SPR vs BLI: how the two label-free techniques differ
Surface plasmon resonance (SPR) and biolayer interferometry (BLI) both follow binding in real time without labels, and both report affinity and kinetics. They measure binding in very different ways, though, and that decides which one fits your samples, your analytes and the detail you need.
How SPR works
A ligand is immobilized on a thin (~50 nm) gold film. Under total internal reflection, light excites surface plasmons in the gold, and the resonance shifts when the refractive index within roughly 200–300 nm of the surface changes as analyte binds. The sample reaches the sensor in one of two ways: flow, where buffer and sample are pumped through a flow cell (kinetic SPR), or static, where the sample is injected and binds by diffusion with no pump. The light never passes through the sample, so opaque matrices such as serum and plasma can be measured.
How BLI works
BLI uses disposable fiber-optic biosensor tips coated with ligand. White light travels down the fiber and reflects from two interfaces at the tip: an internal reference layer and the biolayer. As molecules bind, the biolayer thickens and the interference pattern between the two reflections shifts. The tips are dipped into the wells of a shaken microplate, moving from buffer to sample to buffer. There is no microfluidics, so there are no channels to clog, and the sample stays in its well.
SPR vs BLI at a glance
| Criterion | SPR | BLI |
|---|---|---|
| Measurement principle | Refractive-index change at a gold film, read as a shift in the plasmon resonance | Shift in the white-light interference pattern at a fiber-optic tip as the bound layer thickens |
| Fluidics and clogging | Flow systems use microfluidic channels that particulates and bubbles can block; static-mode SPR has no pump or tubing | No fluidics: tips dip into plate wells, so there is essentially nothing to clog |
| Sample recovery | Sample usually goes to waste after the injection | Sample stays in the well and can generally be recovered |
| Small-molecule sensitivity | Generally higher; widely used for small molecules and fragments | Weaker signal for small analytes; better suited to proteins and antibodies |
| Crude-sample tolerance | Light does not pass through the sample, so serum and plasma can be measured; flow systems usually need filtered or clarified samples to protect the fluidics | Well suited to lysates and culture supernatants; very turbid or opaque samples, such as undiluted serum, can destabilize the signal |
| Throughput and format | Channel-based: a few channels per run, extended by autosamplers | Plate-based: 8 or more tips read in parallel from 96- or 384-well plates |
| Kinetics | Full kon, koff and KD under controlled flow, with the best resolution of fast rates; static mode gives KD at steady state | kon, koff and KD from dip-and-read association and dissociation; very fast rates are harder to resolve |
| Baseline and vibration | Pump pulsation, bubbles, temperature and bulk refractive-index mismatch (e.g. DMSO) show up in the baseline and are handled with reference channels; wavelength readout with fixed optics limits vibration noise | The reference and signal reflections sit in the same tip, so shaking the plate does not disturb the reading; less affected by bulk refractive-index changes; evaporation in open wells can cause drift in long runs |
When to choose BLI and when to choose SPR
Choose BLI when you need
- To run crude samples (lysates, supernatants) without fluidics
- High-throughput screening in 96- or 384-well plates
- To recover sample from the well afterward
- Mostly large analytes, such as antibodies and proteins
Choose SPR when you need
- Higher sensitivity and lower detection limits
- Small molecules, fragments and other low-molecular-weight analytes
- High-resolution kinetics (kon, koff, residence time)
- Measurements in opaque matrices such as serum or plasma
The two are often complementary: many labs use BLI to triage large sample sets and SPR to characterize the shortlist in detail. In one published study featured on our blog, researchers could not get a stable BLI signal in human serum unless it was diluted to 10%, while SPR measured prostate-specific antigen in undiluted serum. Read the SPR vs BLI serum case study.
Where Affinité's SPR instruments fit
A common reason to pick BLI is avoiding microfluidics. Static-mode SPR removes the fluidics while keeping SPR's refractive-index readout.
- P4SPR 2.0 is a static-mode SPR instrument: no pump, no fluidics, no clogging risk. It runs 4 channels at once with 150 µL per injection and gives affinity (KD) and active concentration data in cycles of 10 minutes or less.
- P4PRO + AffiPump runs both static and flow-based experiments on one instrument, adding full kinetics (kon 10³–10⁷ M⁻¹s⁻¹, koff 10⁻⁵–10⁻¹ s⁻¹) with 5–100 µL flow injections.
If your main need is screening hundreds of samples a day in plate format, a plate-based system such as BLI is built for that. Affinité instruments run 4 channels at a time and are designed for labs that want SPR data on their own bench. See how static and kinetic modes compare in our static vs kinetic SPR technical note, or compare the instruments.
Weighing SPR against a plate assay instead? Read SPR vs ELISA: differences and when to use each.
Should you use surface plasmon resonance or isothermal titration calorimetry?
Use SPR when you need binding kinetics (kon, koff) and affinity from small sample amounts. Use isothermal titration calorimetry (ITC) when you need the thermodynamics of binding (ΔH, ΔS) and stoichiometry, measured in solution with no immobilization or labels. Both report the dissociation constant KD, and many labs use them together to cross-check affinity.
What does ITC measure?
ITC measures the heat released or absorbed as one binding partner is titrated into the other in solution. A single titration gives the binding enthalpy (ΔH), the stoichiometry (n) and KD, from which the free energy (ΔG) and entropy (ΔS) follow. Neither partner is immobilized or labeled, but standard ITC does not report binding rates.
What does SPR measure?
