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Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay
Thomas Just Sørensen1, Erling Thyrhaug, Mariusz Szabelski
1Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Nano-Science Center and Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 København Ø, Denmark.
Researchers developed a new red-emitting fluorescent label, azadioxatriangulenium (ADOTA-NHS), for enhanced biomolecule detection. This label enables sensitive fluorescence anisotropy assays for high molecular weight targets, overcoming limitations of traditional dyes.
Area of Science:
- Biochemistry
- Biophysics
- Analytical Chemistry
Background:
- Fluorescence anisotropy assays offer sensitive, ratiometric detection but are limited by fluorophore emission lifetimes matching biomolecule tumbling times.
- Existing labels often require UV/blue excitation and are sensitive to solvent effects, restricting their use with large biomolecules.
- The azadioxatriangulenium (ADOTA-NHS) motif presents a novel solution for long fluorescence lifetime, red-emitting labels.
Purpose of the Study:
- To synthesize and characterize a new long fluorescence lifetime, red-emitting fluorophore based on the azadioxatriangulenium motif.
- To evaluate the utility of ADOTA-NHS as a label for high molecular weight biomolecule assays using fluorescence anisotropy.
- To demonstrate the detection of binding events involving large biomolecules that are challenging for conventional anisotropy assays.
Main Methods:
- Synthesis and conjugation of azadioxatriangulenium NHS ester (ADOTA-NHS) to anti-rabbit Immunoglobulin G (anti-IgG).
- Measurement of correlation time and steady-state fluorescence anisotropy of labeled anti-IgG and its complex with rabbit IgG.
- Application of both steady-state and time-resolved fluorescence anisotropy techniques in solution-phase immunoassays.
- Variable temperature experiments to assess binding-induced changes in correlation time and anisotropy.
Main Results:
- ADOTA-NHS was successfully conjugated to anti-IgG, enabling fluorescence anisotropy measurements.
- The binding of rabbit IgG to ADOTA-NHS-labeled anti-IgG caused a significant increase in correlation time (>75%) and steady-state anisotropy (18%).
- The long fluorescence lifetime of ADOTA-NHS allowed for accurate anisotropy measurements of high molecular weight antibody complexes.
Conclusions:
- The azadioxatriangulenium class of dyes, exemplified by ADOTA-NHS, are suitable for fluorescence anisotropy assays with high molecular weight biomolecules.
- This new class of red-emitting fluorophores overcomes the limitations of traditional labels, expanding the scope of anisotropy-based detection.
- ADOTA-NHS provides a sensitive and robust method for detecting binding events in complex biological systems.
