Flow-induced dispersion analysis
Flow-induced dispersion analysis (FIDA) is an immobilization-free technology used for characterization and quantification of biomolecular interaction and protein concentration under native conditions.{{Cite book|title=Clinical Applications of Capillary Electrophoresis: Methods and Protocols|last1=Morten E|first1=Pedersen|last2=Østergaard|first2=Jesper|last3=Jensen|first3=Henrik|date=2019|publisher=Springer New York|isbn=9781493992126|editor-last=Phillips|editor-first=Terry M.|series=Methods in Molecular Biology|volume=1972|location=New York, NY|pages=109–123|chapter=Flow-Induced Dispersion Analysis (FIDA) for Protein Quantification and Characterization|doi=10.1007/978-1-4939-9213-3|s2cid=23665803}}{{Cite journal|last1=Pedersen|first1=Morten E.|last2=Gad|first2=Sarah I.|last3=Østergaard|first3=Jesper|last4=Jensen|first4=Henrik|date=2019-04-05|title=Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach|journal=Analytical Chemistry|volume=91|issue=8|pages=4975–4979|language=en|doi=10.1021/acs.analchem.9b00537|pmid=30916933|s2cid=85544070 |issn=0003-2700}} In the FIDA assay, the size of a ligand (indicator) with affinity to the target analyte is measured. When the indicator interacts with the analyte the apparent size increases and this change in size can be used to determine the analyte concentration and interaction.{{Cite journal|last1=Jensen|first1=Henrik|last2=Østergaard|first2=Jesper|date=2010-03-31|title=Flow Induced Dispersion Analysis Quantifies Noncovalent Interactions in Nanoliter Samples|journal=Journal of the American Chemical Society|volume=132|issue=12|pages=4070–4071|doi=10.1021/ja100484d|pmid=20201527|issn=0002-7863}} Additionally, the hydrodynamic radius of the analyte-indicator complex is obtained. A FIDA assay is typically completed in minutes and only requires a modest sample consumption of a few μL.
Applications
- Quantification of analytes (e.g. proteins, peptides, DNA, nanoparticles{{Cite journal|last1=Cholak|first1=Ersoy|last2=Bugge|first2=Katrine|last3=Khondker|first3=Adree|last4=Gauger|first4=Kimmie|last5=Pedraz-Cuesta|first5=Elena|last6=Pedersen|first6=Morten Enghave|last7=Bucciarelli|first7=Saskia|last8=Vestergaard|first8=Bente|last9=Pedersen|first9=Stine F.|last10=Rheinstädter|first10=Maikel C.|last11=Langkilde|first11=Annette Eva|date=2020|title=Avidity within the N-terminal anchor drives α-synuclein membrane interaction and insertion|journal=The FASEB Journal|language=en|volume=34|issue=6|pages=7462–7482|doi=10.1096/fj.202000107R|pmid=32277854|s2cid=215742011|issn=1530-6860|doi-access=free}}) in complex solutions (e.g. plasma and fermentation broth {{Cite journal|last1=Poulsen|first1=Nicklas N.|last2=Pedersen|first2=Morten E.|last3=Østergaard|first3=Jesper|last4=Petersen|first4=Nickolaj J.|last5=Nielsen|first5=Christoffer T.|last6=Heegaard|first6=Niels H. H.|last7=Jensen|first7=Henrik|date=2016-09-20|title=Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification|journal=Analytical Chemistry|volume=88|issue=18|pages=9056–9061|doi=10.1021/acs.analchem.6b01741|pmid=27571264|issn=0003-2700}}{{Cite journal|last1=Pedersen|first1=Morten E.|last2=Østergaard|first2=Jesper|last3=Jensen|first3=Henrik|date=2020-04-28|title=In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis|journal=ACS Omega|volume=5|issue=18|pages=10519–10524|doi=10.1021/acsomega.0c00791|pmid=32426609|pmc=7227040|issn=2470-1343|doi-access=free}})
- Determination of
- affinity constants
- binding kinetics
- molecular size (hydrodynamic radius)
- oligomeric state{{Cite journal|last1=Pedersen|first1=Morten E.|last2=Haegebaert|first2=Ragna M. S.|last3=Østergaard|first3=Jesper|last4=Jensen|first4=Henrik|date=2021-02-26|title=Size-based characterization of adalimumab and TNF-α interactions using flow induced dispersion analysis: assessment of avidity-stabilized multiple bound species|journal=Scientific Reports|language=en|volume=11|issue=1|pages=4754|doi=10.1038/s41598-021-84113-z|pmid=33637878|pmc=7910425|issn=2045-2322|doi-access=free|bibcode=2021NatSR..11.4754P }}
- diffusion coefficient
Principle
The FIDA principle is based on measuring the change in the apparent size (diffusivity) of a selective indicator interacting with the analyte molecule. The apparent indicator size is measured by Taylor dispersion analysis in a capillary under hydrodynamic flow.{{Cite journal|last1=Taylor|first1=Sir Geoffrey|last2=S|first2=F. R.|date=1953-08-25|title=Dispersion of soluble matter in solvent flowing slowly through a tube|journal=Proc. R. Soc. Lond. A|language=en|volume=219|issue=1137|pages=186–203|doi=10.1098/rspa.1953.0139|bibcode=1953RSPSA.219..186T |s2cid=97372019|issn=0080-4630}}