Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 90

quencher dye as A RET . This strategy does not generate specifically enhanced red
emission and thus circumvents the task of finding an appropriate A RET with a high
fluorescence quantum yield in the visible/NIR wavelength range, but is aiming at
large signal modulations, which are easier to realize with nonemissive but strongly
absorbing NIR dyes. The system shown in Fig. 36 does not reach that far but
adequately shows the first features, i.e., the metal ion-dependent formation of
different foldamers by a thrombin-binding aptamer probe labeled with FAM (as
donor) and dabcyl (as quencher; for dyes, refer to DNAzyme system shown in
Fig. 23 ) at its 5 0 and 3 0 termini for selective detection of Pb 2+ and Hg 2+ , respectively
[ 212 ]. When binding the metal ions, the DNA strand's conformation can change
into two different folded structures, a G-quadruplex in the presence of Pb 2+ and a
hairpin-like structure in the case of Hg 2+ , respectively, increasing RET-type
quenching (Fig. 36 ). Because the distance between FAM and dabcyl is different
in both complexes, the RET rates differ and fluorescence lifetime measurements
permit discrimination unequivocally between the two complexes. Such dual-ana-
lyte detection has also been realized with conventional supramolecular chemistry
receptors and two fluorophores (see also Sect. 3.2.1) [ 213 ].
FRET with enhanced red emission and FRET with quenching (Q-FRET) are both
mechanisms intensively explored in connection with inorganic nanoparticles in recent
years. Q-FRET applications here mainly rely on the use of gold NPs, because these
quenchers are chemically very attractive for system design, their plasmon bands
usually lie in a spectral range where a large number of organic dyes as possible
D RET are available, and because their extremely high absorption coefficients are
ideal for efficient Q-FRET [ 214 , 215 ]. On the other hand, the particular characteristics
of semiconductor-type absorption bands with high absorption coefficients and usually
narrow emission bands with moderate to high luminescence quantum yield render
semiconductor nanocrystals or quantum dots (QD) attractive D RET partners for sensi-
tive FRET assays using organic dyes as A RET . Alternatively, QDs can be combined
with gold NPs or CPs, offering a wealth of possibilities for system design [ 216 , 217 ].
With regard to target analytes, method development has not only focussed on
metal ions, but the detection of gasses like CO 2 [ 218 , 219 ] and organic compounds
like cocaine [ 220 ] has also been pursued. For the latter analyte, an elegant example
was realized in conjunction with an aptamer-based recognition motif, a QD as D RET
and an Atto 590 dye as A RET , yielding enhanced red emission upon binding
(Fig. 37 ). Figure 37 also contains a sketch of another popular QD-based FRET
format, a competitive assay. Here, system design utilizes a QD (e.g., of CdSe/ZnS
core-shell type) and a quencher dye (e.g., Black Hole Quencher-10, BHQ-10) as
partners. The quencher is attached to a hapten, such as a TNT analog in Fig. 37c ,d,
and the QD carries analyte-responsive antibodies on its surface. In the absence of
the analyte, the quencher-labeled hapten is bound at the receptors, efficiently
deactivating the QD nonradiatively. Once a TNT molecule competes successfully
for the binding site, the quenching conjugate is displaced and the full emission of
the FRET donor is restored. Such competitive formats thus also rely on enhanced
fluorescence signals, though now D RET emission is enhanced. Highly luminescent
D RET thus perfom best in such applications [ 221 ].