Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 71

Fig. 23 (a) DNAzyme-based sensor design with two dabcyl quenchers and a FAM fluorophore
( top ) and mechanism of operation ( bottom ). (b) Fluorescence response before and after complete
cleavage through Pb 2+ ; inset contains the corresponding image of the DNAzyme probe in the
absence ( left ) and presence of Pb 2+ (after 2 min of reaction time, right ). (Reprinted with permission
from [ 150 ]. Copyright 2003 American Chemical Society)
5 0 end of the substrate strand, effectively reducing background fluorescence of the
single strand [ 150 ].
The preparation of DNAzyme sensors is usually done in a combinatorial way by
producing a random set of DNA strands and selecting the analyte-responsive
strands on an affinity column followed by elimination of strands that are sensitive
to a broader group of metal ions. In case of success, a DNAzyme with high
selectivity and sensitivity is obtained after several iterative cycles. DNAzymes
have been found by this process for Cu 2+ ,Pb 2+ , and UO 2+ [ 151 - 153 ].
4.1.3 Systems Based on Chemi- and Bioluminescence
The last systems discussed in this section dealing with the generation of luminescence
signals through chemical reactions are based on chemiluminescence (CL). CL
implies that electromagnetic radiation in the UV, visible, or NIR region is produced
by a chemical reaction that yields electronically excited intermediates or products.
System design is generally possible along two different strategies, i.e., involving (i)
direct CL or (ii) sensitized CL. In the first approach, the reaction directly leads to the
formation of a certain fraction of the products in an electronically excited state,
emitting the photons that can be detected. Indirect or sensitized CL relies on the aid of
a fluorophore. The chemical reaction here also produces an excited species which,
however, does not emit to a sizable degree but relaxes mainly through nonradiative
pathways. The aiding fluorophore added to the system is chosen in such a way that it
can operate as an energy acceptor for the excited reaction product and, after energy
transfer from product to fluorophore, decays by emission of a photon [ 154 - 156 ].
Although CL does not require a lamp as excitation source and thus allows
measurements against a “zero” background, some basic requirements have to be