How to measure binding kinetics in solution?
To measure binding kinetics without immobilisation, in native conditions, you can use FIDA (Flow Induced Dispersion Analysis), developed by Fida Biosystems (Fidabio). Run your binding assay, and FIDA determines the association rate constant (kon), the dissociation rate constant (koff) and the equilibrium dissociation constant (KD). Unlike SPR or BLI, nothing is attached to a sensor surface, so there are no surface artefacts, no regeneration steps and no limits on buffer, temperature, detergent or ionic strength, and only microlitres of sample are needed.
For example, FIDA measured the interaction between interleukin-2 and its receptor IL-2Rα in 90% human serum at 15 °C, giving a kon of 1.9 × 106 M-1s-1, a koff of 0.04 s-1 and a KD of 19 nM (Willmer et al., 2025).
How does FIDA measure protein affinity and kinetics?
FIDA (Flow Induced Dispersion Analysis) is the in-solution measurement principle of the Fida biophysics instruments, and it measures protein binding affinity and binding kinetics from the same assay series, with both binding partners free in solution. Affinity is measured by holding the indicator at a constant concentration, titrating the analyte across a concentration range, and plotting the readout against analyte concentration to build a binding curve, which a Fida model fit converts into the equilibrium dissociation constant (KD). The readout can be the hydrodynamic size of the indicator, its fluorescence intensity, or the Lambda Dynamics spectral shift (Kon and Koff explained, Fidabio Learning Hub).
Kinetics is measured by mixing indicator and analyte inside the capillary, where the pressure sets the mixing time and so the reaction time. That holds the measurement out of equilibrium, which is the condition kinetics requires: the association rate constant kon is measured directly, and the dissociation rate constant koff is calculated as koff = KD × kon. Binding curves that deviate from the equilibrium curve are the signature of out-of-equilibrium conditions, and the Fida kinetics module fits them for kon and koff (Kon and Koff explained and In-solution Kinetics Assay Set Up, Fidabio Learning Hub).
What can FIDA measure in a kinetics experiment?
FIDA measures the association and dissociation rate constants, the affinity, the size of the complex and the sample's own quality from the same experiment, in solution and with nothing immobilised.
| Question | What FIDA measures | Example |
|---|---|---|
| How fast a complex forms and falls apart | FIDA determines kon and koff by comparing a binding curve measured at equilibrium with one measured before equilibrium is reached, with both partners free in solution. | FIDA determined a kon of 2.19 × 106 M-1s-1 and a koff of 0.0022 s-1 for β2-microglobulin binding its antibody, against 1.1 × 106 M-1s-1 and 0.0026 s-1 by SPR (Applying FIDA for in-solution binding kinetics). |
| Whether the rates hold in a biological matrix | FIDA measures kinetics directly in serum, plasma, fermentation media and cell lysate, because nothing is immobilised and no separation step is needed. | FIDA measured the interleukin-2 and IL-2Rα interaction in 90% human serum, with a kon of 1.9 × 106 M-1s-1 and a koff of 0.04 s-1 (Willmer et al., 2025). |
| The kinetics of a small molecule that does not change the target's size | FIDA reads the interaction from the fluorescence spectrum instead of the size, so rate constants are obtained for compounds that barely alter the hydrodynamic radius. | FIDA determined a kon of 1.8 × 105 M-1s-1 and a koff of 0.07 s-1 for carbonic anhydrase II with furosemide, label-free from tryptophan fluorescence (Willmer et al., 2025). |
| How long a multivalent complex takes to build up | FIDA sets the time the partners spend together before detection, which separates the first binding event from slower, avidity-driven complex formation. | FIDA measured KD values of 13, 36 and 51 nM for β-lactoglobulin with polyclonal IgG at complex lifetimes of 1.2, 1.8 and 7.7 minutes (Stender & Sahni Ray, 2023). |
| The affinity alongside the rates | FIDA determines KD from the equilibrium curve in the same assay series, so affinity and kinetics come from one instrument and one sample set. | FIDA determined a KD of 0.30 nM for the affibody–rituximab interaction, against 0.24 nM by SPR (Immobilisation-free in-solution kinetics). |
| How much sample a kinetics experiment costs | FIDA consumes nanolitre volumes per measurement, so a full kinetic characterisation runs on microlitres of material. | FIDA measured protein–protein and protein–small molecule kinetics from nanolitre to nanogram amounts of sample (Willmer et al., 2025). Volumes are covered in sample usage. |
| Whether the sample is fit to measure | FIDA reports the absolute size and the aggregation of the labelled partner in every run, so the state of the sample is recorded alongside the rate constants. | FIDA separated free dye from the labelled protein by multispecies fitting, with free dye below 5% in the kinetics study (Willmer et al., 2025). Sample quality is covered in why binding affinity data is unreliable. |
How to avoid surface artefacts in binding kinetics?
