Is it possible to measure binding kinetics in solution? Now it is.

Scientific progress is driven by continuous evolution, and binding kinetics are no exception.

For decades, immobilization-based methods were the standard for studying molecular interactions. While these techniques provided valuable insights, they also imposed fundamental limitations that researchers have long accepted as trade-offs: surface effects, complex assay setup, and restricted sample compatibility. But science moves forward, and we move with it. Now, in-solution kinetics is reshaping how we measure interactions, offering a more natural, efficient, and precise alternative.

Understanding molecular interactions is at the heart of structural biology, drug discovery, and biophysics. Binding kinetics, the rates at which molecules associate and dissociate, play a fundamental role in drug efficacy, protein function, and biomolecular stability.

How does surface (or lack of it) make a difference?

Traditional methods like Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) have long been used for kinetic studies. Importantly, these methods, albeit highly useful, rely on surface immobilization, which can distort binding behavior. What if you could measure binding directly in solution, preserving native conditions and obtaining absolute kinetic data?

This is where Flow-Induced Dispersion Analysis (FIDA) offers a new perspective. Many biomolecular interactions occur in a complex, dynamic environment, where factors like protein conformation, oligomerization, and aggregation influence binding behavior. However, most kinetic assays require molecular immobilization, which introduces several challenges:

I. Steric hindrance: surface attachment can block or alter binding sites.
II. Non-physiological conditions: immobilized molecules may behave differently than in free solution.
III. Mass transport effects: for fast kinetics, the mass transport to the surface may be the rate-limiting step. Under such circumstances, assessment of binding kinetics becomes difficult or even impossible.

FIDA eliminates these barriers by measuring kinetics in solution, delivering absolute, label-free readouts.

Does FIDA measure kinetics differently?

Yes. Instead of relying on immobilization, FIDA tracks molecular size changes in free solution, using the hydrodynamic radius (R_h) as a direct readout of binding interactions. Here is how it works:

1.  Baseline measurement: the free molecule's hydrodynamic radius (R_h) is determined in its native state.
2.  Titration with binding partner: a series of increasing ligand or analyte concentrations is introduced.
3.  Binding detection: any interaction that causes a size change (due to complex formation, conformational shifts, or oligomerization) is detected with absolute precision.
4.  Binding (K_D) analysis: by capturing size changes at different concentrations in equilibrated samples, the equilibrium interaction constant (K_D) can be determined without immobilization-induced artifacts.
5.  Kinetic (k_on and k_off) analysis: by controlling the reaction time, the kinetic rate constants (k_on and k_off) can be obtained without immobilization-induced artifacts.

Watch the on-demand webinar, in which our CSO Henrik Jensen, Ph.D. explains in-solution kinetics in depth. Or, if you prefer, take a look at the brochure.

Gaining scientific freedom and personal productivity

By removing immobilization-related artifacts and working directly in solution, you get a more physiologically relevant and data-rich approach to kinetics:

Preserve native molecular behavior: no surface immobilization means no artificial constraints and a new level of scientific freedom.
Work with complex samples: measure unpurified samples, multi-subunit assemblies, and even membrane proteins.
Directly measure the hydrodynamic radius: get insights into binding-induced conformational changes.
Minimize sample consumption: use only microlitre-scale volumes, ideal for precious biomolecules.
Get absolute kinetic readouts: remove the need for assumption-based decisions, and use your data for ML and AI models.

Read our user's opinion on in-solution kinetics.

See how FIDA data matches SPR data.

Applications of in-solution kinetics

What is it currently used for? FIDA's absolute kinetic measurements are particularly valuable for:

🔬 Protein-ligand binding: detect even subtle conformational changes upon binding.
🧬 Oligomerization and self-assembly: study how molecular size shifts during complex formation.
💊 Drug discovery and screening: evaluate binding kinetics for lead optimization.
🧪 Structural biology and biophysics: link kinetic profiles to protein stability and aggregation tendencies.

Feeling left with questions or doubts? We are happy to connect.

A closer look: how in-solution kinetics works in practice

The overview above covers why in-solution kinetics matters and how FIDA reads binding from molecular size. The rest of this article goes a step deeper into the practical detail: exactly how the on- and off-rates are measured with in-capillary mixing, how the numbers line up against SPR, what makes the method practical day to day, and the questions researchers ask most often.

Our CSO Henrik Jensen, Ph.D. walks through all of it in the on-demand webinar. Watch it here.

Can you measure binding kinetics, the on- and off-rates of an interaction, without immobilising anything on a surface? Yes. FIDA measures k_on and k_off in free solution by deliberately pushing a reaction out of equilibrium inside a capillary and comparing that with the equilibrium curve. The method is called in-capillary mixing, or CapMix.

Why measure binding kinetics in solution instead of on a surface?

Because keeping the interaction in free solution sidesteps the limits of a surface-based assay and works from very little material. Strong binders are handled without trouble, the measurement runs in essentially any liquid matrix, including complex ones such as serum or plasma, from only nanolitre to microlitre amounts of sample, and because FIDA measures the size of the complex at the same time, every kinetics run also carries structural information that a binding curve alone would not.

