Htrf Read Mode - Molecular Devices SpectraMax Paradigm User Manual

HTRF Read Mode

Homogeneous time-resolved fluorescence (HTRF) is a measurement technique based on
fluorescence resonance energy transfer (FRET) using the advantages of time-resolved
fluorescence (TRF) reading.
Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between
the electronic excited states of two dye molecules in which excitation is transferred from a
donor molecule to an acceptor molecule without emission of a photon.
FRET relies on the distance-dependent transfer of energy from a donor molecule to an
acceptor molecule. Due to its sensitivity to distance, FRET has been used to investigate
molecular interactions. FRET is the radiationless transmission of energy from a donor
molecule to an acceptor molecule. The donor molecule is the dye or chromophore that
initially absorbs the energy and the acceptor is the chromophore to which the energy is
subsequently transferred. This resonance interaction occurs over greater than interatomic
distances, without conversion to thermal energy, and without a molecular collision. The
transfer of energy leads to a reduction in the fluorescence intensity and excited state lifetime
of the donor, and an increase in the emission intensity of the acceptor. A pair of molecules
that interact in such a manner that FRET occurs is often referred to as a donor/acceptor pair.
While there are many factors that influence FRET, the primary conditions that need to be
met for FRET to occur are relatively few:
The donor and acceptor molecules must be in close proximity to each other.
The absorption or excitation spectrum of the acceptor must overlap the fluorescence
emission spectrum of the donor.
The degree to which they overlap is referred to as the spectral overlap integral (J).
The donor and acceptor transition must be approximately parallel.
HTRF uses a donor fluorophore with a long fluorescence lifetime, such as Europium. The
acceptor fluorophore acts as if it also has a long fluorescence lifetime. This lets the time-
gating principle of time-resolved fluorescence be applied to the acceptor emission to
separate specific signal from background and signal caused by compound interference.
Time-gating electronics introduce a delay between the flashes and the start of signal
collection. During the delay, the unspecific fluorescence caused by test compounds, assay
reagents, and the microplate vanishes while only a small portion of the specific fluorescence
from the acceptor fluorophore is sacrificed. Enough of the specific signal remains, with the
benefit of reduced background.
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Chapter 2: Read Modes and Read Types
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