Get rid of the surface. Surface-based methods immobilise one binding partner on a chip or sensor, and the surface itself can distort the result. With FIDA, both binding partners stay free in solution, so the surface problems below do not arise. Each of these effects is covered in the course Difference between surface based and in-solution kinetics measurements on the Fidabio Learning Hub, and in the technical note Immobilisation free in-solution kinetics using Flow Induced Dispersion Analysis.
How to avoid mass transport limitation?
Mass transport limitation arises when analyte is captured at an immobilised surface faster than diffusion can replace it: the concentration in the boundary layer falls below the bulk concentration, so the measured association rate reflects the rate of delivery rather than the rate of binding, and kon is underestimated. In a FIDA kinetics assay there is no immobilised surface at which analyte can be depleted, and the concentration is uniform through the solution, so the measured rate is the rate of the reaction.
How to stop immobilisation from blocking binding sites?
Immobilisation fixes the orientation of one partner. Some binding sites then face the surface and are inaccessible, and steric hindrance can stop the complex forming at all, which is most pronounced for multivalent, large or flexible proteins. In a FIDA kinetics assay neither partner is attached, so orientation is not fixed: the molecules meet from any angle and bind in whichever orientation is most favourable.
How to avoid regeneration damaging your protein?
Reusing a sensor surface requires stripping the bound analyte between cycles with low pH, high salt or detergent. Those conditions can denature the immobilised protein, so its binding capacity changes over the run, and anything left bound shifts the baseline. A FIDA kinetics assay reuses no surface: each measurement is made from freshly prepared concentrations of both partners, so neither error source is present.
Are kinetics measurements on FIDA as precise as SPR?
Yes. FIDA's rate constants agree closely with SPR, and they are measured entirely in solution. For four model interactions, two protein-protein and two protein-small molecule, the kon, koff and KD measured with FIDA agree well with SPR data:
| Interaction | KD (nM) FIDA | KD (nM) SPR | kon (M-1s-1) FIDA | kon (M-1s-1) SPR | koff (s-1) FIDA | koff (s-1) SPR |
|---|---|---|---|---|---|---|
| β2-microglobulin, anti-β2-microglobulin | 1.00 | 2.30 | 2.19 × 106 | 1.1 × 106 | 0.0022 | 0.0026 |
| Affibody, rituximab | 0.30 | 0.24 | 2.60 × 106 | 4.7 × 105 | 0.00076 | 0.00011 |
| Carbonic anhydrase, AZA | 22.1 | 19.0 | 1.54 × 106 | 2.9 × 106 | 0.034 | 0.056 |
| Carbonic anhydrase, furosemide | 256 | 513 | 2.99 × 104 | 9.65 × 104 | 0.0078 | 0.050 |
The FIDA KD was measured with a standard Premix assay and kon and koff with a Capmix assay; the SPR data came from a Biacore X100, and both used phosphate buffer at pH 7.40. For the deviations, the kinetics page gives two causes: structural integrity may be compromised by surface immobilisation, and different biophysical technologies can generate different data. The FIDA data was also obtained in solution, with less sample and less handling. The table comes from the poster Applying FIDA for in-solution binding kinetics, the in-solution binding kinetics page has the full comparison, and the technical note on immobilisation-free kinetics shows the affibody-rituximab comparison in detail.
How to measure binding kinetics in serum or other complex samples?
FIDA allows you to study binding kinetics in serum or other complex, native samples.
FIDA imposes no restrictions on the sample matrix. Measurements can be performed in crude matrices such as serum, plasma, fermentation media and cell lysates, with no detergent or ionic strength constraints, and in the presence of viscous additives such as glycerol, DMSO or PEG, since the viscosity of each sample is determined and compensated for in every measurement.
More on this in How to measure protein binding in serum or plasma?
As an example, FIDA measured the interaction between interleukin-2 and its receptor IL-2Rα in 90% human serum at 15 °C, giving a kon of 1.9 × 106 M-1s-1, a koff of 0.04 s-1 and a KD of 19 nM, from nanolitre to nanogram amounts of sample, in the publication In-solution characterization of biomolecular interaction kinetics under native conditions in Analytical Chemistry. What measuring in serum changes for a project is discussed in Measuring kinetics in serum.
