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Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection

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journal contribution
posted on 27.11.2008, 00:00 authored by Vito Foderà, Minna Groenning, Valeria Vetri, Fabio Librizzi, Salvatore Spagnolo, Claus Cornett, Lars Olsen, Marco van de Weert, Maurizio Leone
The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV−vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the efficiency of the ThT assay for quantitative fibril evaluation at basic pH.