Enzymes fluorescence detection

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Macrocyclic complexes of zinc have inspired interest in varied areas such as supramolecular and biomimetic chemistry including hydrolysis enzymes, such as phosphatases and esterases, and also for the fluorescent detection of zinc. The polyaza macrocycles and their A--functionalized derivatives are particularly well represented. An important aspect of macrocycle synthesis is the use of metal templates to form the ligand. Examples of zinc as a template ion will be discussed where relevant. 

Separation-based assays are preferred in many applications because they allow discrimination of signals due to substrate, product, and interference. When assays that involve fluorescence detection are developed, they are typically carried out by employing plate readers. When separation-based methods are employed for these applications, the influences of interferences (quenchers and other fluorescent compounds) on the final results are minimized because both substrate and product are quantified. With a separation-based approach, the label employed does not need to be placed in close proximity to the site of action of the enzyme, therefore minimizing the effect of the label on the mode of action of the enzyme. Of course, it is often desirable to develop assays that employ substrates free of labels. 

Another method which uses capillary electrophoresis with laser-induced fluorescence detection can also be employed to detect zearalenone (Maragos and Appell 2007). In order to analyse trace amounts of zearalenone in plants, a sensitive, quick and accurate method, the enzyme-linked immunosorbent assay (ELISA) was developed by Chen et al. 1989. 

In addition to these research applications of fluorescence, there is a continuing use of fluorescence detection to replace analytical methods based on radioactivity, as can be judged from the recent books and conferences on fluorescence sensing methods. (7 n) These emerging applications of fluorescence can be seen by the growth and introduction of improved methods for immunoassays, enzyme-linked immunoassays... 

A second possibility is the modification (conjugation) of an antibody by a label, e.g., biotin, which is detected later on by a specific receptor, e.g., (strept)avidin. In each case the last part of such a cascade has to carry a measurable label. Such labels are enzymes, fluorescent dyes, colloids, radioactive isotopes, paramagnetic substances, and others. 

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

Abad, A., M.J. Moreno, R. Pelegrf, et al. 1999. Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection An analytical comparison. J. Chromatogr. A 833 3-12. 

Nunes, G.S., M.P. Marco, M. Farre, et al. 1999. Direct application of an enzyme-linked immunosorbent assay method for carbaryl determination in fruits and vegetables. Comparison with a liquid chromatography-postcolumn reaction fluorescence detection method. Anal. Chim. Acta 387 245-253. 

CL detection is based on the optical emission of an excited species formed in a chemical reaction. For instance, an excited species is formed when luminol (or 3-aminophthalhydrazide) reacted with H202 as catalyzed by various substances, such as metal ions or the peroxidase enzyme. CL detection is achieved as in fluorescent detection, except that no excitation light is needed. 

Figure 9.60 HPLC for the measurement of ferrochelatase activity in human leukocytes with mesoporphyrin and Zn2" as substrates, (a) Enzyme incubation mixture, (b) Blank incubation with boiled leukocytes. Column ODS-Hypersil (250 mm X 5.0 mm i.d.) eluent, 88% (v/v) methanol in 1 M ammonium acetate, pH 5.16. Flow rate, 1.5 mL/ min fluorescence detection, excitation at 403 nm and emission at 574 nm. Peaks 1, Zn-deuteroporphyrin (internal standard) 2, Zn-mesoporphyrin 3, mesoporphyrin. (From Guo et al., 1991.)...

---