2. General Description
16
Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0
2011-09
Fluorescence Resonance Energy Transfer (FRET)
Some microplate applications utilize a sophisticated dual labeling strategy. The
Fluorescence Resonance Energy Transfer effect (FRET) enables you to detect
binding events of various labeled compounds that are in close proximity.
Basically, FRET is a fluorescence intensity measurement of one of the two
fluorescent labels (acceptor). However, the acceptor is not susceptible to the
excitation wavelength of the light source being used. Instead, the acceptor may
receive excitation energy from the other fluorescent label (donor), if both are
spatially close together. As a prerequisite, the excitation wavelength has to apply
to the donor. And secondly, the emission spectrum of the donor has to overlap
the excitation spectrum of the acceptor (resonance condition). Nevertheless, the
transfer of excitation energy from donor to the acceptor is radiation free.
Some FRET-based applications utilize suitable pairs from the fluorescent protein
family, like GFP/YFP (Green/Yellow Fluorescent Protein) (Ref
. Using GFP in
FRET-based applications by Brian A. Pollok and Roger Heim – trends in Cell
Biology (Vol.9) February 1999
). An overview is given in the review article –
Application of Fluorescence Resonance Energy Transfer in the Clinical
Laboratory: Routine and Research by J. Szöllösi, et al. in Cytometry 34 page
159-179 (1998).
Other FRET-based applications take advantage of the use of TRF labels as the
donor, (for example: see.
High Throughput Screening – Marcel Dekker Inc 1997
New York, Basel, Hong Kong – see section 19 Homogeneous, Time-Resolved
Fluorescence Method for Drug Discovery by Alfred J. Kolb, et al.).
B) Fluorescence Time Resolved (TRF)
TRF applies to a class of fluorescent labels (chelates) of lanthanides like
Europium (Ref.
Europium and Samarium in Time-Resolved Fluoroimmunoassays
by T. Stâhlberg, et.al. - American Laboratory, December 1993 page 15)
some of
them having fluorescence lifetimes in excess of 100 microseconds.
The
INFINITE M1000 PRO
uses a flash lamp light source with flash duration
much shorter than the fluorescence lifetime of these species. This offers the
opportunity to measure fluorescence emission at the time when stray light and
prompt fluorescence have already vanished (Lag Time) thus significantly lowering
background fluorescence and improving sensitivity.
The benefits of TRF consequently apply to assays using multiple labels with
different fluorescence lifetimes.
Homogeneous Time Resolved Fluorescence (HTRF)
HTRF technology combines both time-gated fluorescence (commonly referred to
as time-resolved fluorescence = TRF) and fluorescence resonance energy
transfer (FRET). HTRF is based on the energy transfer between two fluorescent
labels, a long-lifetime Eu
3+
-cryptate donor and the XL665 acceptor (chemically
modified allophycocyanin). The main benefit of time-gated measurements is the
efficient reduction of background fluorescence by temporal discrimination. The
addition of energy transfer further minimizes several undesired assay
interferences and side effects (e.g. volume/meniscus, quenching, light scattering,
autofluorescence, molecular size, etc.). Furthermore, the homogeneous format of
these assays, so-called ‘mix and measure’ protocols, satisfies demand from the
industry for one-step, non-separating applications for high throughput screening
(HTS).
