Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 43

intrinsic values of F f are the essential determinants. The same holds for

fluorescent stains, e.g., for cell compartments. Indicators on the other hand have to
express another essential feature, i.e., a change in fluorescence signal upon binding
to the designated analyte that is as pronounced as possible to guarantee optimum
exploitation for sensitive and at best unequivocal detection. The most obvious key
to success for the latter is to choose (a) potentially highly fluorescent dye(s) and to
integrate (a) receptor unit(s) to the chromophore(s) in such a way that the signal is
weak in the absence of the analyte and that only the arrival of the target species
modulates a photophysical process that results in a strong output signal. Important
parameters here are the chemical nature of the analyte, the type of interactions the
binding or recognition event should be accomplished with, and the type of photo-
physical processes that can be installed in a certain (family of) dye(s). In other
words, the primary task is to couple sensitive and selective target recognition with
efficient signal generation. The present chapter will thus not dwell on the best
fluorometric method for signal assessment [ 4 ] or the optimum chromophore [ 5 - 7 ]
or luminescent (bio)macromolecule [ 8 - 12 ] or object [ 13 - 17 ] in terms of achievable
quantum yield, wavelength range of operation, or intrinsic photophysical properties
nor on the details of basic photophysical and photochemical processes such as
excited-state proton, charge and electron transfer (ET) or aggregate formation
[ 18 - 24 ] or versatility in dye synthesis, biochemical coupling, and conjugation
[ 25 - 27 ]. Instead, it will introduce and highlight basic concepts and features that
have been established in the last ca. 20 years for the achievement of strong analyte-
induced signal modulations, covering a broad range of diverse system architectures,
types of analytes, and physical processes.
Before embarking on the description and discussion of actual examples, a few
additional comments are necessary. Because of the limited space available in a
topic article and the large variety of different approaches having been published to
date, we will mainly focus on examples that show strong signal changes connected
with an increase in luminescence. Amplified quenching, though frequently leading
to dramatic signal modulations, will only be discussed for selected systems in
Sect. 4 . The advantage of realizing enhanced fluorescence signals upon analyte
binding is perhaps most obvious from the following two considerations. First, the
measurement of strong signals against a weak background harbors the physical

e l and

designed or not, that responds (often nonspecifically) to a change in a local environmental
parameter (including solvatochromic and solvatokinetic responses); (1a) indicator ¼ specific
(often designed) probe that responds (often selectively) to a certain parameter (e.g., pH or Ca 2+
indicator); (1b) chemosensor ¼ synonym for indicator ; (2) stain ¼ fluorophore that provides a
constant output signal to visualize (a certain region within) a larger object after staining, i.e.,
physical enrichment/accumulation based on hydrophilicity/lipophilicity partioning or electrostatic
forces; (3) label ¼ fluorophore that provides a constant output signal to allow the monitoring/
tracking of an object after (often) selective (usually covalent) chemical attachment to the latter;
(3a) tag synonym for label . Whereas the specificity of the label is imparted by the coupling

reaction, the specificity of the probe is basically imparted by the spectroscopic response, whether a
simple solvatochromic dye is used or an indicator dye that binds only to a certain analyte.

¼