TR-FRET Basics

FRET (Fluorescence Resonance Energy Transfer) uses two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source (e.g. flash lamp or fluorometer laser) triggers an energy transfer to the acceptor if they are within a given proximity to each other. The acceptor in turn emits light at its given wavelength.

Because of this energy transfer, molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. More importantly acceptor emissions, as a measure of energy transfer, can be detected without the need to separate bound from unbound assay compontents. This homogeneous assay format is extremely beneficial, reducing both assay time and costs.

FRET is governed by the physics of molecular proximity, which only allows energy transfer to occur when the distance between the donor and the acceptor is small enough. In practice, FRET systems are characterized by the Förster's radius (R0) distance at which FRET efficiency is 50%. For HTRF, R0 lies between 70 and 90 Å, depending on the acceptor used and the spatial arrangements of the fluorophores within the assay.

These size limitations have not hindered the development of biological assays. Donor and acceptor fluorophores have been conjugated to a variety of biomolecules creating functional assays such as: protein-protein binding, antigen-antibody binding, ligand-receptor binding, DNA hybridization and DNA-protein binding.

Typically the donor and acceptor molecules used in FRET assays are prompt fluorophores which have short half-lives. The limitations of traditional FRET chemistries are caused by background fluorescence from sample components such as buffers, proteins, chemical compounds and cell lysate. Detected fluorescence intensities must be corrected for this autofluorescence which greatly handicaps assay sensitivity and complicates result interpretation. This type of background fluorescence is extremely transient (with a lifetime in the nanosecond range) and can therefore be eliminated using time-resolved methodologies.

Many compounds and proteins present in biological fluids or serum are naturally fluorescent, and the use of conventional, prompt fluorophores leads to serious limitations in assay sensitivity. The use of long-lived fluorophores combined with time-resolved detection (a delay between excitation and emission detection) minimizes prompt fluorescence interferences.

Time-resolved fluorometry (TRF) takes advantage of the unique properties of the rare earth elements called lanthanides. The commonly used lanthanides in TRF assays are samarium (Sm), europium (Eu), terbium (Tb), and dysprosium (Dy). Because of their specific photophysical and spectral properties, complexes of rare earth ions are of major interest for fluorescence applications in biology. Specifically, they have large Stoke's shifts and extremely long emission half-lives (from µsec to msec) when compared to more traditional fluorophores.

It is difficult to generate fluorescence of lanthanide ions by direct excitation, because of the ions' poor ability to absorb light. Lanthanides must first be complexed with organic moieties that harvest light and transfer it to the lanthanide through intramolecular, non-radiative processes. Rare earth chelates and cryptates are examples of light-harvesting devices. The collected energy is transferred to the rare earth ion, which then emits its characteristic long-lived fluorescence. Typical emission spectra of lanthanide complexes are shown in figure 3. The emissions are from terbium(III), dysprosium(III), europium(III) and samarium(III), respectively following excitation at 337 nm.

To be successfully used as labels in biological assays, rare earth complexes should possess specific properties including stability, high light yield and ability to be linked to biomolecules. Moreover, insensitivity to fluorescence quenching is of crucial importance when working directly in biological fluids. Rare earth chelates, although used in heterogeneous fluoroassays, have limitations such as stability, compounds that compete with chelating activities and sensitivity when used in combination with FRET. When complexed with cryptates, however, many of these limitations are eliminated.

HTRF® is a TR-FRET based technology that uses the principles of both TRF and FRET. The HTRF® donor fluorophore is either Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate), fruit of a recent collaboration with Lumiphore Inc.. Both donors have the long-lived emissions of lanthanides coupled with the stability of cryptate encapsulation. XL665, a modified allophycocyanin, is the HTRF® primary acceptor fluorophore. d2 represents a second generation of acceptor characterized by an organic structure 100 times smaller. More info on HTRF donor & acceptor fluors>>>

Example with Europium cryptate and XL665:

When these two fluorophores are brought together by a biomolecular interaction, a portion of the energy captured by the Cryptate during excitation is released through fluorescence emission at 620nm, while the remaining energy is transfered to XL665. This energy is then released by XL665 as specific fluorescence at 665 nm. Light at 665nm is emitted only through FRET with Europium. Because Europium Cryptate is present in the assay, light at 620nm is detected even when the biomolecular interaction does not bring XL665 within close proximity (see figure 4).

HTRF® is a highly flexible chemistry and has been successfully used to measure molecular complexes of many different sizes. This includes assessment of small phosphorylated peptides, immunoassays for quantifying large glycoproteins such as thyroglobulin, receptor tyrosine kinase activity using membrane preparations and indirect detection (via secondary antibodies) of tagged complexes such as CD28/CD86 binding (see figure 5).

Figure 5: HTRF has been used with assays that use multiple toolbox reagents. This binding assay uses Fc-labeled proteins recognized by anti-species antibodies as well as the SA-biotin affinity system.