Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 92

Besides these promising examples of systems for the indication of ions or small-
molecules, FRET still finds more widespread application in bio(macro)molecule
sensing because it operates over distances that are comparable with the size of
biomolecules. In addition, common biomolecules do not affect FRET through less
favorable quenching interactions and the large virtual Stokes shift allows the
suppression of scattering and background signals effectively.
4.5.2 Bioluminescence Resonance Energy Transfer
BL resonance energy transfer is a nonradiative process of energy transfer from a
bioluminescent donor (usually a biomolecule) to an acceptor. The most significant
differences to fluorescence resonance energy transfer, i.e., FRET with a potentially
fluorescent D RET , are that there is no fluorescence background due to coexcitation
of A RET and that these systems can dispense with an external light source, which
avoids photodamaging of biological samples, similar to what we have discussed in
Sect. 4.1.3 . Figure 38 summarizes the differences between BRET and FRET.
As a consequence of the virtual absence of fluorescence background, BRET-
based QDs were developed by coupling carboxylate-functionalized QDs to a
mutant of the bioluminescent protein Renilla reniformis luciferase (Luc8,
Fig. 39a ). On addition of the enzyme's substrate, coelenterazine, a weak lumines-
cence at 480 nm, stemming from Luc8, and a strong signal at 655 nm, significant of
BRET-sensitized QD luminescence, is registered (Fig. 39b ). A unique feature of the
system is shown in Fig. 39c . When monitored in serum, both emission bands are
detectable. However, when employing the same system under identical conditions
in blood, the residual Luc8 BL is absorbed by the matrix though the long-wave-
length QD signal is virtually unquenched. This advantage distinguishes the BL-QD
tandem as a very promising approach toward sensitive in vivo imaging in cells and
deep tissues [ 222 ].
Like FRET, today BRET is predominantly used in biological sciences, especially
in the monitoring of protein-protein interactions such as hormone-receptor interac-
tion [ 223 , 224 ] and protein-DNA interaction in living systems. However, BL
resonance energy transfer can also be applied in immunoassays by using for instance
a peptide-tagged luciferase and a fluorescein-labeled antipeptide antibody [ 225 ].
The development of more BRET assays for small-molecule analytes is thus awaited.
5 Conclusion
After Sousa [ 226 ] and V¨gtle [ 227 ] rediscovered the potential of fluorescent and
chromogenic complex forming reagents in the context of modern supramolecular
photochemistry ca. 35 years ago, the field of the rational development of analyte-
responsive fluorescent reporters saw a tremendous boost and naturally branched out