Surface Plasmon Resonance Sensorgram: A Step-by-Step Guide

Surface Plasmon Resonance (SPR) is a powerful analytical technique used to study molecular interactions in real-time, providing critical insights into binding kinetics, affinity, and specificity. At the heart of SPR lies the sensorgram dynamic plot that visually captures the entire interaction lifecycle between a ligand immobilized on a sensor surface and an analyte in solution. This guide demystifies the sensor program, breaking down its key phases, analysis methods, and troubleshooting strategies. Whether you are investigating protein-protein interactions, optimizing drug candidates, or validating binding mechanisms, understanding how to interpret and utilize sensorgrams is essential. Below, we explore the fundamentals of SPR, decode the four phases of a sensorgram (baseline, association, dissociation, and regeneration), and address common challenges like baseline drift, low binding signals, and non-specific interactions.  

What is SPR and What is A Sensorgram?

SPR is a new analytical technique based on optical principles. It refers to the phenomenon of total reflection of light on the surface of the prism and the metal film, forming a vanishing wave into the photophobic medium, and there is a certain plasma wave in the medium (assumed to be a precious metal), and the resonance phenomenon may occur when the two bands meet on the premise of energy conservation. The technology can detect the interaction between antigen and antibody, DNA and protein, and DNA and DNA, and has been widely used in life science, medical diagnosis, food safety, and environmental detection.

The SPR experiment produces a sensorgram that shows the change in the SPR signal over time. The Biacore system monitors the interaction between two molecules, one of which is attached to the surface of the sensor and the other is free in solution. The sensor map is a map of the response over time, showing the progress of the interaction. During the analysis, this curve will be displayed directly on the computer screen. The association phase shows the binding process between the analyte and ligand fixed on the chip, while the dissociation phase shows the dissociation process of this binding. By analyzing these curves, dynamic information about the intermolecular interactions can be obtained.

Key Phases of A Sensorgram

During sample injection, when the analyte (the interaction partner in solution in Biacore analysis) combines with the ligand (the interaction partner connected to the sensor surface in Biacore analysis), a positive response can be seen in the sensor map. The reaction is weakened during dissociation. After the analysis cycle is complete, the regenerated solution passes through the sensor chip to remove the bound analyte in preparation for the next analysis cycle.

Figure 1: The four main parts of the SPR sensor. (fig from Cytiva Surface Plasmon Resonance (SPR) | Cytiva)

Baseline: Starting point

Refers to the initial stage. This stage is used to check the sensor system for anomalies or instability. A normal baseline should be a flat, straight line. When there is baseline drift, injection spikes, and high buffering response, the system needs to be inspected and cleaned. The baseline is the initial flat line on the sensorgram, representing the system's stability before any analyte is introduced. A stable baseline is crucial for accurate measurements.

Association: Analyte binds

During the association phase, the analyte is injected and binds to the ligand immobilized on the sensor surface. This results in an increase in response units (RU), forming a binding curve.

Association refers to the stage at which the analyte begins to bind to a fixed ligand. The sensor diagram shows a sharp rise in the SPR signal, ideally it is a single exponential curve. This step is controlled by two events, namely mass transfer from the bulk solution to the surface and movement to the surface-fixed ligand: the analyte in the bulk solution must move from the bulk solution to the surface and then bind to the surface-fixed ligand. If the binding of the surface analyte to the ligand is hindered by the movement of the analyte from the bulk solution to the surface (mass transfer constraint), the diffusivity of the analyte and ligand is greater than the kinetic constraint. The correlation curve will be more linear.

Dissociation: Analyte unbinds

After the analyte injection stops, the dissociation phase begins. Here, the analyte gradually unbinds from the ligand, causing a decrease in RU. The rate of dissociation provides information about the stability of the interaction.

Dissociation refers to the breakdown of a specific interaction between the analyte and the ligand when the analyte solution is replaced by a washing buffer. It is represented by a downward-sloping curve after the steady-state stage. Ideally it should be a single exponential decay. In practice, any mass transfer limitations or other factors will affect the curve at this stage.

Regeneration: Resetting the surface

The regeneration phase involves removing any remaining analyte from the surface to prepare it for the next experiment. This is typically done using a regeneration buffer that disrupts the binding without damaging the ligand.

