BIOCHEMICAL AND

BIOPHYSICAL
TECHNIQUES
SPR
SURFACE PLASMON
RESONANCE TECHNIQUES
By Kayshu Grover
SURFACE PLASMON RESONANCE
Surface Plasmon Resonance, shortly known as SPR is a surface sensitive method.
SPR is an optical spectroscopic technique that measures smallest changes in index of refraction of
light.
Refraction is the change in direction undergone by an electromagnetic wave when it crosses the
boundary between media in which its velocity differs.
Index of refraction is the measure of degree of refraction undergone by a wave.
The changes in refractive indices are caused by changes in mass near specific metallic surfaces
and changes in the bulk refractive index of the fluid above the surface.
SPR is expressed in Resonance Units or RU.
1000 RU correspond to about 1 nano-gram milimeter-2 change in mass at the surface of the chip.
SPR can detect changes in the refractive index of less than 104 or changes in layer heights of
about 1 nano-meter.
PRINCIPLE OF SPR
Surface Plasmon Resonance is a sensitive mean to measure the binding of molecules to a thin
gold layer on the underside of a prism.
Light directed through the prism is reflected from the gold surface.
There is one narrow range of angles at which reflection is nearly zero because the thin unbound
gold layer absorbs the light to set up oscillations of the electron cloud in the metal.
These oscillating electrons are known as Plasmons.
When a thin layer of material, such as protein or DNA binds to the side of the gold away from
the prism, the electrical properties of the gold layer are changed and the property of light
reflectivity changes.
A change in the passing wave that develops in a surface when a light beam at the angle of total
reflectance strikes the surface, induces a change in the dielectric constant or relative
permittivity which can be measured.
INSTRUMENTATION
INSTRUMENTATION
Components Of A Surface Plasmon Resonance Spectrometer:
1. Light source
2. Prism
3. Sensor Chip
4. Sample
5. Detector
INSTRUMENTATION
Components Of A Surface Plasmon Resonance Spectrometer:
1. Light source:
 Source of white un-polarized light.
2. Prism:
 Bends incident light according to critical angle making it parallel to the plane of incidence.
 It increases the wave number and focuses polarized light on the sensor chip.
3. Sensor Chip:
 Gold, Silver, Copper, Aluminum chips 50 nano-meters in size are used.
4. Sample:
 Analyte to be tested.
5. Detector:
 An array detector is preferred over a normal single detector.
WORKING OF SPR
Surface Plasmon Resonance Spectrometer analyses the thickness and molecular structure changes which
lead to changes in the path of refracted light.
For SPR, light wavelengths between Infrared and Near Infrared are used.
One component with concentration of 1–5 nano-gram milimeter-2 is immobilized on the sensor chip by
covalent coupling like in affinity chromatography.
 The other component is then injected over the micro-flow cell in the desired buffer with the sensor chip
forming one wall of the cell so that an aqueous solution of the ligand can be pumped at a continuous,
pulse-free rate across the surface which ensures that the concentration of ligand at the surface is
maintained at a constant value.
SPR detection relies on a resonance interaction between the light beam and the thin metal film on the
sensor chip.
Thus, this can detect both the rate of association which increases the mass on the sensor chip as well as
rate of dissociation which decreases the mass on the sensor chip for the particular set of components .
The change in mass attributes the change in refractive index at that point on the sensorgram.
WORKING OF SPR
When the light passes through the metal plate to the aqueous sample it undergoes total internal
reflection.
Total internal reflection (TIR) occurs when a light beam travelling through a medium of higher refractive
index (e.g. glass prism with gold coated surface) meets at an interface with a medium of lower refractive
index (e.g. aqueous sample) at an angle larger than the critical angle.
Since the interface between the prism and the medium is coated with a thin layer of gold, incident
photons excite a vibrational state of the electrons of the conducting band of the metal which propagates
as a longitudinal vibration.
The electrons vibrate with a resonance frequency that is dependent on metal and prism properties, as
well as the wavelength and the angle of the incident beam.
Excitation of the plasmon wave leads to a decreased intensity of the reflected light.
Thus, SPR produces a dip in the reflected light intensity at a specific angle of reflection.
The higher the wavelength of light used in SPR, better is the sensitivity but lower is the lateral resolution
whereas lower the wavelength of light, better is the lateral resolution but lower is the sensitivity.
WORKING OF SPR
ADVANTAGES AND
DISADVANTAGES OF SPR
ADVANTAGES DISADVANTAGES
Very small amount of sample is required for Not very cost-effective.
analysis.
External probes are not required.
No requirement of radio-labeled or
fluorescent-labeled molecules.
It is a non-destructive method.
It can be used with colored and opaque
solutions.
APPLICATIONS OF SPR
Surface Plasmon Resonance Spectrometers are used as biosensors in various fields like
Pharmaceutical and Medical Research as well as Life Sciences.
Pharmaceutical and Medical Applications:
1. Characterization of molecular interactions
Antibody-antigen binding
Ligand- receptor coupling
2. Dynamics of protein interactions
 Binding of proteins to DNA
 Interaction of proteins with carbohydrates
 Interaction of proteins with other proteins
 Reaction of proteins with small molecules
APPLICATIONS OF SPR
Surface Plasmon Resonance Spectrometers are used as biosensors in various fields like
Pharmaceutical and Medical Research as well as Life Sciences.
Research Applications:
1. Determining the thickness of thin films.
2. Monitoring the interaction of two components, like the interaction between an analyte in
solution and its bio-specific partner.
3. Quantification of binding processes between molecules
APPLICATIONS OF SPR
Other than applications in various fields of science Surface Plasma Resonance
Phenomenon is modified and used in combination with imaging of metallic surfaces in
Multi-Parametric Surface Plasmon Resonance Spectroscopy also known as MP-SPR.
Advantages:
1. The path of electromagnetic wave, that is angle of incidence and angle of reflection is
not fixed.
2. Multiple wavelengths are studied simultaneously.
 Applications:
1. The method has been used in biosensor development for electrochemical printed
biosensors, ELISA and SERS.
2. In life and material sciences, it is used in Pharmaceutical drug development and
optimization of thin solid films upto sizes of 100 nano-meters.
APPLICATIONS OF SPR
Other than applications in various fields of science Surface Plasma Resonance
Phenomenon is modified and used in combination with imaging of metallic surfaces in
Surface Plasmon Resonance Microscopy also known as SPRM.
Advantages:
1. High contrast imaging
2. Sensitive to few tenths of nanometer
3. Lateral resolution values are in micrometer scale.
 Applications:
1. Characterization of surfaces like self assembled mono-layers, multi-layer films, metal
nano-particles, oligo-nucleotide arrays and binding and reduction reactions.
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