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TR-FRET Basics
TR-FRET unites TRF (Time-Resolved Fluorescence) and FRET
(Fluorescence Resonance Energy Transfer) principles.
This combination brings together the low background
benefits of TRF with the homogeneous assay format of
FRET. This powerful combination provides significant
benefits to drug discovery researchers including assay
flexibility, reliability, increased assay sensitivity,
higher throughput and fewer false positive/false
negative results. While HTRF® is based on TR-FRET
chemistry it has many properties that separate it from
other TR-FRET products. These include the use of
lanthanides with an extremely long half-life (Europium
and Terbium), a large Stoke's shift, complexing to
cryptate, an entity which confers increased stability,
and the use of a patented ratiometric measurement
enables assay quench and sample interference correction.
Overview of FRET:
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.
Principles of TRF:
 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® Basics:
 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).
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