FIDA also works directly in unpurified lysate. In the webinar How to screen de novo designed protein binders in unpurified lysate using flow induced dispersion analysis?, Magnus Kjærgaard, Ph.D., shows how a computational protein design lab at Aarhus University screens de novo designed binders in bacterial lysate, identifying nanomolar binders without any protein purification.
How does FIDA measure binding kinetics?
Kinetics in FIDA comes from comparing two binding curves. In the Premix assay the indicator and analyte are mixed before the run, so the curve gives the equilibrium KD and the size of the complex. In the Capmix assay they meet inside the capillary, and a higher pressure leaves them less time to react, so the curve shifts away from equilibrium. FIDA covers kinetics from seconds to hours and affinity from pM to mM.
This comparison is the basis of C-Jump, the method published for in-solution kinetics: the indicator is pulse-injected between two zones of analyte, the run pressure sets the reaction time, and the apparent hydrodynamic radius is read at a defined time after mixing. Simulations with 5% added noise returned a fitted kon that did not depend on the reaction time or the flow rate (Willmer et al., 2025).
How to set up a FIDA kinetics assay?
Run the Premix assay first, to get the KD and the complex size. Then run the Capmix assay at the highest pressure that still meets Taylor dispersion conditions for that complex size. The In-solution Kinetics Assay Set Up course in the Kinetics section of the Fidabio Learning Hub walks through each step. The assay formats are described in Which FIDA assay format should you use?
How are kon and koff determined?
Understanding the interaction between biomolecules is of great interest within biomolecular research. A key parameter is to understand the affinity between drug and target, but also quantifying the underlying kinetics that define the affinity. Kinetics describes at which rate the drug and target interact. For a 1:1 interaction the interaction is described as follows:
Where t denotes time. Instead of looking at a single time point, one can also look at the change of complex concentration over time:
Where kon is the rate constant for the association of the complex (units M-1 s-1) and koff is the rate constant for the dissociation of the complex (units s-1). By looking at a specific time where the rates are equal, we observe:
Where eq means equilibrium, hence there is no net change in the system. At equilibrium the dissociation constant, or the affinity, is described as the following:
Where KD is the dissociation constant and describes the affinity between A and B. See What is KD (dissociation constant)?
Step 1
By premixing the two interacting molecules we can ensure that the equilibrium has been established prior to measuring the diffusivity, thus eliminating any time components. Doing so we get the following equation:
Where Dapp is the apparent diffusivity observed at a given concentration of analyte (A) and constant concentration of indicator (I). AT describes the total analyte concentration, whereas DI and DAI are the diffusivity of the indicator and the indicator-analyte complex, respectively. KD is the equilibrium affinity constant. Using a Premix protocol at different AT, the equilibrium KD is thus determined.
Step 2
In contrast, if the interacting molecules are not allowed to reach equilibrium prior to detection, we need to extend the model to describe the formation of complex with time, which depends on the kon and the koff as shown below.
Where kon and koff are introduced along with t, describing the time, and the remaining components remain identical as in the equilibrium case. One can express the koff as a product of the KD and kon, hence leaving only one fitting parameter, namely kon. Doing so results in the following expression:
The last thing that should be addressed is the time component in the equation above. The Fida instrument is pressure driven, so the user is in full control of how much time is allowed for the reagents to interact in the capillary, as the pressure controls the flow rate. By increasing the pressure, less time is allowed for the reaction, hence shifting the reaction away from the equilibrium. How the binding models are fitted is explained in Which binding model should you use to fit FIDA affinity data?, and the Fidabio tech note on kinetics goes into more detail.
How do you measure how long a multivalent complex takes to form?
Because the run pressure sets how long the partners spend together before detection, FIDA allows you to measure the same interaction at several complex lifetimes and so separate the first binding event from slower, avidity-driven complex formation. A complex that keeps growing with time, or an apparent affinity that changes with the time allowed, points to multivalent binding rather than a simple 1:1 interaction.
For example, FIDA measured β-lactoglobulin with polyclonal anti-β-lactoglobulin IgG at complex lifetimes of 1.2, 1.8 and 7.7 minutes, giving KD values of 13, 36 and 51 nM, with the complex growing to 9.23 nm and aggregation increasing with both antibody concentration and time (Stender & Sahni Ray, 2023). For adalimumab with TNF-α, complexes measured within 1.8 minutes of mixing reached approximately 8.0 nm and no higher-order complexes formed, whereas pre-incubated samples formed avidity-stabilised complexes of up to 10 nm, which dissociate slowly (Pedersen et al., 2021). Affinity and avidity for antibodies are covered in how to measure antibody binding affinity in solution.
