Fluorogenic reactions

Figure 2 Structure of fluorescamine (FR) and its fluorogenic reaction with primary amines.

Analytical absorption spectroscopy in the ultraviolet and visible regions of the elechomagnetic spectrum has been widely used in pharmaceutical and biomedical analysis for quantitative purposes and, with certain limitations, for the characterisation of drugs, impurities, metabolites, and related substances. By contrast, luminescence methods, and fluorescence spectroscopy in particular, have been less widely exploited, despite the undoubted advantages of greater specificity and sensitivity commonly observed for fluorescent species. However, the wider availability of spectrofluorimeters capable of presenting corrected excitation and emission spectra, coupled with the fact that reliable fluorogenic reactions now permit non-fluorescent species to be examined fluorimetrically, has led to a renaissance of interest in fluorimetric methods in biomedical analysis. 

Development of Fluorogenic Reactions for Azide-Derivatized Glycans... 

Here, the studies by Xie and coworkers21 and Noji and coworkers70 using the fluorogenic reaction approach are used to illustrate these two experimental designs, both of which studied /3-galactosidase as a model enzyme. 

The fluorogenic reaction approach is also widely applicable so long as suitable fluorogenic reactions are available. With modern synthetic methods, suitable fluorogenic substrates can probably always be designed and synthesized for a particular enzymatic reaction examples are already available in the literature.7... 

To study a particular enzyme using the fluorogenic reaction approach, the first challenge is to find a fluorogenic substrate. As discussed above, with modern synthetic chemistry, the availability of suitable substrates is only limited by researchers creativity in designing molecules. 

For the real-time single-turnover detection of fluorogenic reactions, catalytic reactions with fast turnover rates (or fast product dissociation) pose a time resolution challenge, as single-molecule fluorescence detection often requires detection of hundreds of photons to obtain statistically significant information. For these fast enzymes, one can vary the substrates, use enzyme variants from different organisms, or use mutants to slow the catalysis down. 

Instead of the optical labels, enzymes are also used as a labeling material [7]. ELISA is one of the most popular techniques in the conventional immunoassay and suitable in the microchip format. By using a fluorogenic reaction, the fluorescent signal can be taken at the downstream microchannel without interruption by microbeads. In the case of chromogenic reaction, a TLM is useful [8]. 

Figure 1. Fluorogenic reactions of fluorescamine (Fluram) and MDPF with primary...

Other fluorogenic reagents are available. o-Phthalaldehyde reacts rapidly with primary amino groups in the presence of 2-mercaptoethanol at alkaline pH and room temperature (Roth, 1971). The excitation and emission maxima are at 340 and 450 nm, respectively. As with fluorescamine and MDPF, o-phthalaldehyde is nonfluorescent. The reagent is water soluble and is dissolved and stored in the aqueous buffer (pH 10.0) used for the reaction. The fluorophors are less stable than those formed with fluorescamine (Mendez and Gavilanes, 1976). However, the use of ethanethiol, rather than 2-mercaptoethanol, for the fluorogenic reaction has been reported to yield fluorophors with a half-life of 2 days in aqueous solution at pH 9.1 (Simons and Johnson, 1977). 

Polyacrylamide gel isoelectric focusing of MDPF-labeled peptides has also been demonstrated (Stein, 1977). After the fluorogenic reaction the sample is desalted either with a molecular seiving column, such as Sephadex G-10, or by taking it to dryness and then dissolving the relatively hydro-phobic peptide fluorophor in an organic solvent. 

Alternatively, chromatography columns can be monitored on line. Fluorescamine and o-phthalaldehyde are ideally suited for monitoring columns in this fashion, because they react rapidly and because the reagents are themselves nonfluorescent. Whereas fluorescamine reacts with both the a-amino graoup of the peptide and the -amino group of lysine residues, o-phthalaldehyde interacts only with those peptides that contain lysine residues (Joys and Kim, 1979). For preparative work, the column effluent is split so that only a portion is utilized for the fluorogenic reaction, and the remainder is collected. This can be accomplished with a discontinuous stream-sampling value, which directs aliquots of column effluent, at predetermined intervals, into the detection stream (Fig. 3) (Bohlen et a/., 1975). In this system, the proportion of column effluent taken for the fluorescence assay can be readily... 

Ohkura Y, Kai M, and Nohta H (1994) Fluorogenic reactions for biomedical chromatography. Journal of Chromatography B 659 85-107. 

Other immunoassays No fluorogenic reaction is needed if the radiolabel or the enzyme label is replaced by a fluorogenic label, which can be measured after the bound-free separation. An example of such a label is 4-methylumbelliferone 3-acetic acid. The advantage of these fluoroimmunoassay methods (FIA) is their simplicity. However, a disadvantage is that, as a consequence of the strong background fluorescence, the sensitivity is poor. 

Qian, W. J. Aspinwall, C. A. Battiste, M. A. Kennedy, R. T. Detection of secretion from single pancreatic (3-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy. Anal Chem. 2000, 72, 711-717. 

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