Assays Using Fluorescence

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Two subsequent chemical transformations are necessary. First, the enantiomeric alcohols R)- and ( S)-12 are oxidized separately by horse-liver alcohol dehydrogenase 

Thirty different esterases and lipases were tested. The rate of release of 14 in the wells of standard microtiter plates was monitored by fluorescence (70,86). Control experiments ensured that the apparent rate of release of 14 is directly proportional to the rate of acetate hydrolysis. The predicted and observed E and ee values (as checked by standard HPLC assay on a chiral phase) were found to match within + 20%. Only in one case was a larger discrepancy observed, a result that was believed to be caused by the occurrence of an unusually low value of Km for one of the enantiomers. 

because the test can be carried out on 96-well microtiter plates, high throughput is possible. Of course, the inherent disadvantage noted above for some of the colorimetric tests also applies here, namely, the fact that the optimization of a potential catalyst is focused on a specific substrate 11 modified by incorporation of a probe, in this case the fluorogenic moiety 14. However, one can expect the test to be useful in directed evolution projects in which proof of principle is the goal. Moreover, this kind of approach can be used in very practical applications, namely as a pre-test for the activity of enzymes. 

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An HPLC assay using fluorescence detection was presented for OTC and CTC residues in animal tissues. The sample cleanup was based on SPE on a C18 cartridge followed by an additional cleanup on an SCX cartridge. The TCs were eluted with MeOH 1 M hydrochloric acid solution the extract was alkalinized to pH 12.0 and allowed to stand for 1 hour when the fluorescence of CTC reached the maximal value. The influence of the pH value of the mobile phase on the fluorescence spectra of both OTC and CTC was studied. The reduction of fluorescence yields was explained by the decomposition to their iso-derivatives (38). 

Method B. Bisgaier et al. [78] reported a CETP assay using fluorescent cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenedodecanoate (BODIPY-CE, Fig. 4) in microemulsions. Microemulsions for donor/acceptor contain triolein, BODIPY-CE/cholesteryl oleate, and l-hexadecanoyl-2-[cw-9-octadecenoyl]-s -glycero-3-phosphocholine. The assay mixtures consist of acceptor microemulsions, donor microemulsions, and a test sample in each well of flat-bottom 96-well plates. After preincubation at 37°C for 10 min, the reaction is initiated by addition of CETP solution. The fluorescent intensity in each well of the plates is periodically (every 10 sec) detected at 37°C with a fluorescent 96-well plate reader equipped with 485- and 538-nm bandpass filters in the excitation and emission paths, respectively. 

Parker, G.J. et al. 2000. Development of high-throughput screening assays using fluorescence polarization nuclear receptor-ligand-binding and kinase/phosphatase assays. J. Biomol. Screen. 5, 77-88. 

Marme, N. et al. 2004. Highly sensitive protease assay using fluorescence quenching of peptide probes based on photoinduced electron transfer. Angew. Chem. Int. Ed. 43, 3798-3801. 

Mayer-Almes, F.J. and Auer, M. 2000. Enzyme inhibition assays using fluorescence correlation spectroscopy a new algorithm for the derivation of kcJKM and Kt values at substrate concentrations much lower than the Michaelis constant Biochemistry, 39, 13261-13268. 

Oberheitmann B, Schafer J, Dally H, Garms A, Frentzel-Beyme R and Hoffmann W (1999) The chromosome-based challenge assay using fluorescence in situ hybridisation a possible test for increased cancer susceptibility. Mutat Res 428 157 -164. 

Interferes with digoxin assay using fluorescence polarization immunoassay (ashwagandha, ginseng and Siberian ginseng falsely elevate, Asian ginseng and dan shen T or i levels)... 

Toxiline-DSP is an assay using fluorescence detection. The method is suitable for shellfish species such as mussels, clams, oysters, and scallops. The kit is supplied with all the reagents needed for the assay and allows running up to 26 samples at a time. It has a shelf-life of 3-6 months and is available for purchase. For information or to place orders, please contact Elena Dommguez at Elena zeu-inmunotec.com or phone -t34 976531533-Ext 101. 

Proliferation assays on matched sensitive and resistant cell hnes Transport assays in monolayers of cells expressing P-gp A colorimetric assay for P-gp s ATPase activity Cellular assays using fluorescent or pro-fluorescent P-gp substrates... 

Zyomyx (Hayward, GA) has developed the Zyomyx human cytokine biochip that uses microfluidics to measure 30 different human cytokines simultaneously. Biosite (San Diego, GA) markets commercial microfluidic chip-based immunoassay for clinical applications. Multiplexed assays using fluorescent microspheres/beads in Lab-on-Ghip format manufactured by Becton-Dickinson (Franklin, NJ), Diasorin (Stillwater, MN), and Luminex Gorp. (Austin, TX) are also available for use in clinics. 

Stenroos K, Hurskainen P, Eriksson S et al (1998) Homogeneous time-resolved 1L-2-IL-2R alpha assay using fluorescence resonance energy transfer. Cytokine 10 495 99... 

Yoshida, M. Nishino, N. Non-isotopic histone deacetylase activity assay using fluorescent or chromogenic e-acetyl lysine peptide derivative substrate. Jpn. Kokai Tokkyo Koho JP 2003221398, 2003. 

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