How does FIDA measure k_on and k_off? The CapMix method

The idea is to run two experiments and read the kinetics from the gap between them: an equilibrium measurement and an out-of-equilibrium one. In the equilibrium experiment, the two partners are pre-mixed and left to react to completion, typically incubated for thirty minutes to an hour, then measured to give the equilibrium binding curve.

The out-of-equilibrium experiment is CapMix. Rather than pre-mixing, the reactants are injected as separate bands, one partner, then a very small zone of the other, then the first partner again, so they only meet and react while travelling through the capillary. Because the applied pressure sets how fast the sample reaches the detector, it also sets the reaction time: high pressure means a short reaction time and shifts the binding curve to higher concentrations, while lower pressure lets the system relax back toward equilibrium. The size of that shift between the equilibrium curve and the CapMix curve is what the software turns into the on- and off-rates, k_on and k_off.

How does the kinetics readout compare with SPR?

FIDA's rate constants agree closely with SPR. On beta-2-microglobulin binding an anti-beta-2-microglobulin antibody, the k_on and k_off from FIDA came out very similar to those from surface plasmon resonance (SPR), the surface-based reference method. Because the interaction stays in free solution, though, several problems that come with a sensor surface simply do not arise:

• no surface regeneration between measurements, and none of the run-to-run variability a surface that does not fully recover can introduce;
• no mass-transport limitation at a surface;
• no non-specific binding to a sensor surface, and because size is measured in the same run, specific binding is still easy to tell from non-specific association;
• no immobilisation, so the complex is measured native and freely diffusing; since tethering a protein can change how it folds and binds, the in-solution value can be the more faithful one where the two methods disagree.

What makes FIDA practical for kinetics?

Several things make FIDA practical for routine kinetics:

• Setting up a new assay is fast: optimising the conditions for a new FIDA assay typically takes only a few hours.
• The equilibrium K_D and the on- and off-rates come from the same instrument and largely the same samples, so there is no separate kinetics assay to build.
• Only nanolitre to microlitre volumes are consumed, so kinetics are feasible early in a project, when purified material is scarce.
• The quality-control readouts accompany every run, so aggregation, an unexpected oligomeric state or a viscosity shift is visible in the same measurement, and you know whether the rate constants can be trusted before acting on them.

What about small molecules that barely change size?

For those, the Neo series adds a second readout called FIDA Lambda Dynamics, which detects binding from a small shift in the fluorophore's emission spectrum rather than a change in size, and it supports kinetic measurements too. It works by splitting the emission into two spectral regions and following their ratio, a self-referenced signal sensitive enough to resolve very small changes in the emission spectrum, as in a carbonic anhydrase titration against furosemide. If your interest is small-molecule work, the companion articles on FIDA Lambda Dynamics and measuring a small-molecule affinity in about a minute go into it in depth.

Frequently asked questions

What is FIDA, and what does it measure?

FIDA (Flow Induced Dispersion Analysis) is an in-solution technique that measures a molecule's hydrodynamic radius, its effective size, from which binding can be followed directly. A thin band of fluorescent sample, only about 40 nanolitres, is loaded by pressure into a 75-micrometre fused-silica capillary and pushed through under laminar flow. The flow is fastest in the middle and slowest at the walls, so the band disperses into a Gaussian profile whose shape depends on how fast the molecule diffuses: small molecules diffuse quickly and give a narrow profile, larger ones diffuse slowly and give a wider one. The whole run, including data analysis, is automated.

Titrating one partner against another and following the hydrodynamic radius gives an equilibrium binding curve, and its midpoint is the dissociation constant, K_D. Every measurement also returns a set of quality-control readouts at the same time, size, aggregate count, polydispersity, viscosity, labelling quality, sample loss and surface stickiness, so you can see immediately whether the sample is behaving. Kinetics builds directly on this equilibrium measurement.

Can FIDA measure fast kinetics?

Yes. Dedicated kinetics capillaries let you follow interaction half-lives down to roughly 5 to 10 seconds. For fast systems, the pre-mix experiment is recommended alongside CapMix to confirm you are genuinely in the kinetic regime rather than close to equilibrium.

Can it measure weak binders?

Yes, into the millimolar range. Because viscosity is measured on every run, the high concentrations that weak binders require can be compensated for automatically in the analysis, avoiding viscosity artefacts.

Does kinetics work label-free?

Yes, using a protein's intrinsic tryptophan fluorescence, though the sensitivity is lower, typically hundreds of nanomolar. Labelling one partner is recommended for very sensitive work and for complex matrices such as serum or plasma.

What maintenance does the instrument need?

Very little. The only parts that contact the sample are the capillary, a consumable changed after about 400 measurements, and the well plate, so routine maintenance is minimal.

Key takeaways

• On- and off-rates in free solution: CapMix reads k_on and k_off from the shift between an equilibrium and an out-of-equilibrium curve.
• No surface: no mass-transport limits, no regeneration, and no immobilisation artefacts.
• Two experiments, shared samples: an equilibrium run and one CapMix run, reusing most of the equilibrium samples.
• SPR-comparable rate constants, measured entirely in solution.
• Fast or weak, labelled or label-free: half-lives to 5 to 10 seconds, millimolar affinities, and intrinsic tryptophan detection when needed.

Curious whether FIDA fits your interaction? Explore the FIDA technology and the Fida instrument, or browse the peer-reviewed literature. You can also watch the full webinar with Henrik Jensen.