surface, this changes the SPR conditions and produces a double-changing sensor signal. This allows for highly sensitive and real-time monitoring of molecular interaction processes.

INSTRUMENTATION OF SURFACE PLASMON RESONANCE (SPR)

Surface Plasmon Resonance (SPR) spectroscopy typically uses a light source with wavelengths ranging from 500 nm to 800 nm. The most commonly used wavelengths are usually around 620 nm to 800 nm, as this range can generate strong surface plasmon effects and allows for the detection of changes in the refractive index of the surrounding medium.

Figure 2. The main parts of an SPR system

LSM LEDs are available in discrete wavelengths ranging from 310-880 nm and in a warm white option with color temperature of 3000K. The innovative optical design of the LSM LED family provides highly efficient coupling into an optical fiber. LSM LEDs accommodate multiple mounting options (DIN rail, optical bench, rack) and are supplied with a rugged plastic case for carrying multiple LEDs and accessories.

Figure 3. LSM LEDs of Ocean Insight

The spectrometer was used to record the reflected light spectrum by the sample that passed through the fiber optic. The USB2000 spectrometer is unique combination of technologies providing users with both an unusually high spectral response and good optical resolution in a single package. The electronics have been designed for considerable flexibility in connecting to various USB2000 series modules as well as external interfaces.

Figure 4. USB2000 spectrometer of Ocean Optics

Ocean Insight instruments are trusted to provide these researchers with the perfect balance between high resolution and high sensitivity while designing systems and components to suit the user's purpose.

APPLICATIONS OF SURFACE PLASMON RESONANCE

SPR has become a widely used tool in drug discovery, protein engineering, and biomolecular interaction studies. Here are some examples of its applications:

1. Drug Discovery

SPR analyzes the binding kinetics and affinity of drug candidates to target proteins in order to find and improve therapeutic candidates. It can also be used to check a vast library of chemicals for a target protein-binding capability. In the process of creating a cancer treatment, SPR was employed to examine the binding kinetics and affinity of a small molecule inhibitor to the target protein kinase.

2. Antibody Characterization

SPR is used to assess the affinity and kinetics of antibodies’ binding to their target antigens. SPR was employed to investigate the kinetics and affinity of an antibody’s binding to its target antigen in the process of creating a therapeutic antibody for autoimmune disorders.

3. Protein-Protein Interactions

Protein interactions, which are crucial for many biological processes, are studied using SPR. It can be applied to investigate the mechanics of known interactions as well as discover new protein-protein interactions. SPR was used to investigate how proteins interact with their binding partners, which control how genes are expressed.

4. Enzyme Kinetics

The kinetics of enzyme-catalyzed reactions are investigated using SPR. The kinetics of an enzyme-catalyzed process that is crucial in the liver’s drug metabolism was investigated using SPR.

CONCLUSION

Surface Plasmon Resonance (SPR) is a potent and adaptable analytical method that has completely changed the world of molecular biology and biochemistry. SPR is a promising method for analyzing protein-protein, protein-DNA, and protein-ligand interactions since it can detect biomolecular interactions in real-time and without labeling. Additionally, SPR has a wide range of uses in environmental monitoring, medical diagnostics, and drug development. It is anticipated that SPR will become more significant in both basic and practical research as technology develops.

The size of the particles on the surface of the material is determined by the localized surface plasmon resonance shift. The LSPR is different for particles of different sizes.