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Introduction to Surface Plasmon Resonance

Surface plasmon resonance or SPR is an optical effect that can be utilized to measure the binding of
molecules in real-time without the use of labels. SPR instruments are primarily used to measure the binding
kinetics and affinity of molecular interactions. SPR can be used, for example, to measure the binding between
two proteins, a protein and an antibody, DNA and a protein, and many more. SPR is unique because it is one
of the few techniques that allows determination of binding kinetics and not just binding affinity, as you would
get from traditions techniques like ELISA. The binding kinetics, or the on and off rates, can only be
determined with a biosensing technique that gives real-time binding data of both the association and
dissociation phases of the interaction. This data gives detailed insight into the binding strength and stability
of the interaction, which is critical for many industries and research areas. It helps researchers determine
which molecules interact, why they interact, and how strongly they interact.

What are Binding Kinetics?

Determining binding kinetics is one of the most common applications of surface plasmon resonance. Some
researchers and individuals just look for yes/no binding, or equilibrium constants, but binding kinetics can
give you much more detailed information about the molecules and systems you are studying, including the
on rate, the off rate, and the affinity constant for the interaction being analyzed. Below you will see a typical
binding curve. A typical binding curve consists first of an association phase, during which an analyte sample is
passing through the system and binding to the ligand. Following this phase is the steady state, or equilibrium
phase during which the on and off rates for analyte binding are equal, and the system is in equilibrium. This
phase gives data on both the on, the ka, and off rate, the kd. Following this phase is the dissociation phase,
during which only running buffer is run through the system, and off rate, or kd only is measured. To get
accurate binding constants, you will usually need to use a few binding curves using analytes at different
concentrations. The final phase on this curve, is called regeneration. Unless you have an interaction with a
high off rate, you will probably need a regeneration solution to remove the analyte from your ligand prior to
the injection of the next analyte concentration. During regeneration, the analyte is removed from the ligand
without damaging the ligand using a regeneration buffer.

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 Association Dissociation
phase phase

Response (pm)

Time
Fluid flow over Running Analyte Running Regeneration Running
sensor chip buffer Sample buffer solution buffer

Applications of SPR

The data from SPR is critical in a number of industries, and has been in use for over 25 years by companies
like Pfizer, Roche, and GSK and by many universities throughout the world. Some examples of its applications
include:

• Affinity and kinetic analysis • Quality control in bioprocess monitoring

• Concentration determination • Developing new diagnostic assays
• Thermodynamics • Basic research such as discovering and
• Stoichiometry characterizing protein function, disease
• Ligand fishing mechanisms, etc.
• Epitope mapping • Sensors food safety and environmental
• Screening and developing new analysis
pharmaceuticals and new biotherapeutics

SPR has a number of advantages over other common assay techniques:

• 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

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How Does Surface Plasmon Resonance Work?

Surface plasmon resonance can be used to measure binding events because it is very sensitive to changes in
refractive index. SPR instrument are made up of an optical measurement system to measure the refractive
index, a fluid handling system for sample delivery, and a sensor chip.

The sensor chip is a gold film coated on a glass substrate that has been chemically modified to make it easier
to immobilize one of the binding partners onto the surface of the sensor. The molecule that is immobilized is
known as the ligand, and the molecule in solution is known as the analyte. The sensor

chip is interface with the fluidic system through the use of a small flow cell, so that the analyte can be
injected at different concentrations in a very repeatable manner.

The fluidic system provides a continuous stream of buffer flowing through the flow cell and across the sensor
chip, maintaining a very well controlled environment. The analyte is easily injected as a “plug” into the
flowing buffer so that it will interact with the sensor surface for a specific amount of time. The fluidic system
is also used for ligand immobilization, analyte regeneration, cleaning, and conditioning.

The optical system consists of a light source and a detector, and varies depending on the exact way in which
the instrument is designed. The light source illuminates the gold film, and the detector measures the unique
optical spectrum produced by the SPR phenomenon. Most SPR instruments utilize a laser that shines through

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a prism beneath the sensor chip, generating a total-internal-reflection condition. When this occurs, a
plasmonic wave is generated on the surface of the gold, with the electrical field of this wave extending
hundreds of nanometers into the space above the surface of the sensor. The reflected light will have a
characteristic dip in intensity at a certain angle due to the plasmonic wave, which is measured with a
detector. When a molecule binds to the surface of the sensor chip, the refractive index of the space in which
the plasmonic wave is propagating through changes, and the reflectance angle will shift. The amount it shifts
depends on how much mass of material has bound to the surface, and the shift can be measured in near real
time.

