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

Abstract

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 ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r0 = 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 (ϕfl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕfl, a ~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.

Fluorescence pH Probes Based on Photoinduced Electron Transfer Quenching of Long Fluorescence Lifetime Triangulenium Dyes

“…The fluorescence lifetimes measured at pH 2 for 3 a-3 cPF 6 ( Table 1) are significantly reduced but correlate with the quantum yields as the radiative lifetimes (τ 0 ) are comparable to those measured for similar ADOTA derivatives [16] and 3 d. Thus, the significantly reduced fluorescence lifetimes and quantum yields compared to 3 d, reveal that the phenol group quenches the ADOTA fluorescence by 50-90 % even at pH = 2. Thus, we conclude that the ADOTA chromophore is not ideal as fluorophore motif in combination with the phenol/phenolate on/off PET quenching pair, since the on-state is already significantly quenched.…”

Diazaoxatriangulenium: synthesis of reactive derivatives and conjugation to bovine serum albumin

“…The properties of 5c are compared to those of the corresponding N -(4-carboxyphenyl)-ADOTA tetrafluoroborate 4c . 46, 47 The spectra displayed in figure 1 show that DAOTA ( 5c ) has more disable absorption and emission wavelengths in the red region of the visible spectrum for measurements on biological systems as compared to ADOTA ( 4c ). 49, 53, 65 Table 1 summarises the photophysical properties of 4c and 5c in acetonitrile, dimethyl sulfoxide (DMSO), and phosphate buffered saline at pH = 7.4 (PBS).…”

“…This reaction is expected to predominately result in conjugation of the triangulenium based probe to the N -terminus of the protein. 46 The labelling protocol was optimised to give a low degree of labelling (DOL) to ensure that complications arising from multiple labels, such as energy transfer between labels, was minimal. When developing a fluorescence polarisation assay, these effects can be probed by looking for Weber’s red-edge effect.…”

“…DAOTA has r 0 = 0.38, 65 which is very close to the maximal value (0.4), while the fluorescence lifetime allows for probing biomolecules with a molecular weight up towards 1000 kDa. 46 …”

Nanoparticle metrology of silica colloids and super-resolution studies using the ADOTA fluorophore

“…In order to test the susceptibility to aggregation, photochemical robustness of the ADOTA fluorophore, and to see if it was suitable for tracking the whole sol-gel process from particle to gelation, we irradiated its acid derivative linked to dextran with laser excitation in order to populate dark states in the manner required for dSTORM measurements, and monitored the recovery of its fluorescence. The power dependence, that reflects the generation of dark states following increase in fluorescence, is shown in Figure 7 for Acid-ADOTA-Dextran (structure shown in figure 3(c)), which has also recently been used [29] to label and record the rotation of Bovine Serum Albumin using fluorescence anisotropy ( r ~ 37 ns). Acid-ADOTA-Dextran was found to demonstrate excellent recovery of its native fluorescence properties following laser irradiation and in this regard is at least comparable to other dyes that have been assessed for SMLM [30].…”