Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 48

the acceptor fragment thus also leads to a formal positive charge on the donor in the
excited complex, leading to electrostatic repulsion between the bridgehead atom
and the cation. The net effect is the reduced influence of the guest in the S 1 state and
on its deactivation, which is reflected by generally smaller shifts

in fluorescence
than absorption, see Fig. 3 . 10 For further elaborations on the topic, the interested
reader is referred to [ 53 ]. The important point in terms of this chapter, however, is
the fact that, besides rather small shifts in fluorescence, the fluorescence intensity
modulations expressed as the ratio of the fluorescence quantum yield of free
(superscript fp) and bound probe (superscript bp) or the fluorescence enhancement
factor FEF ( 1 ) typically found for ICT probes is also comparatively small, usually
ranging between quenching and enhancement by a factor of 2, i.e., 0.5

D

2
(the data in Fig. 3 are also representative in this respect). These moderate changes in
conjunction with opposite signs are connected to the intrinsic complexity of the
excited-state reaction mechanisms of donor-acceptor-substituted chromophores,
especially when the fragments are connected by a series of single and double
bonds such as in 7. A detailed discussion would go beyond the scope of this chapter
so that [ 54 , 55 ] are recommended for further reading. For rational design of
fluorescence “light-up” ICT probes, however, a profound knowledge of the photo-
physics of the parent chromophore is indispensible. If such data are not available,
development can quickly result in success by serendipity or entail tedious prelimi-
nary mechanistic studies. A few rational approaches that led to success will be
discussed in Sect. 3 .

< FEF <

bp
f
F

¼ F FEF (1)

fp
f

Although the aforementioned might suggest that ICT probes are generally not the
best performers in terms of strong fluorescence output or high FEF, their architecture
is ideally suited for the design of ratiometric probes. Ratiometric measurements rely
on pronounced spectral separation of the absorption and/or the emission spectra of
free and bound probe and can principally be realized in two ways, i.e., using two
different excitation and a single observation wavelength or using a single excitation
and two different observation wavelengths (see also Sect. 2.2.2 ). According to the
mechanism of operation sketched above, i.e., usually strong shifts in absorption and
weak shifts in fluorescence, ICT probes like 7-9 would mainly qualify for the former
type. However, instrument operation with two different excitation settings and one
emission channel is less favorable than the single excitation/double emission

ICT probes thus do not play a role as pH-indicators. However, this fact can be utilized for powerful
sensing schemes in a different context, see Sect. 4.1.1 .
10

In principle, the trends should be exactly opposite for ICT probes carrying the receptor in the
acceptor fragment. However, as can be seen for 9 in Fig. 3 and some other probes of this type (e.g.,
in [ 50 - 52 ]), the relationship is not straightforward. Moreover, since the literature on acceptor-type
ICT probes is much less abundant than on their donor counterparts, the database for a comprehen-
sive analysis and discussion is still rather weak and further conclusions will not be drawn here.