Authors from the US Naval Research Laboratory in Washington, from George Mason University in Manassas, Va., and from the National Research Center on nanoStructures and bioSystems at Surfaces in Modena, Italy, recently wrote a review in which they discuss the benefits and limitations of various fluorophores that can be used as donors and acceptors in bioanalytical Förster resonance energy transfer (FRET) techniques.

FRET uses an excited donor fluorophore to transfer energy to an acceptor in a nonradiative process that takes place through dipole-dipole interactions. Optimal interactions usually occur in the 10 to 100 Å range. Because the FRET process is distance-dependent on the nanoscale, it has proved to be a powerful technique for monitoring myriad biomolecular interactions.

Traditional FRET dyes are organic compounds that emit in the ultraviolet to near-infrared range. Another organic dye is the photochromic type that can be switched between two different structural forms when illuminated with certain wavelengths. This changes the dye’s acceptor functionality and allows it — essentially — to be turned on and off.

Naturally occurring biomolecules such as tryptophan and fluorescent proteins are increasingly being used as donors and acceptors in FRET. They are easily introduced into proteins and can be used in vivo for cell labeling. However, fluorescent proteins can be large and susceptible to changes in environmental conditions.

Inorganic materials also are increasingly being used as donors and acceptors. For example, gold nanoparticles have been shown to exhibit increased sensitivity over some other dye combinations for FRET-based DNA sensing. Silicon nanoparticles are difficult to produce but are being considered as a nontoxic alternative to semiconductor materials for in vivo imaging. Quantum dots have versatile excitation wavelengths with emissions ranging from the ultraviolet to the infrared and may have applications in photodynamic cancer therapy.

It may even be possible to produce unique emissions using multilabeled DNA structures with different combinations of molecules and a single excitation wavelength. This technology could have applications in information encoding.

The authors conclude that the growing array of materials available for FRET will expand its applications. They point to the study of protein- and peptide-folding kinetics, elucidation of macromolecular interactions and multicolor analysis as some applications that may particularly benefit from FRET advances. (Angewandte Chemie Int. Ed. 2006, 45, pp. 4562-4588.)