Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays.
In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.
Rantanen
Terhi
T
Department of Biotechnology, University of Turku, Tykistökatu 6A, FIN-20520 Turku, Finland. terhi.rantanen@utu.fi
Päkkilä
Henna
H
Jämsen
Laura
L
Kuningas
Katri
K
Ukonaho
Telle
T
Lövgren
Timo
T
Soukka
Tero
T
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D010799:Phycoerythrin
D005456:Fluorescent Dyes; D010799:Phycoerythrin