Terbium(III) chelate as an efficient donor for multiple-wavelength fluorescent acceptors

Area of Science:

• Biochemistry and Biophysics
• Analytical Chemistry
• Immunochemistry

Background:

• Lanthanide chelates possess versatile emission spectra, enabling their use as energy donors for various acceptor molecules.
• Fluorescence Resonance Energy Transfer (FRET) is a powerful technique for studying molecular interactions and analyte detection.

Purpose of the Study:

• To evaluate the functionality of terbium(III) chelate as an efficient energy donor for multiple acceptors emitting across the visible to far-red spectrum.
• To investigate the application of FRET using terbium(III) chelate-labeled antibodies and Alexa Fluor-conjugated 17beta-estradiol in a competitive immunoassay.

Main Methods:

• Utilized a terbium(III) chelate-labeled antibody Fab fragment as the donor.
• Employed 17beta-estradiol conjugated to Alexa Fluor 488, 555, 594, or 680 as acceptors.
• Measured sensitized acceptor emission at specific wavelengths to quantify FRET efficiency in a competitive homogeneous E2 assay.

Main Results:

• All tested Alexa Fluor dyes served as efficient acceptors, demonstrating successful FRET with the terbium(III) chelate donor.
• Alexa Fluor 680 yielded the highest signal-to-background ratio and best assay performance due to minimal donor emission in the far-red region.
• Simultaneous measurement of Alexa Fluor 488 and 680 sensitized emissions was achievable without significant spectral cross-talk.

Conclusions:

• Terbium(III) chelates are versatile donors for a range of acceptors, facilitating FRET-based assays.
• The competitive homogeneous E2 assay utilizing FRET is effective, with Alexa Fluor 680 offering optimal performance for 17beta-estradiol detection.
• Simultaneous detection capabilities using specific Alexa Fluor dyes are feasible, enhancing assay multiplexing potential.