How do you measure amyloid fibril length and binding in solution?
Amyloid fibril length and binding to fibrils can be measured in solution with Flow Induced Dispersion Analysis (FIDA), developed by Fida Biosystems (Fidabio). FIDA measures the size of a small fluorescent probe, its hydrodynamic radius (Rh): when the probe binds a fibril, it takes on the size of the fibril, and because fibrils are built from protein layers with a known spacing, that size can be converted into fibril length. The same size readout tells monomers, oligomers and fibrils apart and measures how tightly antibodies, nanobodies and chaperones bind each of them.
As an example, the fluorescent peptide FibrilPaint1 measured 1.7 nm on its own, did not change size with Tau monomer, and grew to 45 nm when it bound Tau fibrils, so FIDA reported the fibrils and not the monomer. The method sized Tau fibrils from 4 to 1,100 layers long, in buffer, in 50% cell lysate and in 50% human serum (Aragonès Pedrola et al., 2025).
What can FIDA measure in amyloid fibril research?
FIDA measures the presence, size, length, binding, growth and stability of amyloid fibrils from the same size readout, in solution and with nothing immobilised.
| What you want to know | What FIDA measures |
|---|---|
| Whether fibrils are present | FIDA detects fibrils with a fluorescent probe that binds fibrils and not monomers, so a size increase shows that fibrils are there. |
| How large the species are | FIDA measures the absolute size (hydrodynamic radius) of monomers, small oligomers and fibrils in the same sample, without a calibration standard. |
| How long the fibrils are | FIDA converts the measured fibril size into fibril length, using the known spacing between the protein layers of a fibril. |
| How tightly a binder binds | FIDA measures the binding strength (KD) of antibodies, nanobodies, peptides and chaperones to monomers, oligomers and fibrils. |
| How fibrils grow over time | FIDA measures fibril size at successive time points during aggregation, so growth is followed as an absolute size rather than a relative signal. |
| How stable the fibrils are | FIDA measures the free energy of fibril stability by dissolving fibrils in urea and measuring how much monomer is released. |
| How much sample and which buffer | Each FIDA measurement uses about 40 nL of sample, in any buffer including cell lysate, and runs automatically from 96-well plates or vials. |
Prof. Alexander Buell from the Technical University of Denmark (DTU) and colleagues, who developed the FIDA method for fibril stability, describe it as “rapid, fully automated, amenable to high throughput, label-free” and say it “uses minute amounts of sample” (Farzadfard et al., 2024). How fibril stability is measured is covered in how to measure protein stability in solution.
What makes amyloid fibrils hard to measure, and how does FIDA handle them?
| Challenge with amyloid samples | How FIDA handles it |
|---|---|
| Monomers, oligomers and fibrils are mixed in the same sample | FIDA measures a different size for each species, and a fibril-specific probe binds only the fibrils. |
| Fibrils are long and vary in length | FIDA measures species up to about 500 nm in hydrodynamic radius, and the measured size can be converted into fibril length. |
| Fibrils are scarce in patient fluids | FIDA follows only the fluorescent probe, so fibrils can be measured in serum, blood and cerebrospinal fluid without isolating them. |
| Fibrils grow and change over time | FIDA measures the same sample repeatedly in minutes, so fibril growth can be followed as a curve over time. |
| Binders must be selective for oligomers or fibrils | FIDA measures binding to monomers and to oligomers or fibrils separately, so the selectivity of an antibody or chaperone is measured directly. |
| Large aggregates can distort the result | FIDA counts large aggregates as separate spikes in every run, so they are reported rather than hidden in the average size. |
How does FIDA measure the length of amyloid fibrils?
FIDA measures fibril length by measuring the hydrodynamic radius of a fluorescent probe bound to the fibril and converting that size into a length, using the known spacing of 4.7 Å between the protein layers of an amyloid fibril. The conversion uses the size the Fidabio software predicts from the fibril's structure (structure-based size prediction).
Example: Prof. Stefan Rüdiger from Utrecht University used FibrilPaint and FIDA to measure the length of Tau, amyloid-β, α-synuclein and huntingtin fibrils. As a cross-check, FIDA measured a fibril length of 425 nm against an average of 430 nm by electron microscopy (Measuring amyloid fibril size and length with FIDA: a conversation with Prof. Stefan Rüdiger).
