Surface plasmon resonance (SPR) is a bioanalytical technique that enables the study of biological and chemical interactions in real time, without labelling the analyte. SPR allows researchers to observe binding rates and binding levels between molecules, so that the specificity, kinetics, and affinity of an interaction can be determined.
This technical note provides new and existing users a foundational understanding of SPR as an analytical tool. The focus is on four key concepts:
- Principle of the SPR effect
- SPR instrument configuration and implications
- Classes of SPR sensors and considerations
- SPR sensor functionalization for selectivity
Concept 1
Principle of the SPR Effect
A typical SPR sensor is a thin metallic film — usually gold (Au) — coated on a glass substrate from which light is reflected to generate the SPR effect. The sample solution containing targets such as biomolecules or chemical compounds sits on top of the sensor surface.
The sensitive region is generated by a coupling effect between the incident light in the glass and the free electrons at the metal surface. This evanescent wave extends approximately 200 nm out-of-plane, into the sample solution — and it is highly responsive to refractive index (RI) changes in that region.
Binding events, chemical concentration changes, and polymer conformational shifts all produce RI changes detectable by SPR. The spectral signal is characterized by a sharp drop in reflectance at a specific wavelength or angle, depending on the interrogation mode used.
Concept 2
SPR Instrument Configuration
SPR instrumentation is defined by how the sensor is interrogated — specifically, the type of light source and detector used to track the SPR signal. Two modes exist for thin-film sensors:
Wavelength interrogation (fixed-angle SPR) — a polychromatic light source (broadband LED or tungsten lamp) illuminates the sensor at a fixed angle. The reflected spectrum is acquired, and binding is tracked by monitoring shifts in the SPR band wavelength over time. This is the approach used in all Affinité Instruments systems.
Angular interrogation — a monochromatic laser is swept across multiple angles to locate the SPR minimum. Requires high mechanical precision for angular scanning and alignment between the moving light source and detector.
| Parameter | Wavelength (Fixed-Angle) | Angular |
|---|---|---|
| Robustness | High — no moving parts | Lower — precision mechanics required |
| Miniaturization potential | High | Low |
| Sensitivity | High | Very high |
| Throughput | High — multi-channel simultaneous | Lower |
| Instrument cost | Low (< $100K) | High (> $100K) |
| Maintenance cost | Low | High — often exceeds annual instrument cost |
| Portability | Suitable for field and bench use | Lab-only |
Wavelength interrogation SPR offers a compelling path to accessible, affordable SPR — reducing the cost barrier that has historically limited adoption to well-funded core facilities. The miniaturization potential of fixed-angle systems also enables new use cases: portable field testing, point-of-care diagnostics, and process monitoring integration.
Affinité Instruments takes this further with a lensless SPR architecture — eliminating the optical components entirely and achieving the same measurement with a smaller, more robust instrument. This approach, protected by US patent, extends the advantages of wavelength interrogation while removing the remaining alignment constraints.
Concept 3
Classes of SPR Sensors
Thin-film SPR sensors belong to one of two broad classes, the second being nanoparticle-based sensors. SPR using nanoparticles is often called localized SPR (LSPR) — the free electron oscillation is confined within the boundaries of the nanoparticle. In contrast, free electrons on a thin-film sensor propagate along the film axis over several microns.
| Parameter | Thin-Film SPR | Nanoparticle LSPR |
|---|---|---|
| Excitation mode | Total internal reflection (TIR) — light never crosses the sample path. Direct detection in complex or opaque media possible. | Transmission — light must cross the sample path. Limited to clear samples; preparation often required. |
| Sensor format | Thin film on glass — scalable, reproducible manufacturing. Easy to functionalize. | Nanoparticles in suspension or immobilized on glass — multi-step process, higher variability, fragile when immobilized. |
| Evanescent depth | 200–300 nm — greater flexibility for complex sandwich assays and larger targets. | 20–30 nm — highly surface-sensitive, but limited sensing strategies. |
| Sensitivity (nm/RIU) | 2000–3000 | 500–600 |
| Diffusion mode | Planar — consistent with the vast majority of SPR kinetics literature. | Radial — different kinetic profile; fewer reference publications; may produce less accurate rate constants. |
Concept 4
Surface Functionalization for Selectivity
The SPR effect is intrinsically responsive to all refractive index changes and indiscriminate of their source. Most applications investigate a specific response — binding of a biomolecule or chemical compound to a defined target. Two complementary strategies make SPR sensors selective:
Increasing specificity — using a recognition element (antibody, DNA probe, aptamer, receptor) with high affinity or selectivity for the target of interest.
Reducing non-specificity — minimizing interactions with molecules other than the target. This involves surface chemistry choices (SAM layers, PEG, hydrogel), biofunctionalization strategies, and buffer optimization.
Common surface functionalization approaches include:
- Covalent coupling — NHS-EDC chemistry to immobilize proteins directly via amine groups. High surface density, permanent attachment.
- Capture surfaces — protein A/G for antibody orientation; NTA-Ni²⁺ for His-tagged proteins; streptavidin for biotinylated ligands. Enables controlled orientation and regeneration.
- SAM layers — self-assembled monolayers (e.g., MHDA) as the base chemistry for further functionalization. Controls surface density and non-specific binding.
- AffiCoat sensors — pre-functionalized sensors from Affinité Instruments optimized for direct use, reducing surface preparation time and variability.
The choice of functionalization approach directly affects data quality: surface orientation, ligand density, and non-specific binding all influence sensorgram shape and the accuracy of fitted kinetic constants. For a detailed guide, see TN-01: SPR Sensor Surface Functionalization & Coupling Approaches.
SPR with Affinité Instruments
Affinité Instruments builds portable, multi-channel SPR instruments designed to make the full analytical power of thin-film SPR accessible — on the bench and in the field.
The P4SPR 2.0 uses wavelength interrogation SPR in a lensless architecture. Four channels run simultaneously — three active and one reference — enabling reference-subtracted data, triplicate measurements, and robust results from a single chip. Manual injection via syringe enables steady-state (KD) analysis without any accessories.
The P4PRO integrates fluidics (AffiPump) for kinetic measurements — delivering full kon, koff, and KD from automated multi-concentration runs. AffiLabs software handles acquisition, reference subtraction, and kinetics fitting in a single session.