Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 74

Fig. 26 Mechanism of the ATP- and Mg 2+ -dependent firefly luciferase catalyzed bioluminescence
oxidation reaction of D -luciferin ( D -LH 2 ) to oxyluciferin (oxy-L)

leads to an intermediate with a four-membered dioxetane ring, which decomposes
to CO 2 and the excited form of oxy-L that finally decays by emission. The
efficiency of the reaction is

0.41 at pH 8.5 which is the highest known
efficiency for a BL reaction. Being ATP-dependent, the firefly luciferase/ D -luciferin
system can be and has of course been used to detect and quantify ATP and even
allows the monitoring of changes in ATP concentration in other ATP producing or
consuming catalytic cycles.

F CL ¼

4.2 Displacement of Fluorophores from Binding Sites

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent
indicators, F-IDAs - are based on the next alternative concept described here.
A receptor with an affinity for a given analyte is loaded with an indicator, usually
a fluorescent or colored dye, whose spectral properties undergo a change upon
complexation with the receptor. Treatment of this indicator-receptor complex with
the analyte results in the displacement of the indicator from the receptor and a
restoration of the indicator's original spectral properties, indirectly reporting ana-
lyte coordination (Fig. 27 ). For effective detection, two main requirements have to
be fulfilled: (1) the receptor/indicator interaction must be reversible and weaker
than the interaction of the receptor with the designated analyte and (2) the indicator
must show significantly different optical properties when bound to the receptor and
when freely dissolved in solution.
The use of synthetic receptors for such displacement assays was pioneered by
Metzger and Anslyn in 1998, based on the concept of competitive immunoassays.
The authors developed a method for the determination of citrate in beverages and
chose receptor 66 as the host, consisting of three guanidinium groups for hydrogen
bonding and electrostatic interaction with citrate, and 5-carboxyfluorescein (67)as
the indicator, because it has one carboxylate group less available than the analyte
for binding with 66 (Fig. 27 ). In this case, the fluorescence of 67 is increased when
forming a complex with 66. Upon citrate addition, 67 is displaced and a decrease in