Fluorescent Sensors and Probes

Much of the recent activity has focused on fluorescent sensors and probes and on therapy, particularly photodynamic therapy. 

Cyanines are by far the most popular and extensively researched chromo-phore for use in biological and medical imaging applications [70,72], They are used almost exclusively in fluorescence-related protocols. Their popularity may be attributable to factors such as their excellent synthetic flexibility and fluorescence properties, the latter being very high in some instances. Cyanines are the only synthetic near-infrared fluorophores which are commercially available (for 

Squarylium dyes such as (83) [75] have probably received less attention than cyanine dyes due to the fact that the majority of syntheses furnish symmetrical species which are difficult to monofunctionalize in reactions such as the formation of peptide conjugates. The unsymmetrical types have been reported but seem to suffer from about 50% decrease in extinction coefficient. Squaiyliums are also more difficult to handle due to their low solubility. Very few water-soluble systems have been reported. These compounds are also used exclusively as fluorophores, but quantum yields are highly dependent on substituents and environment. 

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The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

Wardle B (2009) Principles and applications of photochemistry, Wiley. This book includes some excellent chapters on fluorescence sensors and probes, as well as a detailed description of more advanced fluorescence spectroscopy and imaging techniques. 

Lee JS, Kim YK, Vendrell M et al (2009) Diversity-oriented fluorescence library approach for the discovery of sensors and probes. Mol BioSyst 5 411—421... 

The design of fluorescent sensors is of major importance because of the high demand in analytical chemistry, clinical biochemistry, medicine, the environment, etc. Numerous chemical and biochemical analytes can be detected by fluorescence methods cations (H+, Li+, Na+, K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc.), anions (halide ions, citrates, carboxylates, phosphates, ATP, etc.), neutral molecules (sugars, e.g. glucose, etc.) and gases (O2, CO2, NO, etc.). There is already a wide choice of fluorescent molecular sensors for particular applications and many of them are commercially available. However, there is still a need for sensors with improved selectivity and minimum perturbation of the microenvironment to be probed. Moreover, there is the potential for progress in the development of fluorescent sensors for biochemical analytes (amino acids, coenzymes, carbohydrates, nucleosides, nucleotides, etc.). 

The development of fluorescent probes for anion recognition has been very limited so far in comparison with those for cations. Most of the presently available methods of detection of anions based on fluorescence involve quenching, redox reactions, substitution reactions, ternary complex formation(15) and thus cannot be considered as recognition methods. For instance, the fluorescent sensors that are used for the determination of chloride anions in living cells are based on collisional quenching of a dye by halide ions 6-methoxy-iV-(sulfopropyl)quinoliniuni and... 

An NIR biosensor coupled with an NIR fluorescent sandwich immunoassay has been developed. 109 The capture antibody was immobilized on the distal end of an optical fiber sensor. The probe was incubated in the corresponding antigen with consecutive incubation in an NIR-labeled sandwich antibody. The resulting NIR-labeled antibody sandwich was excited with the NIR beam of a laser diode, and a fluorescent signal that was directly proportional to the bound antigen was emitted. The sensitivity of the technique increased with increasing amounts of immobilized receptor. There are several factors involved in the preparation of the sandwich type biosensor. A schematic preparation of the sandwich optical fiber is shown in Figure 7.14. 

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