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Short answer
Bio-layer interferometry (BLI), also spelled biolayer interferometry, is a label-free method for measuring how biomolecules bind each other. One partner is immobilised on the tip of a disposable biosensor, the tip is dipped into a solution of the other partner, and binding is read as a shift in the interference pattern of white light. The shift traces association and dissociation in real time, from which rate constants and the equilibrium constant KD are derived.
What is BLI (biolayer interferometry)?
BLI systems are built around disposable biosensors that carry a layer of a recognition molecule, such as an antibody or a capture protein, on the sensor tip (Murali et al., 2022). The method is described as label-free because neither partner needs a fluorescent or enzymatic tag for the binding to be detected; only molecules binding to or leaving the biosensor surface shift the interference pattern (BiophysX core resource).
In practice this makes BLI a bench tool for questions such as how tightly an antibody binds its antigen, how fast it binds and how fast it lets go. It sits alongside other binding methods covered on CASRAI, including surface plasmon resonance, isothermal titration calorimetry and fluorescence polarization assays.
How does BLI measure affinity?
White light is directed down the length of the biosensor and reflected from two surfaces. Binding at the tip changes the optical depth of the binding surface, which shifts the wavelength of the reflected light in proportion to that depth (Murali et al., 2022). The instrument plots this shift against time as a sensorgram.
In a typical run the biosensor carrying the immobilised partner is dipped into a solution of the other partner to record association, then moved back into buffer to record dissociation. Because the signal comes from the sensor tip rather than from the bulk solution, it is not affected by changes in the refractive index of the surrounding medium or by flow rate (BiophysX).
What is KD in BLI?
KD is the equilibrium dissociation constant, the summary number for how tightly two partners bind: a smaller KD means tighter binding. BLI reports it together with two rate constants, the association rate constant (ka) and the dissociation rate constant (kd), which are defined in the review by Murali et al. (2022).
The reading path from a sensorgram is:
- Fit the dissociation phase to obtain kd, the rate at which the complex falls apart in buffer.
- Fit the association phases from a series of analyte concentrations to obtain ka. The BiophysX core resource notes that generally 10 concentrations are needed, 5 each above and below the KD.
- Combine them as KD = kd / ka. The worked calculation, with units, is in the CASRAI guide to calculating KD from a sensorgram, which uses SPR data.
A fitted KD is only meaningful if the interaction behaves as simple 1:1 binding. Vendor assay-development notes for Octet systems list proper assay development to ensure 1:1 binding, an appropriate range of analyte concentrations and optimisation of ligand loading to avoid binding artefacts among their core topics (Sartorius).
What are the key differences between SPR and BLI?
Both methods follow binding in real time without labels. The differences described here concern format, cost and throughput:
| Feature | BLI | SPR |
|---|---|---|
| How the sample meets the surface | Biosensors are dipped into sample wells | Sample flows through flow channels |
| Apparatus cost and throughput | Described as less expensive, with higher throughput potential | Uses a flow-channel setup, which the review describes as the more expensive apparatus |
Both rows come from Murali et al. (2022), who describe BLI as advantageous because of the simplicity of dipping biosensors as opposed to using flow channels. For lab planning, note that the biosensors are described as disposable, so they recur as a consumable in an assay budget.
Planning a BLI experiment
The BiophysX core resource gives several planning notes that are easy to miss when comparing methods:
- Plan an entire day for the first experiment.
- Generally 10 concentrations are needed, 5 each above and below the KD.
- Run a negative control: perform each run also on a biosensor with nothing immobilised.
- If long incubations are used, 200 µl of each concentration is the volume the core resource cites.
Artefact checklist: what a BLI trace can mislead you about
Murali et al. (2022) list the following as limitations of BLI. Each is worth considering before reporting a KD:
- Sample evaporation over time.
- Agitation is required to prevent re-binding.
- Regeneration needs optimisation.
- Non-specific binding.
- Only one binding partner is immobilised.
Common misunderstandings
BLI is not a flow-based method
BLI uses dip-and-read biosensors; it does not rely on flow channels the way SPR does (Murali et al., 2022).
Label-free does not mean artefact-free
The method avoids tags on the binding partners, but non-specific binding, evaporation and re-binding can still distort a sensorgram, which is why a control biosensor with nothing immobilised is recommended.
KD is not measured directly
The instrument records a wavelength shift over time. KD is calculated from fitted rate constants, so the quality of the fit and the concentration series matter.
FAQ
What is BLI?
BLI is a label-free technique that measures biomolecular interactions by analysing the interference pattern of white light reflected from a biosensor tip as molecules bind to it.
What is KD in BLI?
KD is the equilibrium dissociation constant, derived from the association and dissociation rate constants fitted to the sensorgram. Smaller values mean tighter binding.
How does BLI measure affinity?
Binding at the tip shifts the wavelength of reflected light in proportion to optical depth. The instrument records the shift during association and dissociation, and fitting those phases gives ka, kd and KD.
What are the key differences between SPR and BLI?
BLI dips disposable biosensors into samples, while SPR uses flow channels. BLI is described as having a less expensive apparatus and higher throughput potential (Murali et al., 2022).
Sources
- Murali S, Rustandi RR, Zheng X, Payne A, Shang L. Applications of Surface Plasmon Resonance and Biolayer Interferometry for Virus-Ligand Binding. Viruses. 2022;14(4):717. pmc.ncbi.nlm.nih.gov/articles/PMC9027846 (checked 2026-10-29).
- BiophysX core resource, BLItz page. biophysx.ismb.lon.ac.uk/blitz.html (checked 2026-10-29).
- Sartorius, biomolecular binding kinetics assays on the Octet platform. sartorius.com Octet BLI resources (checked 2026-10-29).
Related reading: Hill coefficient in binding curves and protein purification, for related lab techniques.
