title: Combined thioflavin T-Congo red fluorescence assay for amyloid fibril detection.
abstract: Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535-540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT-CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.
authors.affiliation: Department of Nuclear and Medical Physics, V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61022, Ukraine. Department of Neuroscience and Biomedical Engineering, School of Science and Technology, Aalto University, FI-00076, Espoo, Finland. Author to whom any correspondence should be addressed. 19/2 Tankopiya Str., ap. 47, Kharkov 61091, Ukraine.
authors.forename: Mykhailo
authors.initials: M
authors.lastname: Girych
authors.forename: Galyna
authors.initials: G
authors.lastname: Gorbenko
authors.forename: Ivan
authors.initials: I
authors.lastname: Maliyov
authors.forename: Valeriya
authors.initials: V
authors.lastname: Trusova
authors.forename: Chiharu
authors.initials: C
authors.lastname: Mizuguchi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
authors.forename: Paavo
authors.initials: P
authors.lastname: Kinnunen
mesh_terms: D000682:Amyloid; D052160:Benzothiazoles; D003224:Congo Red / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes; D007700:Kinetics; D011485:Protein Binding; D015203:Reproducibility of Results; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red