SPR measures binding as it happens at a sensor surface where one partner, the ligand, is immobilized. Flow-based SPR gives the association and dissociation rate constants (kon, koff) and KD; static-mode SPR gives KD at steady state and analyte concentration. SPR typically needs much less sample than ITC, but requires an immobilization step.
SPR vs ITC at a glance
| Criterion | SPR | ITC |
|---|---|---|
| Measurement principle | Refractive-index change at a gold film as analyte binds an immobilized ligand | Heat released or absorbed with each injection of one partner into a cell containing the other |
| Main outputs | kon, koff and KD (flow); steady-state KD and concentration (static) | KD, ΔH and stoichiometry (n), with ΔG and ΔS derived |
| Kinetics | Yes, in flow mode | No in standard ITC; kinetic analysis of the titration signal (KinITC) is a specialized exception |
| Thermodynamics | Indirect: from KD measured at several temperatures (van’t Hoff analysis) | Direct: ΔH is measured in a single experiment |
| Format | One partner is immobilized on the sensor surface; the other is label-free in solution | Both partners free in solution; no immobilization or labels |
| Sample consumption | Low: ng to low-µg amounts; 150 µL static or 5–100 µL flow injections on Affinité instruments | Higher: micromolar concentrations in the cell and a more concentrated titrant in the syringe for each titration |
| Complex samples | Serum and plasma can be measured, with reference channels to subtract non-specific signal | Best with purified proteins in matched buffers; buffer mismatch and side reactions add heat that masks binding |
When to choose ITC and when to choose SPR
Choose ITC when you need
- The thermodynamic signature of binding (ΔH, ΔS)
- The stoichiometry (n) of the complex
- Both partners in solution, with no immobilization or labels
- An immobilization-free check of a KD measured by SPR
Choose SPR when you need
- Association and dissociation rates (kon, koff, residence time)
- Low sample consumption for scarce proteins
- Analyte concentration as well as affinity
- Measurements in complex matrices such as serum or plasma
SPR and ITC are complementary rather than competing. ITC confirms affinity and stoichiometry in solution, free of any surface effect, and shows what drives binding energetically; SPR shows how fast the complex forms and how long it lasts. When an SPR KD agrees with an ITC KD, that is good evidence that immobilization has not changed the interaction.
On Affinité instruments, the P4SPR 2.0 (static mode) gives KD and concentration, and the P4PRO + AffiPump (static and flow) adds full kinetics. See our static vs kinetic SPR technical note or compare the instruments.
SPR vs BLI and ITC: frequently asked questions
What is the difference between SPR and BLI?
Both are label-free, real-time optical methods for measuring biomolecular binding. SPR (surface plasmon resonance) detects refractive-index changes at a thin gold film, with the sample either flowed through a flow cell (kinetic mode) or injected and left to bind by diffusion (static mode). BLI (biolayer interferometry) detects a shift in the white-light interference pattern at the tip of a fiber-optic biosensor that is dipped into microplate wells, so it uses no microfluidics. SPR generally offers higher sensitivity and kinetic resolution; BLI offers plate-based throughput and tolerance of crude samples.
Is SPR more sensitive than BLI?
Generally, yes. SPR typically reaches lower noise and lower detection limits than BLI, which matters most for small molecules and other low-molecular-weight analytes, where BLI signals are weak. For large analytes, such as antibodies binding an immobilized antigen, both techniques usually give ample signal, and the choice depends more on throughput, sample type and how much kinetic detail you need.
Why is BLI less sensitive to vibration than SPR?
In BLI, the two reflections that create the interference signal come from surfaces inside the same fiber tip, so moving or shaking the tip shifts both together. This is why BLI instruments can shake the plate during a measurement. SPR reads a resonance through external optics and, in flow-based systems, through fluidics, so mechanical disturbances, pump pulsation, bubbles and temperature changes can appear as baseline noise. How much this matters depends on the instrument: SPR systems that read the resonance by wavelength with fixed optics, as Affinité's do, are designed to limit vibration-related noise, and reference channels subtract disturbances common to all channels.
When should I choose BLI instead of SPR?
BLI is a good fit for crude samples such as cell lysates and culture supernatants, for screening many samples in 96- or 384-well plates, and when you want to recover sample from the well afterward. SPR is the better choice when you need higher sensitivity, small-molecule or fragment work, or high-resolution kinetics. Many labs use both: BLI to triage large sample sets and SPR to characterize the shortlist.
Can SPR be run without microfluidics or clogging?
Yes. In static-mode SPR, the sample is injected by syringe and binds by diffusion, with no pump or microfluidic tubing to clog. Affinité's P4SPR 2.0 is a static-mode SPR instrument (no pump, no fluidics, no clogging risk) that gives affinity (KD) and concentration data. The P4PRO with AffiPump runs both static and flow-based experiments, adding full kinetics (kon, koff, KD).
Should I use surface plasmon resonance or isothermal titration calorimetry?
Use SPR when you need binding kinetics (kon, koff) and affinity from small sample amounts. Use isothermal titration calorimetry (ITC) when you need the thermodynamics of binding (ΔH, ΔS) and stoichiometry, measured in solution with no immobilization or labels. Both report the dissociation constant KD, and many labs use them together to cross-check affinity.
Can ITC measure binding kinetics?
Not in its standard form. ITC reports equilibrium parameters: KD, binding enthalpy (ΔH), entropy (ΔS) and stoichiometry, measured in solution without immobilization. Specialized analyses of the titration signal, such as kinetic ITC (KinITC), can extract rate information in some cases, but when you need association and dissociation rates (kon, koff), SPR is the more direct choice.