What Happens in an SPR Experiment?

In a typical SPR experiment, the first step is to immobilize the ligand to the sensor surface. There are a
number of different types of sensor surfaces and methods to do this. Once the ligand is immobilized the
remaining binding sites are blocked, and the surface often conditioned with injections of regeneration buffer.
Running buffer is being continuously run over the surface of the sensor chip, and once the surface is stable,
the analyte is injected at a specific flow rate for a specific amount of time (usually 2-5 minutes) and at a
specific concentration. The time in which the analyte is passing through the flow cell is known as the
association phase, and the analyte will accumulate on the surface as it binds to the ligand. The rate of binding
will depend on the on-rate and off-rate of the interaction, as well as the mass transport of the analyte from
the bulk to the surface. This why it is important to maintain a high flow rate and use a low ligand density, so
that the concentration of analyte at the surface will remain high. Binding of the analyte to the ligand will
cause the signal to increase until equilibrium is reached (if it is reached). Once the analyte passes through the
flow cell and is replaced with buffer, the analyte will start to come off the surface, and the signal will begin to
decrease. This is known as the sensorgram. If the ligand and analyte bind strongly, a regeneration buffer will
be injected to break up the ligand-analyte complex, leaving the ligand on the surface. The analyte injection is
repeatable multiple times for 3-5 different concentrations, which is needed in order to obtain reliable kinetic
constants.

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 Association Dissociation
phase phase
Response (pm)

Time
Fluid flow over Running Analyte Running Regeneration Running
sensor chip buffer Sample buffer solution buffer

Once the experiment is complete, the user has a set of binding curves that can be mathematically fit with
binding models to extract the on rate, off rate, and affinity. Good binding curves should be exponential. A 1:1
binding model is the simplest, but corrections for mass transport and more complex binding models can be
added. The set of analyte curves are globally fit, so a single k on and koff are generated for the entire data set.

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How to Optimize Binding Kinetics

Being able to differentiate between good and bad data is key for getting binding kinetics. Accurate binding
constants come from simple exponential binding curves, so it is important that the sensorgrams have
curvature, and are not just straight lines. Some scientists try to fit bad data to more complex binding models
just because they fit better, which is often a mistake. You should avoid using complex models unless you have

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evidence that the interaction is in fact more complicated than a 1:1. Here we will discuss how to optimize the
various aspects of your experiment in order to have the most success with SPR.

Sensor Chips
The first step to optimizing binding kinetics is to make sure you are using the correct sensor chips for your
application. Plain gold sensor chips are available for immobilization of thiolated ligands. Carboxyl coated
chips are used for EDC/NHS coupling to any amine group. NTA coated chips for immobilization of his tagged
targets, and streptavidin coated chips are used for immobilization of biotinylated targets. Hydrophobic chips
are used for immobilization of lipids and membrane proteins. Antibodies can also be immobilized onto COOH
chips to allow for oriented capture of the ligand.

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 Figure 0.1 - Covalent coupling of ligand to COOH sensor chips using EDC/NHS activation.

Figure 0.2 - Capture coupling of ligand to Streptavidin sensor chip.

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 Figure 0.3 - Capture coupling of ligand with His-tag on NTA sensor chip.

Figure 0.4 - Capture coupling of ligand onto Antibody-coated sensor chip.

Choosing the right sensor chip allows you to bind you ligand to the sensor, however, you’ll want to make sure
you avoid non-specific binding. Non-specific binding (NSB) occurs when the analyte interacts non-specifically
with the sensor chip. NSB is caused by molecular forces (charge interactions, hydrophobic interactions,
hydrogen bonding, etc) between the analyte and the sensor surface. In order to test for NSB, a preliminary
binding test with only the bare sensor (sensor without the ligand) and the analyte should be completed. If
there is significant response when the analyte is passed over the bare sensor (false positive), do not stress, as
there are a variety of steps you can take to reduce NSB for SPR experiments.

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 Figure 0.5 - Specific binding of analyte to the ligand vs non-specific binding of analyte to the surface.

The first step to reduce nonspecific binding, is to immobilize the molecule that has the least non-specific
binding with the sensor. You will also want to immobilize the largest target as well. If you experience non-
specific binding after this step, don’t worry as there additional steps you can take. Adding BSA, Tween-20
and/or salt and adjusting the pH level (to the running buffer and samples) are a few options that can allow
you to reduce nonspecific binding, and we have a great article on this topic in our blog that explains how, and
why you should use each of these techniques.

