Indian Institute of Technology, Bombay. Lecture-3. An Overview of Surface Plasmon Resonanace (SPR)
Indian Institute of Technology, Bombay. Lecture-3. An Overview of Surface Plasmon Resonanace (SPR)
Welcome to MOOC intractomics course. In today’s lecture we will talk about one of the very
promising label-free technique, surface Plasmon resonance. It is an optical method based on
surface plasmon resonance and evanescent waves, which provides kinetic resolution of
binding reactions in a label-free manner. The SPR spectroscopy bio-sensors are popular
technology because of their simple instrumentation and high sensitivity, they are also in great
demand because they can provide label-free real time detection of various bio-molecular
interactions. SPR is used in research for various application of biology including the drug
discovery, clinical diagnostics as well as security applications.
These are very sensitive to any changes on this boundary like adsorption of bio-molecules to
the metal. In SPR, a light beam impinges at the interface between metal and media at a
defined angle called as resonance angle. The resonance angle depends upon the refractive
index in immediate vicinity of the gold surface. When metal binds to the gold surface the
refractive index increases and the SPR curve shift toward the higher angles. So, these changes
in the angle of refraction of light caused due to the binding of probe to the immobilized
proteins are measured for the characterization of bio-molecular interactions in real time.
(Refer Slide Time: 4:15)
Now, in this slide let us look at the SPR angle, how it depends on refractive index near the
surface and the SPR angle which is directly related to the amount of bio-molecules binding
on the gold surface. The real time label-free detection of binding events can be detected by
measuring changes in SPR reflectivity. These changes in refractive index are continuously
monitored to obtain the kinetic data in real time manner making it a remarkable label-free
detection technique.
(Refer Slide Time: 5:33)
Let us now discuss SPR sensorgrams. The sensorgrams describe the changes in SPR signal
verses time as molecules bind and dissociate from the sensor surface, the resulting change in
resonance signal creates a sensorgram. Let us look at the various steps involved in these
sensorgram. As you can see this slide, there is prism, light source, a gold coated chip and
ligand immobilized on the chip surface, initially the running buffer is injected on to the
immobilized surface, it generates a baseline. The baseline is straight until the query molecule
or the analyte is injected in the medium.
(Refer Slide Time: 6:38)
So, initially the surface is washed with the running buffer followed by injection of the query
molecule in the same running buffer, as the analyte starts interacting with the ligand
association phase can be observed in SPR sensorgram from which association rate Kon or Ka
can be derived. In this slide, you can see some of the query molecules have started to bind to
the ligand immobilized on the chip surface, note, sometime when the association achieves a
saturation level, they reach to a state known as stochastic steady state.
Injection of running buffer at this point helps in analyte dissociation from which the
dissociation rate Koff or KD can be derived. As shown here in the right panel initially, you
generate a base line followed by an association phase and then a dissociation phase. The left
panel is also showing proteins being dissociated from bound molecules. So, after the run is
completed, the same chip can be reused for further runs but one need to perform mild acid
treatment on the surface and further washing with the running buffer. This process of
complete removal of analyte from the chip surface is known as regeneration.
So, now, we will view an SPR animation to understand some other basic concepts. Surface
Plasmon resonance (SPR) is a highly sensitive, spectroscopic tool that is increasingly being
used for label-free detection studies. Test proteins such as antibody are immobilized on to the
gold coated glass array surface incident light is striking the surface is constantly reflected at a
particular angle in this state.
The SPR angle or the angle at which minimum intensity of reflected light is obtained is
indicative of the amount of biomolecule binding to the surface. The graph shown on the right
side represents change in reflection intensity before and after the antigen binding.
Now, the graph is showing association or on rate Ka, after sometime the binding reaches to a
saturation level known as stochastic steady state. When running buffer is further added, the
bound proteins are dissociated which can be seen as a dissociation rates in the graph which is
represented by off rate or Koff. After the SPR run is finished, same chip can be reused by
applying a mild acid treatment and further washing steps by a process known as regeneration.
The immobilization procedure may require lot of optimization with different type of surface
chemistries, often non-specific interaction can also result in fast SPR signal. So, there is need
to ensure that the signals obtained are specific and unique to the experiments, the bulk effect
can also interfere with the actual data which needs to be avoided. Lastly, refractive index is
also temperature dependent. So, these are some of the limitations associated with SPR
however, it is a still one of the very promising label-free detection platform.
(Refer Slide Time: 17:23)
Let us now discuss the guidelines for performing SPR experiments and its data analysis.
Performing a good SPR experiment and accurate interpretation of binding reactions from
these biosensor is always very challenging. David Myszka from university of Utah in United
Sates has provided very detailed guideline for biosensor analysis. I will briefly describe some
of these guidelines which can be used for performing SPR experiments and data analysis.
First of all, experimental preparation. It is very important to start with quality reagents that
ensure high quality data. Reagents should be homogeneous; there should not be any
aggregates or precipitation. Filtration and degasing of buffers is important because even a
small bubble can ruin the whole experiment. Additionally, instrumental cleaning is very
important, any dust particle or any type of contaminant can result in artifacts.
(Refer Slide Time: 19:15)
Ideally, the analyte and the ligand should be monomeric in solution, and able to form one to
one complex for the data to fit into the simple reaction model. Surface capacity is another
important consideration. What kind of controls need to be used? What reference empty
surface needs to be used? Single component binding and baseline checks, all of these are
important points to consider.
Low sensor surface capacity is preferable in many cases which can help in minimizing issues
such as mass transport, aggregation and steric hindrance. There are various experimental
parameters which one needs to consider, for example, how fast or how slow the injection rate
should be? Ideally, the fast flow rate can minimize mass transport type of effects, analyzing
blanks of running buffer periodically is critical to ensure reliable results and stable baseline.
Now, how to fit the data to selection of appropriate fitting models is always very critical. The
experiment must be designed accurately. As an example, for an antigen and antibody
interaction, for an antibody being able to bind to two different antigens, it should be used as a
ligand and antigen should be flown over as analytes to study their biomolecular interactions.
If binding data has to be correlated to interaction modules, experiments should be designed
critically to be able to fit to the data by using appropriate fitting models.
In summary, today we talked about surface Plasmon resonance which monitors bio-molecular
interactions in label-free manner moreover, it provides quantitative information for the
binding kinetics. We also discussed about its strengths and weakness of the approach and
guidelines to follow the SPR assays; we will continue our discussion on SPR imaging in next
lecture. Thank you.
(Refer Slide Time: 22:10)