Fluorophore-gold nanoparticle FRET/plasmonic lasers with the streptavidin-biotin complex as the acceptor–donor linkage

J A Rivera,K V Desai,J G Eden

doi:10.1063/5.0068168

Abstract

Several Förster resonance energy transfer (FRET) lasers have been realized by employing the robust and versatile streptavidin-biotin (SPB) biocomplex as the acceptor–donor linkage. SPB offers a fixed acceptor–donor separation (“ruler”) of &lt;6 nm, which lies within the Förster radius for a broad range of donors and acceptors. A Cy3-SPB-Cy5 conjugate laser (where Cy3 and Cy5 are cyanine dyes) peaking at λ ∼ 708 nm has been observed, and its bandwidth and threshold pump energy (at 532 nm) have been measured to be ∼4.5 nm and 118 µJ (corresponding to a pump energy density of 179 ± 5 µJ/mm2), respectively. Depolarization of the linearly polarized pump optical field by this FRET process is found to be &lt;12%. To tether the acceptor and donor, the SPB complex requires only that either be conjugated, thereby allowing FRET processes to be examined among an extensive set of biomolecules, inorganics, and nanoantenna acceptors, for example. As a result, fluorophore-nanoparticle lasers having characteristics of both FRET lasers and plasmonic emitters have been demonstrated. Laser spectra and the phase shift induced by a 10 or 100 nm gold nanoparticle tethered to the Cy3-SPB complex suggest that both the fluorescent protein and nanoparticle are able to act as an acceptor. The brightness associated with this new class of fluorophore/nanostructure FRET lasers will broaden the scope of accessible biomedical diagnostics, including cellular imaging and the detection of DNA and proteins.

Highlights

In 1948, Förster reported the molecular energy-exchange process that has proven to be an invaluable tool in chemistry and biology for more than 70 years, known as Förster resonance energy transfer (FRET).1,2 A milestone in the development of FRET as a biochemical diagnostic was the demonstration by Stryer and Haugland3 in 1967 of a “spectroscopic ruler,” a synthesized oligomer of poly-L-proline that served to separate donor and acceptor fluorophores by 1.2–4.6 nm
Binding of Cy3 and a 100 nm-diameter, biotinylated gold nanoparticle (AuNP) to streptavidin yields peak lasing at ∼617 nm, and the peak intensity in the longitudinal mode spectrum is red-shifted by ∼1.4 cm−1 (∼42 GHz) with respect to the Cy3-streptavidin spectrum
The experimental results reported here demonstrate that the streptavidinbiotin conjugate provides a versatile and robust acceptor–donor linkage for realizing efficient FRET lasers with reproducible characteristics

Summary

Introduction

In 1948, Förster reported the molecular energy-exchange process that has proven to be an invaluable tool in chemistry and biology for more than 70 years, known as Förster (or fluorescence) resonance energy transfer (FRET).1,2 A milestone in the development of FRET as a biochemical diagnostic was the demonstration by Stryer and Haugland3 in 1967 of a “spectroscopic ruler,” a synthesized oligomer of poly-L-proline that served to separate donor and acceptor fluorophores by 1.2–4.6 nm. The affinity constant for the SPB complex has been measured to be 1015 M−1.9 In addition, the geometry of streptavidin (volume of 4.2 × 4.2 × 5.6 nm3) provides up to four binding (“docking”) sites per molecule and sets the donor–acceptor separation at ∼6 nm, a value comparable to (or less than) the Förster radius for the well-known fluorophores Cy3 and Cy5 (cyanine dyes).10 Tethering a moiety such as a fluorescent protein or nanoparticle to streptavidin requires only that the molecular acceptor or donor be conjugated.

Results

Conclusion