Fluorescent biosensor for the detection of hyaluronidase: intensity-based ratiometric sensing and fluorescence lifetime-based sensing using a long lifetime azadioxatriangulenium (ADOTA) fluorophore

Abstract

In this report, we have designed a rapid and sensitive, intensity-based ratiometric sensing as well as lifetime-based sensing probe for the detection of hyaluronidase activity. Hyaluronidase expression is known to be upregulated in various pathological conditions. We have developed a fluorescent probe by heavy labeling of hyaluronic acid with a new orange/red-emitting organic azadioxatriangulenium (ADOTA) fluorophore, which exhibits a long fluorescence lifetime (∼20 ns). The ADOTA fluorophore in water has a peak fluorescence lifetime of ∼20 ns and emission spectra centered at 560 nm. The heavily ADOTA-labeled hyaluronic acid (HA-ADOTA) shows a red shift in the peak emission wavelength (605 nm), a weak fluorescence signal, and a shorter fluorescence lifetime (∼4 ns) due to efficient self-quenching and formation of aggregates. In the presence of hyaluronidase, the brightness and fluorescence lifetime of the sample increase with a blue shift in the peak emission to its original wavelength at 560 nm. The ratio of the fluorescence intensity of the HA-ADOTA probe at 560 and 605 nm can be used as the sensing method for the detection of hyaluronidase. The cleavage of the hyaluronic acid macromolecule reduces the energy migration between ADOTA molecules, as well as the degree of self-quenching and aggregation. This probe can be efficiently used for both intensity-based ratiometric sensing as well as fluorescence lifetime-based sensing of hyaluronidase. The proposed method makes it a rapid and sensitive assay, useful for analyzing levels of hyaluronidase in relevant clinical samples like urine or plasma. Graphical Abstract Scheme showing cleavage of HA-ADOTA probe by hyaluronidase and the change in the emission spectrum of HA-ADOTA probe before and after cleavage by hyaluronidase.

Design, synthesis, and time-gated cell imaging of carbon-bridged triangulenium dyes with long fluorescence lifetime and red emission

“…Functional derivatives 9 and 10 can easily be converted into maleimides and thus allow for efficient bioconjugation of the CAOTA + and CDATA + dyes to cystines. 40 Reacting 6 with an amino acid, instead of a diamine, would allow for synthesis of activated ester ( e.g. NHS) derivatives of CAOTA ( 7 ) and CDATA ( 8 ) targeting free amines, as previously reported for aza/oxa-triangulenium dyes.…”

“… 34 However, the combination of long fluorescence lifetime, high quantum yields, and chemical stability makes them unique probes in FLIM and time-gated fluorescence imaging, 23 , 24 , 35 , 36 and for fluorescence lifetime and polarization assays. 36 – 40 A redshifted congener of DAOTA + is the dimethoxyquinacridinium (DMQA + ) system ( Chart 1 ). This [4]-helicenium precursor of DAOTA + , 32 , 41 displays absorption and emission in the attractive range from 600 nm–700 nm.…”

“…In the search for new organic fluorophores with long fluorescence lifetimes and emission beyond 600 nm, we turn our attention to modification of the aza/oxa triangulenium dyes, with the expectation that the planarity and rigidity of these systems will keep the rate of non-radiative deactivation low and thus allow long fluorescence lifetime and high quantum yields. In this regard, the DAOTA + dye ( Chart 1 ) is particularly interesting as it display higher chemical stability 32 and less susceptibility to quenching 40 , 52 compared to ADOTA + . In the present work, structural variations are introduced in the DAOTA + chromophore motif, which redshift the absorption and fluorescence spectra as compared to those of DAOTA + and with only moderate increase in the rate of non-radiative deactivation, thus conserving the desired long fluorescence lifetime.…”

Responsive fluorophore aggregation provides spectral contrast for fluorescence lifetime imaging

“…Lifetime can be altered chemically, such as by coordination with metal ions 3, 4 or through acid-base equilibrium with environmental pH 5, 6 ; it is also exquisitely sensitive to small changes in environmental factors like temperature 7 and solvent viscosity 8, 9 . The lifetime of a fluorophore is influenced by its capacity to undergo non-radiative emission 10 , which is altered by immobilizing factors such as tethering to macromolecules 11 , entrapment in aggregated proteins 12 , or intercalation into DNA 13 . In these examples, the lifetime of the free fluorophore is altered when exposed to a macromolecular aggregate.…”

“…Aside from FITC, other small molecule fluorophores have reported faster lifetimes when associated with large polymers, aggregates, or biomacromolecules, than when measured as smaller entities 11, 16, 17 . While the reverse trend has also been reported 13, 18 , these findings suggest that fluorescence lifetime is sensitive to local aggregation.…”