Optimization of the Coupling of Target Recognition and Signal Generation - Advanced Fluorescence Reporters in Chemistry and Biology - page 42

1 Introduction

There are many techniques among the portfolio of analytical methods that allow the
sensitive determination of a variety of different substances after clean-up and
separation in one experimental run or that permit the identification of compounds
unequivocally by their intrinsic fingerprint. Gas and liquid chromatography, mass
spectrometry, and NMR spectroscopy as well as a number of other methods -
ranging from more traditional electroanalytical techniques to miniaturized rendi-
tions of established methods or hyphenated methodological combinations - all are
powerful tools to tackle today's analytical problems. Why then bother with the
often tedious development of a dye molecule that has to be inherently (and at best
highly) fluorescent and that has to show a specific change in its fluorescence signal
upon interaction with a designated target molecule? The answer is twofold. First, in
contrast to all the methods mentioned above, analyte-responsive fluorophores and
fluorescence measurements as such do not necessarily require a laboratory setting.
Fluorophores simply need light to be excited and a detector (if not the naked eye)
for the signal to be registered, whether addressed and accessed directly or remotely,
through space or through tissue, with fiber optics or with a microscope objective.
Chemi- and bioluminescent protocols in addition can even dispense with a light
source. Second, fluorescence or more general luminescence is a very sensitive detec-
tion technique allowing the counting of single photons and tracking or observation of
single molecules and, if required, at a rather high spatial resolution. Fluorescent
reporters thus unfold their truly unique advantages as analytical tools on site -
whether as a squad of individual indicator molecules hunting for a certain target
analyte in a live cell or as a crowd of indicators immobilized in a solid porous matrix
at the tip of a fiber, waiting for a certain target analyte to pass by the sensor head, in
real time - whether for continuous monitoring of a specific parameter in a bioreactor
or as a quick dip stick test for qualitative analysis in the hands of a food inspector, and
in many diverse imaging applications, ranging from TIRF and FISH to FRAP and
FLIM 1 and providing distinct spatial information on the object of interest [ 1 - 3 ].
Besides instrumental features such as the wavelength range of excitation and
emission and the decay time of the luminescence signal, two quantities are espe-
cially important in the design of functional fluorophores, i.e., the molar absorption
coefficient at the excitation wavelength ( e l ) and the fluorescence quantum yield

( F f ). A high molar absorption coefficient is a prerequisite for efficient transforma-

tion of a molecular emitter from the ground into its excited state and a high
fluorescence quantum yield means that the absorbed photons are efficiently con-
verted into emitted photons. For a fluorophore as a label 2

for a biomolecule, the

1

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization
(FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging
microscopy (FLIM).
2 In this chapter, terms often used inconsistently in the literature for various types of functional
fluorophores or fluorescent reporters are defined as follows: (1) probe ¼ fluorophore, whether