Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

作者：Bogh Sidsel A^*; Bora Ilkay; Rosenberg Martin; Thyrhaug Erling; Laursen Bo W; Sorensen Thomas Just

来源：Methods and Applications in Fluorescence, 2015, 3(4): 045001.

DOI：10.1088/2050-6120/3/4/045001

• fluorescence polarisation
• fluorescence anisotropy
• time-resolved fluorescence
• fluorescent dyes
• long fluorescence lifetime dyes

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is similar to 20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r(0) = 0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (phi(fl)) of the free dyes in PBS solution are determined to be similar to 55%, which is reduced to similar to 20% in the ADOTA-BSA conjugates. Despite the reduction in ffl, a similar to 20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

• 出版日期2015-12