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.
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.
10. Principle :
The SPR system consists of three core component Namely
The Sensor chip
Micro fluid system
SPR detection unit
11. 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 .
12. 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 .
14. 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.
15. 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
16. 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).
