Photophysical study of Thioflavin T as fluorescence marker of amyloid fibrils

Abstract

Thioflavin T is a highly sensitive fluorescent marker of amyloid fibrils that has been widely used for in vitro biomedical assays. However, neither its complex photophysical behavior nor its binding mode to amyloid fibrils are still well understood. We present a detailed analysis of the photophysical properties of Thioflavin T in various media, including solvents and solvent mixtures of different viscosities as well as fibrillar and globular proteins. We propose a model that explains the strong wavelength dependency of the Thioflavin T fluorescence and the large fluorescence enhancement in certain environments. We determine the binding affinities and the fluorescence properties of Thioflavin T bound to amyloid-β (1-42) fibrils and to bovine serum albumin and discuss the sensitivity and the specificity of this probe to amyloid aggregates. These results allow us to assess the suitability of Thioflavin T for quantitative determinations in biomedical studies.

Biophysical insight into the anti-amyloidogenic behavior of taurine

“…The concentration of ThT was measured using molar extinction coefficient ε M = 36,000 M −1 cm −1 at 412 nm [25]. The protein samples (10.0 M), in the absence as well as presence of varying concentration of Taurine, were incubated for 120 h at 65 • .…”

Fluorescent dyes in the context of DNA‐binding: The case of Thioflavin T

“…In that case, the photoexcited state cannot relax into the twisted one and a fluorescence light‐up is observed. Due to this characteristic, ThT has become a “gold standard” for visualizing and quantifying the presence of amyloid fibrils and it has been successfully used as a G‐quadruplex specific fluorescent sensor . Recent studies have also investigated the affinity of ThT binding to single and double stranded DNA …”

Ultrafast fluorescence spectroscopy reveals a dominant weakly-emissive population of fibril bound thioflavin-T

“…It should be noted that the similar multiple binding mode for ThT has also been observed for Aβ42 fibrils where most of the ThT were bound to weak binding sites and contributed marginally to the observed fluorescence enhancement. 19 Effect of ionic strength on the probe-macromolecular binding is usually employed to verify if there is a significant electrostatic interaction between the probe and the macromolecule. Thus, to investigate if charged interactions are involved in these two widely different binding modes, we performed the transient decay trace measurements, for ThT in fibril, in presence of salt (sodium chloride).…”