Sample Prep

The quality of sample prep is key for good binding kinetics. For SPR applications, buffer should be at the
proper pH, degassed and filtered. You want to make sure that everything is completely dissolved in you
sample and your buffer and you want to avoid any aggregates. If you are adding any unstable additives, you
should make fresh buffer every day. Additionally, make sure to degas the buffer first before you add the
unstable additives.

The most important thing to remember is that you want your buffer to match the sample as closely as
possible to avoid background refractive index changes. This means dissolving your sample in the running
buffer and making sure they are at the same temperature (if you store your buffer in the fridge). If the
sample and buffer do not match, you will notice a sharp increase in signal at the start and end of the injection

Mass Transport

Limiting mass transfer effects is key to getting good data. In surface plasmon resonance, the reactant in a
solution must first diffuse from the bulk to the surface to interact with the immobilized ligand. If the diffusion
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rate is slower than the association rate, mass transfer effects can be observed in the data. Mass transfer
limitations are most common for fast binding reactions, as diffusion limits the association rate. Diffusion
limited binding will often look linear rather than exponential. If you do experience mass transfer effects, the
easiest way to limit them is to use higher flow rates. Increasing the flow rate will deliver the analyte to the
immobilized ligand faster, without affecting the kinetic data. Injecting the analyte at a few different flow
rates is also a quick way to determine if you have mass transfer effects. If the ka decreases at lower flow
rates, the interaction is mass transport limited. Another way to reduce mass transfer effects is to reducing
the amount of ligand immobilized to the sensor. This will help reduce diffusion-limited effects as less analyte
needs to diffuse for the interaction to occur. Finally, you can use a fitting model that includes mass transport
in the overall reaction equations to mathematically account for mass transport effects. Most kinetic
processing software, including the Tracerdrawer software offered by Nicoya, includes this model as an option
when fitting data. To read more about limiting mass transfer effects, you can again, head over to our blog
after this presentation!

Flow
ANALYTE

km km

ka kd

LIGAND

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Optimizing Binding Curves

In order to get accurate binding constants, you will usually need to use have a few binding curves using
analytes at different concentrations. In order to determine which concentration of analyte you will start with,
you will want to make sure you do a preliminary binding test to determine the lowest concentration of
analyte that will give you a nice curve. Once you have this concentration value, labelled as “1” in the diagram
below, you can increase the analyte concentration, usually aiming for a 3-fold difference in concentration to
ensure adequate spacing between your curves. For example, if you start with 3nM, the next concentration
would be 9nM, then 27nM, and so on. You want to use 2 to 5 concentrations of analyte to generate your
binding curves. You will not want to use more than 5 analyte concentrations, as this can stress the fitting
models. Of course, as with any scientific experiment, you’ll want to repeat each analyte concentration 3
times, in order to have confidence in your results.

5
4
3
2
1

Regeneration

Regeneration conditions are very important for optimizing binding kinetics. To determine accurate binding
constants, 2-5 binding curves of analyte at different concentrations are required, and the analyte will need to
be removed from the ligand in between these steps. Whether you need a regeneration step or not depends
on the off rate of the ligand-analyte complex. If the off rate is high, the analyte will dissociate from the
surface in a short amount of time, for example, 5 minutes, so a regeneration step is not needed in order to
have multiple analyte injections. If the off rate is low, the analyte will take a very long time to dissociate,
sometimes hours. In order to get multiple analyte injections in a reasonable amount of time, you need to
regenerate. The most effective regeneration buffer is specific to the types of molecules being studied, and
their affinity for each other. The regeneration buffer must be harsh enough to remove all of the analyte, but
mild enough so that it does not severely damage the functionality of the ligand, otherwise the analyte
response will decrease significantly during future injections. When testing regeneration conditions, always

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start with the least harsh conditions, because you also don’t want to damage the sensor during the
regeneration process.

We have summarized the most common regeneration buffers in the table below. The optimal buffer to use is
specific to the type of molecules being studied and their affinity.

Below we have listed common regeneration conditions for different types of molecular interactions. This is a
great starting point when you are unsure of the type of regeneration to use.

1. Acid 5-150 mM

• Proteins
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 • Antibodies

2. SDS 0.01 – 0.5%

• Peptides
• Proteins/Nucleic acids

3. NaOH 10 mM

• Nucleic acids/Nucleic acids

4. IPA:HCl 1:1

• Lipids

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