Nile red compensates for thioflavin T assay biased in the presence of curcumin

Abstract

Amyloid fibrillary aggregates, which are gradually formed under predisposing conditions in the human body, are the main sign of conformational diseases such as type 2 diabetes, Alzheimer's disease and Parkinson's disease. How the aggregates are formed, what characteristics they have, and how their formation can be inhibited, have been targeted in many studies thus far. In all of such studies, thioflavin T (ThT) fluorescence assay is used to qualify the amyloid aggregates and the inhibitory effect of polyphenolic small molecules. Among the proteins with the potential of the aggregation in vitro, hen egg white lysozyme (HEWL) is much used, and on the other hand, curcumin, a known polyphenol, has a potential to inhibit the aggregation process. However, whether curcumin affects the aggregation process or not, ThT fluorescence is biased in the presence of curcumin because it displays an enhanced absorption at the excitation wavelength of ThT. To address this problem and to qualify the aggregation more accurate, it is recommended to use Nile red (NR) anisotropy as an alternative fluorescence-based method which considers molecular sizes, not emissions. Since NR, as bound to amyloid fibrils, fluoresces significantly beyond the range of curcumin, NR anisotropy can further minimize the interference effect of curcumin.