What is an efficient method for screening binding and characterising molecules?
Flow Induced Dispersion Analysis (FIDA) is an efficient, in-solution method for screening molecular binding and characterising molecules, because a single run yields eleven readouts from one nanolitre-scale sample under native conditions. Rather than running a separate assay for affinity, another for size and another for quality, FIDA returns binding, size and sample quality together from the same measurement, which is what makes it efficient for both screening interactions and characterising a molecule.
What does FIDA measure?
FIDA takes three measurements from the fluorescence signal in each run, and the eleven readouts are derived from them. The first is molecular size, the hydrodynamic radius, from Taylor dispersion and the Stokes-Einstein relation, measured absolutely and without calibration (see what hydrodynamic radius is). The second is Lambda Dynamics, the ratio of fluorescence at two wavelength regions, which detects a shift in emission wavelength when binding changes the environment of the fluorophore, and is well suited to protein-small molecule interactions. The third is the Binding-Related Intensity Change, the change in fluorescence intensity on binding or a conformational change, used when the size or wavelength change is small. Every readout below comes from these three.
What can you screen and characterise in one run?
Binding. FIDA screens binding affinity in free solution as the dissociation constant KD (see what KD is), across protein-protein, protein-small molecule, antibody-antigen and oligomerisation interactions, and it measures binding kinetics in solution without immobilising a partner on a surface, so the on and off rates reflect the free complex. See the affinity readout.
Size and structure. FIDA characterises absolute molecular size in solution, reading folding, oligomerisation and complex formation as a change in size; it reports the polydispersity index, the spread of sizes that shows whether a sample is uniform or mixed; and through the PDB correlator it compares the measured size with the size expected from a crystal or AlphaFold structure. See the molecular size readout.
Sample quality. From the same run FIDA reports the checks that decide whether data can be trusted: aggregation, so unstable samples are caught during measurement; stickiness, the non-specific adsorption that skews results; sample viscosity, measured and compensated on every run so size is not distorted; absolute concentration; sample loss; and labelling quality, the amount of free label in a labelled sample.
What are the FIDA readouts?
In full, the eleven FIDA readouts derived from those three measurements are molecular size, binding affinity, binding kinetics, viscosity, aggregation, stickiness, quantification, sample loss, polydispersity index, labelling quality and the PDB correlator. They are shown together on the FIDA readouts product page.
Can you screen and characterise in one run?
Yes, and it is an efficient way of working especially in early stages. Getting binding, size and quality from a single in-solution assay reduces complexity, saves precious sample material and speeds up decisions, because one FIDA run replaces several separate techniques and every result describes the same sample under the same conditions. The measurement is absolute and needs no calibration, which is what makes FIDA an efficient way to screen interactions and characterise molecules with confidence.
| # | Readout | What it is | Why it matters | Learn more |
|---|---|---|---|---|
| 01 | Molecular size | The absolute hydrodynamic radius (Rh) of a molecule in solution. Unlike calculated molecular weight, it reflects how the molecule behaves in its native environment. | Precise study of conformational changes, oligomerization and protein-ligand interactions. | Read more → |
| 02 | Binding affinity | Strength of the interaction between two molecules at equilibrium, expressed as the dissociation constant (KD), the balance between association and dissociation rates. | Low KD indicates strong binding; high KD indicates weaker interactions. | Read more → |
| 03 | Binding kinetics | Rates at which molecules associate (Kon) and dissociate (Koff). | Mechanistic insight into molecular recognition and complex stability. To our knowledge, FIDA is the only technology that measures kinetics in solution, without surface immobilization. | Read more → |
| 04 | Sample viscosity | A fluid's internal resistance to flow. In biological samples, it reflects molecular interactions, crowding and the presence of large biomolecules. | Affects diffusion, mobility and binding kinetics. Key for formulation, protein stability and excipient studies, tracked directly in small volumes for research and QC. | Read more → |
| 05 | Aggregation | Association of individual molecules into larger assemblies, from small oligomers to insoluble fibrils. Reversible or irreversible; often driven by hydrophobic interactions, misfolding or stress. | Directly impacts protein stability, therapeutic safety and disease mechanisms. Study protein stability, amyloid fibrils and biomolecular condensates. | Read more → |
| 06 | Sample stickiness | Nonspecific attractive interactions between biomolecules, or between biomolecules and surfaces, from electrostatics, hydrophobic patches or exposed domains. | Affects solubility, stability and assay reliability. Quantified in solution to flag problematic formulations and unstable proteins early. | Read more → |
| 07 | Quantification | Absolute concentration of a molecule in solution, derived from physical parameters such as diffusion and hydrodynamic radius. | No calibration curves required, unlike relative methods. | Read more → |
| 08 | Sample loss | Material that is degraded, aggregated or otherwise unavailable during measurement. | Reported automatically for every assay, an extra layer of experimental control and insight into sample integrity. | Read more → |
| 09 | Polydispersity index (PDI) | The width of a sample's molecular size distribution. | Shows whether a sample is uniform or contains size variants such as aggregates or oligomers. | Read more → |
| 10 | Labelling quality (free label) | Direct detection of free label in the sample that has not been conjugated to the protein. | Free label signals suboptimal labelling that can compromise accuracy. Reported automatically to confirm sample integrity before further analysis. | Read more → |
| 11 | PDB correlator | Links measured hydrodynamic radius to structural models in the Protein Data Bank by comparing measured Rh with values derived from atomic coordinates. | Validates structural states, detects conformational changes and confirms folding models. | Read more → |
Common questions
What is an efficient method for screening binding affinity?
FIDA screens binding affinity by measuring the dissociation constant KD in free solution, on a nanolitre-scale sample, from the same run that reports molecular size and sample quality, so a screen and a characterisation happen together.
How can you characterise a molecule and screen its binding at the same time?
FIDA does both from one in-solution run, reporting binding, absolute size and sample quality together, under native conditions and without immobilising or separating the sample.
Why does FIDA need only one run for several parameters?
Because all eleven readouts are derived from the same three measurements, size, Lambda Dynamics and the intensity change, so binding, size and quality all come from a single measurement rather than from separate assays.
Related resources
The application note on selective quantification of IgG directly in fermentation media is a worked example of measuring in a complex matrix, and the white paper Label-free biophysical assays using nanoliter sample volumes shows several of these parameters measured together, label-free, from nanolitre samples.
Read next: the full FIDA readouts list, What is FIDA? and What is hydrodynamic radius?
