Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 72

met. The production of an electronically excited state that emits in the visible region
requires a strongly exothermic reaction (ca. 150-300 kJ mol -1 ), which is usually
only accomplished by redox reactions with strong oxidants like oxygen, hydrogen
peroxide, or potassium permanganate. Furthermore, the quantum yield of P *
formation ( P *

excited reaction product) of the reaction must be favorable as
well as the luminescence quantum yield of P *. For sensitized CL, the efficiency of
the energy transfer to the fluorophore and the fluorescence quantum yield of the
latter are the crucial parameters.

¼

F CL ¼ F ex F f

(2)

F CL ) is usually expressed as the product of the efficiency
of the production of P *(

The efficiency of CL (

F f ) of the excited
species ( 2 ). Efficiencies found in CL reactions are usually rather low with ca.
0.001-0.1; higher yields are only found in bioluminescence (BL). Although these
values sound rather inefficient, it should be kept in mind that CL setups are very
simple yet very sensitive. As mentioned above, no external light source is needed so
that the experiment can be conducted in a light-tight cell. Furthermore, since
background emission equals virtually zero, the reaction cell can be mounted directly
in front of the detector with no dispersive elements or filters being required. None-
theless, there are some important difficulties for CL analysis which have to be taken
into account or circumvented. Because the reactions strongly depend on experimen-
tal parameters, these must be controlled meticulously for comparable results. More-
over, since CL reactions are usually performed with strong oxidants that show very
low selectivity, today, most analytical CL applications are postseparation, i.e., CL is
used as detection technique for HPLC and gel chromatography, involving for
instance the injection of the reaction mixture into a flow-through cell [ 157 ].
A well known example of a CL reaction is the oxidation of luminol (63) with
strong oxidants like permanganate, hypochlorite, and especially hydrogen peroxide
in alkaline medium (Fig. 24 ). A representative example of sensitized CL is the
oxidation of oxalates with hydrogen peroxide in the presence of a fluorophore [ 158 ].
A special case of CL is electrochemiluminescence (ECL) which will only be
briefly mentioned here. In ECL, a redox reaction is conducted by switching the
voltage of an electrode instead of mixing reactants like in CL, converting electrical
energy into light [ 159 ]. The first compound that was found to show ECL is Ru II -tris
(bipyridine) [ 160 ] which is also the best studied and most applied ECL system
(Fig. 25 ). It has an ECL efficiency of 0.05 [ 161 ] and is usually taken as a reference
for ECL efficiency measurements [ 162 ]. With respect to ECL sensing, Ru II -tris
(bipyridine) can for instance be employed to indicate strong reductants such as
oxalate [ 163 ]. The reaction scheme, however, is potentially more complicated since
after oxidation of Ru II to Ru III at the electrode surface, reaction of the Ru III species
with oxalate can generate various highly reactive radical anion intermediates and
proceed through various reaction sequences. Nonetheless, if conditions are properly
controlled the ECL response is linear and allows for example the determination of
oxalate in urine at relevant micromolar concentrations [ 163 ].

F ex ) and the luminescence efficiency (