SURFACE PLASMON RESONANCE (SPR).pptx

The presentation has brief explanation of SPR and it's applications.

1. SURFACE PLASMON RESONANCE R. HARINI III – B.SC BIOTECHNOLOGY BON SECOURS COLLEGE FOR WOMEN

2. SYNOPSIS : Definition Components Principle Application Advantage Disadvantage

3. Definition : Surface – Surface area ( metal surface ) Plasmon – group of electrons undergoing oscillation Resonance – vibration Surface plasmon resonance (SPR) is an optical-based, label-free detection technology for real-time monitoring of binding interactions between two or more molecules. Surface Plasmon resonance is a phenomenon that occurs where electrons in a thin metal sheet become excited by light that is directed to the sheet with a particular angle of incidence, and then travel parallel to the sheet.

4. Components : Flow cell system Light source Free antigen Bounded antibody Antigen – Antibody complex Glass slide Gold film Prism Incident light Reflected light Change in Angle of reflection

5. Flow cell system: A fluidic device that allows entry of antigens and continuously removes unbound antigens from the system. Light source : Laser is used as a light source in SPR . Free antigen: Antigens that have not bound to their complimentary antibody are in their free state.
Bound antibodies: Test proteins such as antibodies that are capable of specifically capturing the desired target protein with high affinity are immobilized on to the gold coated glass microarray slide.

6. Antigen-Antibody complex:
The complex formed due to binding interaction between the free antigen and its corresponding bound antibody.
Glass slide: The array surface most commonly used for SPR applications. It is suitably coated with a metal film like gold or silver. Gold film: A thin film of gold is used to coat the glass array surface due to its favourable electronic inter-band transitions which fall in the visible range. In most other metals, these transitions lie in the ultraviolet region, thereby making them unsuitable for SPR. Prism: The prism placed in contact with the glass slide surface helps in reflecting the incident light from the surface.

7. Incident light:
Light falling on the gold-coated array surface with its immobilized antibodies has a particular wavelength and is known as the incident light. Reflected light:
Some of the energy of the light incident on the array surface gets absorbed for molecular transitions while the remaining light of lower energy (and higher wavelength) gets reflected from the array surface at a specific angle.
Change in angle of reflection:
Any changes in the angle of reflected light are indicative of biomolecular binding interactions on the array surface. The angle at which minimum intensity of reflected light is obtained is known as the SPR angle and serves as a quantitative measure of biomolecules binding to the array surface.

8. SPR FLOW DIAGRAM :

9. Principle : The SPR system consists of three core component Namely The Sensor chip Micro fluid system SPR detection unit

10. The SPR-based binding method involves immobilization of a ligand on the surface of a sensor chip which has a monolayer of carboxymethylated dextran covalently attached to a gold surface. The ligand of interest is immobilized on the surface of the sensor chip using well-defined chemistry allowing solutions with different concentrations of an analyte to flow over it and to characterize its interactions to the immobilized ligand. The SPR signal originates from changes in the refractive index at the surface of the gold sensor chip. The increase in mass associated with a binding event causes a proportional increase in the refractive index, which is observed as a change in response. These changes are measured as changes in the resonance angle (δθ) of refracted light when the analyte, flowing in a microfluidic channel, binds to the immobilized ligand and increases in density at the sensor chip. Importantly, for protein-protein interactions the change in refractive index on the surface is linearly related to the number of molecules bound. The response signal is quantified in resonance units (RU) and represents a shift in the resonance angle. When a steady-state is achieved (all binding sites occupied), the maximum RU is determined .

11. Monitoring the change in the SPR signal over time produces a sensorgram, a plot of the binding response (RU) versus time which allows different stages of a binding event to be visualized and evaluated. During the injection of an analyte, the binding response increase is due to the formation of analyte–ligand complexes at the surface and the sensorgram is dominated by the association phase. After a certain time of injection, a steady state is reached, in which binding and dissociating molecules are in equilibrium. The decrease in response after analyte injection is terminated is due to dissociation of the complexes, defining the dissociation phase. Fitting the sensorgram data to an appropriate kinetic binding model allows calculation of kinetic parameters such as the association (ka) and dissociation (kd) rate constants, and the binding affinity of the tested interactions .

12. SPR SENSOGRAM :

13. Application : SPR is a powerful label-free method widely used to study binding between two macromolecules . SPR spectroscopy is mostly used in biosensing, especially when investigating binding affinities, such as antibody-antigen interactions. Screening and developing new pharmaceuticals and new biotherapeutics
Quality control in bioprocess monitoring
Developing new diagnostic assays
Basic research such as discovering and characterizing protein function, disease mechanisms.

14. Advantage : Real-time monitoring, label-free detection, small sample size requirement, reusable sensor chips, use of complex samples, and shorter experimental runs. Label-free (less expensive and easier to perform)
Small sample volumes (100-200uL)
High sensitivity (can be used for small molecules to large proteins)
Real-time (giving deeper insight into the binding kinetics compared to yes/no binding or affinity techniques)
Quantitative

15. Disadvantage : One potential limitation of the SPR method is that the ligand may not maintain its native configuration upon immobilisation on the sensor chip surface. Low sensitivity Thickness of the metal film (thin film is preferred).

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