Can FIDA measure amyloid fibrils in serum, blood or cerebrospinal fluid?
Yes. FIDA measures amyloid fibrils in serum, blood and cerebrospinal fluid, because it only follows the fluorescent fibril-binding probe, and the other proteins in the fluid do not change its size. How FIDA works in crude samples is covered in how to characterise proteins in complex biological matrices.
Example: Dr. Françoise Dekker, co-founder of NeurotidalDx, measures amyloid fibril length directly in patient blood and cerebrospinal fluid as a route to the early detection of Parkinson's disease. The samples are measured essentially as they are, using about 10 µL of the 100 µL requested (how a start-up is turning fibril length into a biomarker). In the same research line, FIDA measured patient-derived fibrils from Alzheimer's disease, frontotemporal dementia and corticobasal degeneration, tagged with fluorescent ubiquitin, at 130 nm, 70 nm and 80 nm (Aragonès Pedrola et al., 2024).
How does FIDA tell oligomers apart from monomers and fibrils?
FIDA tells oligomers apart from monomers and fibrils by their size: each species has its own hydrodynamic radius, measured in solution with nothing immobilised, so oligomers are measured without being stuck to a surface.
Example: FIDA measured cytotoxic α-synuclein oligomers at a hydrodynamic radius of 10 nm, against 3.0 nm for the monomer, and used this size to show that several compounds from a drug-repurposing screen blocked oligomer binding to liposomes without breaking the oligomers apart (Somavarapu et al., 2023).
How do you measure antibody or nanobody binding to oligomers and fibrils?
FIDA measures antibody or nanobody binding to oligomers and fibrils by following the size of the labelled binder or the labelled amyloid species as the partner is titrated in, which gives the binding strength (KD). Running the same titration with monomer shows whether the binder is selective for the disease-relevant species.
Example: FIDA measured monoclonal antibodies against α-synuclein oligomers with KD values of about 3–5 nM for the best four, and one antibody bound the oligomer 559-fold more tightly than the monomer (Nielsen et al., 2024). Two nanobodies raised against the same oligomer bound it with KD values of 31 nM and 41 nM and did not bind the monomer at all (Nielsen et al., 2025).
Can FIDA measure chaperone binding to amyloid species?
Yes. FIDA measures chaperone binding to monomers, oligomers and fibrils separately, so it shows which species a chaperone recognises and how tightly.
Example: FIDA showed that the chaperone domain Bri2 BRICHOS did not bind α-synuclein monomers but bound α-synuclein oligomers with a KD of 78 nM, and also bound fibrils (Adam et al., 2023).
Can FIDA follow fibril growth and early assembly over time?
Yes. FIDA follows fibril growth by measuring the size of fibril-bound probe at successive time points, from the earliest assemblies a few nanometres in size to long fibrils (Aragonès Pedrola et al., 2025), and it reports early oligomers as a separate size population.
Example: During Tau aggregation, the size of fibril-bound FibrilPaint grew from 1.7 nm to 5 nm in 2 hours and reached 45 nm at about 8 hours. The Thioflavin T signal had already levelled off by about 4 hours, while FIDA showed the fibrils still growing from 10 to 45 nm (Aragonès Pedrola et al., 2025).
In another study, FIDA measured the C-terminal domain of TDP-43, the protein implicated in ALS and frontotemporal dementia, directly in yeast cell lysate. Below a threshold concentration it was monomeric, above it a mix of monomer and oligomers appeared, and the oligomers survived a 250-fold dilution (Wu et al., 2026).
Frequently asked questions
Can you measure amyloid fibril length in solution?
Yes. FIDA measures amyloid fibril length in solution from the hydrodynamic radius of a fluorescent probe bound to the fibrils. For example, FIDA with the peptide FibrilPaint1 sized Tau fibrils from 4 to 1,100 layers long, in buffer, cell lysate and human serum (Aragonès Pedrola et al., 2025).
Can FIDA tell α-synuclein oligomers from monomers?
Yes. FIDA tells oligomers from monomers by their size in solution. For example, α-synuclein oligomers measured 10 nm and the monomer 3.0 nm in hydrodynamic radius (Somavarapu et al., 2023).
