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(bioluminescence RET), where the donor is an intrinsically bioluminescent
molecule.
13
4.5.1 F¨rster/Fluorescence Resonance Energy Transfer
F¨rster Resonance Energy Transfer (FRET) with a potentially fluorescent donor is
unique in generating dramatically red-shifted fluorescence signals. Besides the
mechanistic prerequisites as outlined before (high
F
f
of
D
RET
; large overlap
integral, the latter implying both, good spectral overlap between
D
RET
and
A
RET
,
and a high molar absorption coefficient of
A
RET
), for strong fluorescence signals the
RET acceptor also has to possess at best a high fluorescence quantum yield. In
addition, if strong fluorescence modulations upon analyte binding are pursued, the
FRET efficiencies between unbound and bound state of the signaling system have
to be largely different. This means that analyte binding has to induce a distance
change between
D
RET
and
A
RET
that is as pronounced as possible.
Among the chemical species discussed in this chapter, again metal ions and in
particular notorious quenchers such as Cu
2+
,Pb
2+
,Hg
2+
,orCr
3+
are primary targets
for FRET signaling system development. One of the hopes here is that the FRET
concept holds better promises for achieving “light-up” responses upon binding of
these quenching species, simply because it operates over comparatively long dis-
tances. Related to the excimer probes introduced in Sect.
2.2.2
, the traditional
approach uses a metal chelating unit conjugated with two (different types of)
fluorophores as the RET donor and acceptor units at the terminal ends of a linear
receptor. Recognition and binding of the metal ion then leads to a folding of the
receptor around the metal ion according to its preferred coordination geometry and
the architecture of the ligand, bringing closer together
D
RET
and
A
RET
and produc-
ing an enhanced RET signal. However, it turned out that in line with the considera-
tions mentioned for most of the concepts in Sect.
2
, special care has also to be taken
for FRET systems to avoid a direct interaction of quencher and FRET system. A
typical example shows why. If a FRET system is constructed with a rather long yet
flexible Cu
2+
-chelating peptide sequence which is flanked by a tryptophan (donor)
and a dansyl chloride (acceptor), FRET indeed occurs upon Cu
2+
complexation
though at the same time the overall fluorescence signal intensity is reduced because
13
The correct and IUPAC-approved term for FRET is F¨rster RET [
209
], particularly motivated by
a statement given under the entry 'fluorescence resonance energy transfer':
Term frequently and
inappropriately applied to resonance energy transfer in the sense of Fo¨rster-resonance-energy
transfer (FRET), which does not involve the emission of radiation
in [
209
]. The literature, on the
other hand, uses both terms, F
¨
rster RET and fluorescence RET with the latter even prevailing in
the biochemically and bioanalytically oriented communities. However, the dilemma becomes
apparent when discussing FRET and BRET, both F
¨
rster-type processes which differ only in the
properties of the donor. Interestingly, the IUPAC Photochemistry Commission does not mention
BRET in their recommendations. To distinguish between the two different types of donors,
however, it seems more adequate for us to use FRET for a RET involving a potentially fluorescent
donor and BRET for a RET involving a potentially bioluminescent donor here.
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