Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 70

Fig. 22 (a) Oxymercuration reaction for Hg 2+ sensing with 58.(b) Oxidation state-selective Pd/Pt
catalytic production of fluorophores 61 and 62 from 60. Nonfluorescent 58 is selectively converted
into strongly green fluorescent 59 and nonfluorescent 60 is either converted into strongly green
fluorescent 61 or strongly greenish-yellow fluorescent 62
derivative 61 by Pd(0) and, to a lesser extent, Pt(II) and to the fluorescein dye 62 in
a catalyzed Claisen rearrangement by Pd(II) and Pt(IV), enabling not only Pd and
(to a lesser extent) Pt detection at trace concentrations, but also a spectral distinc-
tion of the oxidation state [ 147 , 148 ].
A rather new approach for detecting metal ions with very high sensitivity and
selectivity utilizes DNAzymes. DNAzymes are a special class of enzymes formed
from DNA nucleotides. Compared to proteins and ribozymes, they are more stable,
structurally simpler, and therefore cheaper. As DNAzymes often require metal ion
cofactors, they are interesting sensing platforms for these metal ions [ 149 ].
DNAzymes are usually composed of a substrate strand that forms a stable duplex
with an enzyme strand. The substrate strand possesses a particular cleavage site at a
defined position. The indication reaction then involves the cleavage of a certain
substrate strand by the designated cofactor or metal ion, breaking the duplex and
releasing the fragments. The cleavage sites are specific to certain metal ions, e.g., a
ribo-adenosine (rA) for Pb 2+ and UO 2+ .Cu 2+ on the other hand is capable of
directly cleaving the strand at a guanidine position.
An example is displayed in Fig. 23 . For sensory applications, the substrate strand
is tagged with a fluorophore (e.g., 6-carboxyfluorescein, FAM) at the 3 0 end and the
enzyme strand with a quencher (e.g., 4-(4 0 -dimethylaminophenylazo)benzoic acid,
dabcyl) at the 5 0 end. As long as the duplex is formed, the emission of the
fluorophore is quenched and no fluorescence is registered. Only after activation
of the catalytic activity by the target ion, the substrate strand is cleaved and the
fluorophore liberated, enabling the detection of the unquenched fluorescence.
However, because of the bulged structure of the DNAzyme (Fig. 23 ), the melting
temperature of the duplex is comparatively low. Thus, for optimum performance,
i.e., for optimum suppression of background signals at relevant temperatures such
as ambient or body temperature, a second internal quencher was attached to the