The sensorgram is the core output of every SPR experiment — a real-time plot of binding signal versus time. Understanding what each phase represents is the foundation of interpreting SPR data correctly.
What Is a Sensorgram?
When molecules bind to a surface, they change the local refractive index near that surface. SPR detects this change optically and reports it as a signal, typically expressed in Response Units (RU) or millidegrees. Plotting this signal continuously over the course of an experiment produces the sensorgram.
Because the measurement is continuous and label-free, the sensorgram captures the full kinetics of the interaction — not just an endpoint. This is what makes SPR fundamentally different from techniques like ELISA, which only report whether binding occurred, not how fast it formed or fell apart.
The Five Phases of a Sensorgram
1. Baseline. Before any sample is introduced, a running buffer flows over the sensor surface. This establishes a stable reference signal. A flat, stable baseline is essential — drift or noise here will propagate through the entire experiment.
2. Association phase. The analyte sample is injected and flows over the ligand-coated sensor surface. As binding occurs, the refractive index increases and the signal rises. The rate at which the signal rises reflects both the association rate constant (ka) and the analyte concentration. The steeper and faster the rise, the higher the affinity.
3. Equilibrium / steady state. If the association and dissociation rates balance out during the injection window, the signal plateaus. This plateau level is concentration-dependent and is used in steady-state affinity analysis to determine the equilibrium dissociation constant (KD).
4. Dissociation phase. Running buffer replaces the sample, and the bound analyte begins to dissociate from the surface. The signal decreases at a rate determined by the dissociation rate constant (kd). A slow dissociation (flat curve) indicates tight binding; a rapid drop indicates a weaker or transient interaction.
5. Regeneration. In most experiments, a regeneration solution is briefly applied to remove any remaining bound analyte and return the surface to baseline — ready for the next injection. Choosing the right regeneration condition (pH, salt, detergent) without damaging the ligand surface is a critical part of assay development.
What Can You Extract from a Sensorgram?
A well-designed SPR experiment produces sensorgrams at multiple analyte concentrations. Fitting these curves to a binding model yields:
- ka (association rate constant, M−1s−1) — how fast the complex forms
- kd (dissociation rate constant, s−1) — how fast the complex breaks apart
- KD (equilibrium dissociation constant, M) — overall binding affinity, calculated as kd / ka
- Rmax — the maximum binding capacity of the surface, useful for surface quality assessment
- Concentration — via calibration curve, the analyte concentration in an unknown sample
Kinetic vs. Steady-State Analysis
There are two main approaches to extracting affinity data from sensorgrams. Kinetic analysis fits the full shape of the association and dissociation curves simultaneously to extract ka and kd individually — this gives the most complete picture of binding behavior. Steady-state analysis uses only the equilibrium plateau levels at each concentration and fits them to a binding isotherm to get KD directly. Steady-state is simpler and works well for fast-dissociating interactions where true equilibrium is reached during the injection window.
Reading the Shape: What Can Go Wrong
Not every sensorgram looks like a textbook curve. Common issues include a drifting baseline (surface instability or temperature fluctuation), a signal that does not return to baseline after dissociation (incomplete regeneration or covalent-like binding), a hook effect at high concentrations (analyte-analyte aggregation), and biphasic dissociation curves (heterogeneous surface or multiple binding sites). Recognizing these shapes and knowing their causes is an essential skill in SPR data interpretation.
Conclusion
The sensorgram packs a remarkable amount of information into a single time-resolved plot. Once you understand the five phases and what each parameter means, you can move quickly from raw data to meaningful conclusions about binding affinity, kinetics, and surface quality. The Affinité SPR platform is designed to generate clean, reproducible sensorgrams with minimal setup — so you can focus on interpreting the biology, not troubleshooting the instrument.