Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 65

4 Strategies of Signal Amplification

The examples discussed in the previous sections have shown how rational design of
fluorescent reporters can yield strongly enhanced fluorescence signals upon target
binding. In certain architectures, this enhancement can even reach factors of more
than a thousand. However, all the examples rely on the stoichiometric interaction of
reporter and analyte and generate a stoichiometric amount of photons, sometimes
with a higher and sometimes with a lower probability, i.e., generate higher or lower
fluorescence enhancement. The question is now if and how we can go beyond such
traditional enhancement. One of the most obvious keys to success might lie in the
consideration of signal amplification in natural systems, most of all enzymes. In a
stimulated process, a single co-factor can induce the activation of an enzyme which
then processes its substrates almost limitlessly or, more precisely, until inhibition
occurs or it runs out of educts. These, in a general sense, catalytic approaches are
very appealing and have also been realized in other areas of analytical chemistry,
for instance, in the polymerase chain reaction (PCR) or in rolling circle amplifica-
tion (RCA)-based techniques. However, many other strategies are possible. The
following section will shed light on the most prominent concepts of fluorescence
amplification for the indication of charged inorganic and small-molecule organic
analytes.

4.1 Chemical Reactions

Perhaps the most obvious strategy for a chemist is to use an actual chemical
reaction involving covalent bond formation rather than the interplay of supramo-
lecular forces. The following section thus illustrates the use of chemical reactions in
the context of luminescence signaling, concentrating on two different phenomena:
(i) the production of a fluorophore in a chemical reaction, which still requires a
conventional fluorescence measurement setup, and (ii) chemiluminescence (CL),
where photons are produced by a chemical reaction, but which only needs a detector
for registration of the emitted light.

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Fig. 15 Metal complex-based anion probes. (a) Stable Cd 2+ complex of ICT probe 41 undergoes
recoordination of the metal ion in the presence of pyrophosphate, reinforcing ICT. (b) Elaboration
of the concept shown in (a) to a peptide probe (see c) able to assess protein kinase activity; (d) time
course of the corresponding bathochromic shift in fluorescence excitation spectra during enzyme
activity according to (b). (e) Ligand exchange at the Eu 3+ emitter in 42 leads to strong emission
increase (sensitizer in magenta). (f)Ru II -tris(bipyridine) probe quenched by the quinoid calix[ 4 ]
arene subunits ( brown ); anion insertion into the binding site ( blue atoms ) leads to suppression of
quenching. (Part d) adapted from [ 129 ]; graphical material kindly provided by the authors of the
original publication)