How do you check that an antibody binds oligomers and not monomers?
You check that an antibody binds oligomers and not monomers by measuring its binding to each species separately with FIDA. For example, two nanobodies bound α-synuclein oligomers with KD values of 31 nM and 41 nM, while the monomer caused no change in size (Nielsen et al., 2025).
Can amyloid fibrils be measured in cerebrospinal fluid or blood?
Yes. FIDA measures amyloid fibrils in cerebrospinal fluid and blood with a fluorescent fibril-binding probe, without isolating the fibrils. For example, NeurotidalDx measures fibril length in patient samples from about 10 µL each (how a start-up is turning fibril length into a biomarker).
Can FIDA follow amyloid aggregation kinetics?
Yes. FIDA follows amyloid aggregation by measuring the absolute size of fibril-bound probe over time. For example, FIDA showed Tau fibrils still growing from 10 to 45 nm after the Thioflavin T signal had levelled off (Aragonès Pedrola et al., 2025).
How large an amyloid species can FIDA measure?
FIDA measures species up to about 500 nm in hydrodynamic radius, or 1 µm in diameter (size range and precision). For example, a fibril length of 425 nm measured with FIDA matched the 430 nm average from electron microscopy (Prof. Stefan Rüdiger, Utrecht University).
References
Adam, L., Kumar, R., Arroyo-Garcia, L. E., Molenkamp, W. H., Nowak, J. S., Klute, H., Farzadfard, A., Alkenayeh, R., Nielsen, J., Biverstål, H., Otzen, D. E., Johansson, J., & Abelein, A. (2023). Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS. Protein Science. https://doi.org/10.1002/pro.5091
Aragonès Pedrola, J., Dekker, F. A., Garfagnini, T., Mayer, G., Koopman, M. B., Bergmeijer, M., Förster, F., Hoozemans, J. J. M., Jensen, H., Friedler, A., & Rüdiger, S. G. D. (2025). FibrilPaint to determine the length of Tau amyloids in fluids. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.2502847122
Aragonès Pedrola, J., Dekker, F. A., Rüdiger, S. G. D., et al. (2024). Fibril Paint targets amyloid fibrils for ubiquitination. bioRxiv. https://doi.org/10.1101/2024.08.27.609886
Farzadfard, A., Kunka, A., Mason, T. O., Larsen, J. A., Norrild, R. K., Torrescasana Dominguez, E., Ray, S., & Buell, A. K. (2024). Thermodynamic characterization of amyloid polymorphism by microfluidic transient incomplete separation. Chemical Science. https://doi.org/10.1039/D3SC05371G
Fidabio [Wasilczyk, M.]. (2026). Measuring amyloid fibril size and length with FIDA: A conversation with Prof. Stefan Rüdiger [Blog post]. fidabio.com
Fidabio [Wasilczyk, M., & Dekker, F.]. (2026). How can you measure amyloid fibril length directly in cerebrospinal fluid or blood? [Blog post]. fidabio.com
Nielsen, J., et al. (2024). Molecular properties and diagnostic potential of monoclonal antibodies targeting cytotoxic α-synuclein oligomers. npj Parkinson's Disease. https://doi.org/10.1038/s41531-024-00747-6
Nielsen, J., Pedersen, J. N., Kleijwegt, G., Nowak, J. S., Otzen, D. E., et al. (2025). Nanobodies raised against the cytotoxic α-synuclein oligomer are oligomer-specific and promote its cellular uptake. npj Biosensing. https://www.nature.com/articles/s44328-025-00042-1
Somavarapu, A. K., Kleijwegt, G., Nagaraj, M., Alam, P., Nielsen, J., & Otzen, D. E. (2023). Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions. Chemical Science. https://doi.org/10.1039/D2SC05534A
Wu, J., Venkatesan, S., Jensen, J., et al. (2026). Intracellular TDP-43 amyloid nucleates from arrested nascent condensates. bioRxiv. https://doi.org/10.64898/2026.02.10.705006
Related resources
Read next: How to measure protein stability in solution? for fibril stability, What is protein aggregation? and What size range can FIDA measure, and how precise is it? The poster Peptide-based strategy to target Tau fibrils shows fibril-binding peptides measured with FIDA.
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