What Is a Sensorgram?
An SPR sensorgram is a plot of SPR response versus time. It reveals whether a binding event occurs between an analyte and a ligand, whether the binding is specific, and contains kinetic (kon, koff), affinity (KD), and concentration information. The analyte is the molecule being investigated; the ligand is the recognition element immobilized on the sensor surface.
Overview of SPR
Surface plasmon resonance is an optical, surface-sensitive technique used for screening, characterization, and bio- and chemical sensing. In a typical experiment, the interaction between an analyte and a ligand is characterized by kinetic and affinity data. Ligands are immobilized on the sensor surface, which is exposed to a flowing solution of analytes in a microfluidic channel.
Plane-polarized light shines through a glass prism coated with a thin gold film under total internal reflection conditions (Kretschmann configuration). Surface plasmons — charged oscillations at the metal surface — create an evanescent field (~200 nm) extending into the sample. When analytes bind to surface-immobilized ligands, the refractive index changes, shifting the resonance wavelength. This shift is detected in real time.
The Five Phases of a Sensorgram
1. Baseline
Running buffer conditions the surface and establishes a flat reference signal. Any drift, injection spike, or high buffer response indicates the system should be checked. Standard buffers include PBS and HEPES-NaCl.
2. Association
Analytes begin binding to immobilized ligands, producing a sharp rise in SPR signal. Ideally a single exponential curve. If the curve is linear, mass transport limitation may dominate over binding kinetics.
3. Steady-State
The net rate of bound analytes is zero — association and dissociation are balanced. This is not necessarily saturation (all ligands occupied), but an equilibrium response.
4. Dissociation
Analyte solution is replaced by wash buffer, causing specific interactions to break. Ideally a single exponential decay. Mass transport limitation or other factors can affect the shape.
5. Regeneration
A low-pH buffer (e.g., glycine) resets the baseline. A steady signal confirms the surface is free of bound analytes, ligands are intact, and the sensor is ready for the next measurement cycle.
P4SPR: Steady-State Measurements
The P4SPR uses wavelength interrogation — detecting the shift in the absorption band minimum (Δλ) as binding changes the refractive index near the surface. The resulting sensorgram plots Δλ versus time.
In a typical P4SPR experiment, increasing analyte concentrations are manually injected in a single cycle with no regeneration between injections. This produces a continuous series of association steps from lowest to highest concentration. The P4SPR does not flow buffer at a fixed rate, so there is typically no dissociation curve between injections. KD is determined by fitting a binding curve model (SPR response vs. concentration) at equilibrium.
Regeneration (e.g., with glycine) is optional and applies when the dissociation constant is in the higher µM or mM range. For tight binders, a single injection series without regeneration is typical.
Sensorgram Shapes
Concentration Optimization
Analyte concentrations must be optimized to obtain meaningful sensorgrams. Concentrations that are all too high will saturate the surface immediately; concentrations that are all too low will produce negligible signal. The ideal range spans approximately 0.1–10× the expected KD, producing well-separated curves that enable accurate fitting.
High Quality vs. Poor Quality Sensorgrams
| Feature | Poor Quality | High Quality |
|---|---|---|
| Ligand density | High | Low |
| Baseline | Drift | Flat |
| Association shape | Linear (mass transport limited) | Single exponential with curvature |
| Saturation | Not reached | Demonstrated by multiple analyte concentrations |
| Concentration range | Narrow | Wide (0.1–10× KD) |
| RI jumps and spikes | Present | Negligible jumps, no spikes |
| Dissociation decay | Short | Long |
| Replicates | Absent or non-superimposable | Present and superimposable |
From SPR Signal to Sensorgram on the P4SPR
The P4SPR workflow for generating a sensorgram:
- The detector measures the change in wavelength (Δλ) of the absorption band minimum as analyte binds to the surface.
- Raw data is saved as Δλ over time.
- The software plots Δλ versus time to produce the sensorgram.
- Kinetic and affinity data are calculated by fitting the sensorgram into a suitable binding model.
Conclusion
SPR sensorgrams are the primary data output of any SPR experiment. Understanding the five phases, recognizing quality features, and optimizing analyte concentrations are essential for producing meaningful kinetic and affinity data. The P4SPR provides real-time wavelength-mode data acquisition suited for both steady-state affinity measurements and, with regeneration, multi-cycle kinetic analysis.
Related Resources
- Your Sensorgram Is Telling You Something — interactive guide to interpreting real sensorgram patterns and troubleshooting common artefacts
- Sensorgram Cheat Sheet — quick visual reference for sensorgram phases and shapes
- TN-01: Sensor Surface Functionalization & Coupling Approaches — choosing the right sensor chip and coupling method
- Static vs. Kinetic SPR — comparing P4SPR 2.0 (steady-state) and P4PRO (kinetic) measurement modes
References
- A. Marquart, "SPRpages", 2006–2020.
- R.B.M. Schasfoort, "Introduction to Surface Plasmon Resonance," in Handbook of Surface Plasmon Resonance (2nd ed.), 2017, pp. 1–26.
- R.L. Rich and D.G. Myszka, "Survey of the year 2007 commercial optical biosensor literature," J. Mol. Recognit., 21, 355–400 (2008).
TN-03 — Affinité Instruments Technical Note Series