Regeneration means resetting the SPR baseline signal with a flowing low-pH buffer (such as glycine) at the beginning of the next set of SPR measurements. Similarly, the signal needs to be a flat curve to indicate that the sensor system has no bound analytes and non-specifically adsorbed molecules, has stability (with intact and functional ligands), and has no temperature or surface chemical changes, etc.

Analyzing A Sensorgram

A sensorgram is a graphical representation of the interaction between molecules over time, typically obtained from surface plasmon resonance (SPR) or similar biosensing techniques.

In the SPR experiment, the change in the refractive index of the sensing layer caused by the combination of molecules will cause the surface structure to change, and the inclination will move, indicating that the Angle or wavelength of the resonance has changed. The descent (lowest point) of the reflectance spectral curve is used to visually show the Angle or wavelength at which light is absorbed by the surface plasmon. The increase in refractive index leads to an increase in resonance wavelength shift. The change in the reflection Angle or wavelength is proportional to the mass of the newly combined analyte at the surface. The SPR sensor map is generated by plotting the change in the SPR response over time.

Watch for binding curves

The shape of the binding curve during the association and dissociation phases provides valuable information about the interaction. A steep curve indicates fast binding, while a gradual curve suggests slower binding.

Calculate binding rates (ka, kd)

Ka (Association Rate Constant): Measures how quickly the analyte binds to the ligand.

Kd (Dissociation Rate Constant): Measures how quickly the analyte unbinds from the ligand.

KD (Equilibrium Dissociation Constant): KD = Kd/Ka, representing the binding affinity (units: M).

Regenerate surface for next use

Regeneration of the sensor surface is a key step in SPR or similar biosensing experiments. It involves removing the bound analyte from the ligand on the sensor surface so that the surface can be reused in subsequent experiments. Proper regeneration ensures consistent and reliable results across multiple binding cycles.

Common Issues and Solutions

Baseline Drift: Check contamination

Baseline drift in SPR or similar biosensing experiments refers to a gradual increase or decrease in the baseline signal over time, which is not caused by specific binding events. Contamination is one of the most common causes of baseline drift and can significantly affect the quality of your data.

Baseline drift may be caused by residual analytes or impurities on the sensor surface, contaminants in the running buffer or sample, bubbles in the fluid system that cause sudden spikes or drifts, changes in temperature that affect the refractive index of the buffer, deterioration or scaling of the sensor surface, evaporation or degradation of the running buffer.

You can solve these problems by cleaning the sensor chip, cleaning the fluid system, replacing the contaminated buffer, checking the sample preparation, and ensuring that the sample does not contain aggregates or particulate matter.

Low Binding: Adjust concentrations

Low binding signals in SPR or similar biosensing experiments can result from insufficient interaction between the analyte and the ligand. Adjusting the concentrations of the analyte or ligand is a key strategy to enhance binding signals and improve data quality.

The cause of the problem may be low analyte concentration, insufficient ligand fixed on the sensor surface resulting in reduced binding capacity, the interaction between the analyte and ligand may have a low affinity, suboptimal pH, temperature, or buffer composition which all affect weak binding.

You can try to increase the analyte concentration, optimize ligand fixation, increase the ligand density, optimize the fixation conditions (e.g., pH, coupling chemistry), and verify the fixation level to ensure that there are enough ligands present to solve the above problems.

Non-specific Binding: Use controls

Non-specific binding (NSB) is a common issue in SPR or similar biosensing experiments. It occurs when the analyte interacts with the sensor surface or other components in a non-specific manner, rather than binding specifically to the ligand. Using appropriate controls is essential to identify and minimize non-specific binding, ensuring accurate and reliable data.

This may be due to hydrophobic or charged surfaces that attract analytes non-specifically, impurities in the analytical solution, improper sensor surface obstruction after ligand fixation, unsatisfactory pH, ionic strength, or additives.

You can solve the problem by using hydrophilic and neutral surfaces, removing aggregates and impurities from the analytical solution, and adjusting the buffer conditions.

When these recommendations don't exactly solve your problem, you can turn to our Surface Plasmon Resonance service, which provides accurate and reliable real-time binding data whether you're exploring protein-protein interactions, screening for small molecule drugs, or validating antibody affinity.