Combined thioflavin T-Congo red fluorescence assay for amyloid fibril detection
Mykhailo Girych1, Galyna Gorbenko, Ivan Maliyov
1Department 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.
Methods and Applications in Fluorescence
|March 30, 2017
Summary
This study introduces a combined thioflavin T (ThT) and Congo red (CR) fluorescence assay for more reliable detection of amyloid fibrils. The novel method enhances accuracy by integrating the strengths of both traditional amyloid detection dyes.
Area of Science:
- Biochemistry
- Biophysics
- Analytical Chemistry
Background:
- Amyloid fibrils are pathogenic protein aggregates implicated in various diseases.
- Traditional detection methods like thioflavin T (ThT) and Congo red (CR) assays have limitations and can yield false results individually.
- Combining ThT fluorescence and CR absorbance/fluorescence offers potential for improved amyloid detection and characterization.
Purpose of the Study:
- To develop and validate a novel, combined fluorescence assay using ThT and CR for enhanced amyloid fibril identification.
- To investigate the spectral behavior of ThT and CR upon binding to different amyloid fibrils.
- To establish reliable criteria for distinguishing between fibrillar and monomeric protein states using a dual-dye approach.
Main Methods:
- Simultaneous application of thioflavin T (ThT) and Congo red (CR) dyes.
- Analysis of fluorescence spectral changes of ThT and CR in the presence of amyloid fibrils from lysozyme, insulin, and apolipoprotein A-I.
- Monitoring fluorescence intensity changes and spectral shifts upon dye-protein interactions.
Main Results:
- A significant increase in ThT fluorescence intensity was observed upon binding to amyloid fibrils.
- Addition of CR caused a sharp decrease in ThT fluorescence.
- CR excitation spectra showed a maximum at 535-540 nm, and CR fluorescence intensity increased at 610 nm.
- The combined assay demonstrated distinct spectral signatures for fibrillar versus monomeric protein states.
Conclusions:
- The novel combined ThT-CR fluorescence assay significantly enhances the reliability of amyloid detection compared to individual assays.
- This integrated approach provides new avenues for the structural characterization of amyloid fibrils.
- The developed assay offers a more robust tool for routine screening and research involving amyloidogenic proteins.
