Fluorophores general structure

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

An understanding of specific and general solvent effects can provide a basis for interpreting the emission spectra of fluorophores that are bound to macromolecules or, in general, are located in different sites of structurally heterogeneous system. 

One way to reduce the number of independent variables in the FRET-adjusted spectral equation is to use samples with a fixed donor-to-acceptor ratio. Under these conditions, the values of d and a are no longer independent, but rather the concentration of d is now a function of a and vice-versa. This approach is typical for the situation of FRET-based biosensor constructs. These sensors normally are designed to have a donor fluorophore attached to an acceptor by a domain whose structure is altered either as a result of a biological activity (such as proteolysis or phosphorylation), or by its interaction with a specific ligand with which it has high affinity. In general, FRET based biosensors have a stoichiometry of one... 

The solubility in water of many fluorophores is achieved by grafting sulfonate groups. Fortunately, these groups only slightly affect the fluorescence characteristics of the parent molecule. In general, there is a small red-shift of the fluorescence spectrum, whose vibrational structure is somewhat blurred, and the fluorescence quantum yield is slightly decreased. 

The first pH indicators studied possessed the acid-base site (phenol, aniline, or carboxylic acid) as an integral part of the fluorophore. Structurally, in the most general sense, pH sensitivity is due to a reconfiguration of the fluorophorets re-electron system that occurs on protonation. Consequently, the acid and the base forms often show absorption shifts and also, when the two forms fluoresce, emission shifts or at least, when only one form emits, a pH-dependent fluorescence intensity. This class of compounds has been reviewed 112 and the best structures have to be designed according to the medium probed and the technique used. After a short consideration of physiological pH indicators we will describe the main photophysical processes sensible to protonation. 

The fluorescence emission maximum, quantum yield, and lifetime of a fluorophore are very sensitive to its immediate environment. A blue shift in the emission maximum and an increase in the fluorescence quantum yield or lifetime is generally observed when a fluorophore is transferred form a polar solvent to a nonpolar one or when it binds to a hydro-phobic protein site. Furthermore, fluorescence quenching or enhancement may result from interactions of the fluorophore with various structural elements in its vicinity. 

Fig. 2 Schematic cartoon (a), and chemical structures (b) of the general fabrication procedure of a sensitive fluorescent monolayer on glass i) silanation of the glass slide with N-[3-(trimethoxysilyl)propyl]ethylenediamine to form the amino-terminated monolayer, ii) reaction with an amino-reactive fluorophore, iii) covalent attachment of a binding molecule...

Small-molecule extrinsic fluorophores are generally used in structural and folding studies of RNA molecules (Fig. 8.5). There are a number of factors that will influence the choice of donor—acceptor fluorophores... 

The fluorophores show different fluorescence spectra. A fluorescence spectrum reflects the electronic distribution of the molecule in the excited state Si and thus the structure of the molecule in this state. We notice also that ANS does not fluoresce in a phosphate buffer (i.e., a polar medium), which is not the case when it is bound to serum albumin. Thus, fluorescence depends not only on the structure of the fluorophore but also on its environment. Each fluorophore has its own spectral properties, and one should be careful not to generalize a rule for all fluorophores, although most of the fluorophores share common rules. 

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