Frequently asked questions
What are binding kinetics?
Binding kinetics describe the rate at which two molecules, such as a drug and its target, associate and dissociate. Equilibrium binding tells you how much binding occurs; kinetics tells you how fast binding and unbinding happen. Fast-on, slow-off drugs can bind quickly and remain bound longer, improving efficacy.
What are kon and koff?
The association rate constant kon describes how quickly the complex forms (units M-1 s-1), and the dissociation rate constant koff how quickly it falls apart (units s-1). Their ratio is the equilibrium dissociation constant: KD = koff/kon.
Can FIDA measure kinetics?
Yes, in solution, from seconds to hours. For example, FIDA determined a kon of 2.19 × 106 M-1s-1 and a koff of 0.0022 s-1 for β2-microglobulin binding its antibody, in agreement with SPR (Applying FIDA for in-solution binding kinetics).
Can binding kinetics be measured without immobilisation?
Yes. FIDA measures kon and koff with both partners free in solution, so there is no sensor surface, no regeneration step and no immobilisation chemistry. For example, FIDA determined rate constants for protein–protein and protein–small molecule interactions in solution, in agreement with SPR values (Immobilisation-free in-solution kinetics).
Can binding kinetics be measured in serum?
Yes. Because nothing is immobilised and the sample is not separated, kinetics can be measured directly in serum. For example, FIDA determined a kon of 1.9 × 106 M-1s-1 and a koff of 0.04 s-1 for interleukin-2 binding IL-2Rα in 90% human serum (Willmer et al., 2025).
Can the kinetics of a small molecule be measured if the target's size does not change?
Yes. The interaction is then read from the fluorescence spectrum rather than the size. For example, FIDA determined a kon of 1.8 × 105 M-1s-1 and a koff of 0.07 s-1 for carbonic anhydrase II with furosemide, label-free from tryptophan fluorescence (Willmer et al., 2025). Small molecule assays are covered in how to measure small molecule binding in solution.
How do FIDA rate constants compare with SPR?
They agree closely. For example, for β2-microglobulin binding its antibody FIDA gave a koff of 0.0022 s-1 against 0.0026 s-1 by SPR, and for carbonic anhydrase with acetazolamide a KD of 22.1 nM against 19.0 nM by SPR (Applying FIDA for in-solution binding kinetics). Why methods can disagree is covered in why binding affinity data is unreliable.
How much sample does a kinetics experiment need?
Each measurement consumes nanolitre volumes, so a full kinetic characterisation runs on microlitres of material. For example, FIDA measured kinetics for protein–protein and protein–small molecule pairs from nanolitre to nanogram amounts of sample (Willmer et al., 2025).
References
Pedersen, M. E., Haegebaert, R. M. S., Østergaard, J., & Jensen, H. (2021). Size-based characterization of adalimumab and TNF-α interactions using Flow Induced Dispersion Analysis: assessment of avidity-stabilized multiple bound species. Scientific Reports. https://doi.org/10.1038/s41598-021-84113-z
Stender, E. G. P. V., & Sahni Ray, K. (2023). Characterising multivalent complex formation using controlled complex lifetime in Fida 1 [Poster]. fidabio.com
Willmer, P., Stender, E. G. P., Sahni Ray, K., Hundahl, A. C., Marie, R., & Jensen, H. (2025). In-solution characterization of biomolecular interaction kinetics under native conditions. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.5c02164
Related resources
New to in-solution kinetics? The In-solution Kinetics Ebook is a guide to measuring affinity and on and off rates without immobilisation, and the In-solution Kinetics Webinar shows how Capmix controls the reaction time, with results validated against SPR.
Want to see the data? The technical note Immobilisation Free In-solution Kinetics Using Flow Induced Dispersion Analysis (FIDA) compares kon, koff and KD with SPR, and Applying FIDA for in-solution binding kinetics shows the method in use. Already running SPR? A powerful duo in molecular characterization: FIDA System + SPR Biacore System shows how the two work together.
Curious what users think? Read why one researcher says in-solution kinetics changed his workflow in User's opinion on FIDA kinetics, and how FIDA gets around the limits of a sensor surface in How to overcome the limitations of Surface-Based Kinetic assessments?
Want to know if in-solution kinetics fits your project? Book a discovery call. Already a FIDA user? The full Kinetics courses are waiting for you on the Fidabio Learning Hub.
