Fluorimetric Assays

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

PROCEDURE 8.6 Fluorimetric assay of u-amino acid oxidase (fixed time)... 

Burke, M. D. and Mayer, R. T. Ethoxyresorufin direct fluorimetric assay of a microsomal 0-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Dispos. (1974) 2 583-588. 

Another approach of using fluorimetric assays for screening purposes is the use of coupled enzyme systems. McElroy et al. presented an assay for glutamate... 

Stirling JL, Robinson D, Fensom AH, Benson PF, Baker JE (1978) Fluorimetric assay for prenatal detection of Hurler and Scheie homozygotes or heterozygotes. Lancet 1 147... 

Van Diggelen OP, Voznyi YV, Keulemans JLM, Schoonderwoerd K, Ledvinova J, Mengel E, Zschiesche M, Santer R, Harzer (2005) A new fluorimetric assay for the diagnosis of Niemann-Pick A/ , with specificity of natural sphingomyelinase substrate. J Inherit Metab Dis 28 733-741... 

Example Fluorimetric Assay of Selenium in Brazil Nuts... 

There are many ways to estimate microbial growth. The simplest is visual inspection of colonies growing on agar plates, though this method is difficult to adapt for HTS. There is a well-known correlation between cell density and optical density, which can be exploited in a 96-well microtiter plate format (e.g., [43]). Measurement of the incorporation of radioactive nutrients is an excellent quantitative method, but has fallen from favor due to concerns about spills and contamination. Finally, both spectrophotometric and fluorimetric assays are conveniently adapted to HTS formats, and Alamar blue is only one example of the tools available for this purpose. As mentioned, we have even found it convenient to use simple visual inspection of Alamar blue plates to identify wells of interest. However, quantification obtainable with a microplate reader is attractive in many settings. 

Fig. 9.6 Recombinant Factor C (rFC) is activated by endotoxin into rFC . (a) A single-step activation of rFC which hydrolyses a substrate to yield either a colored (colorimetric assay) or fluorescent (fluorimetric assay) product, hence, (b) reporting on the presence and quantity of endotoxin present in samples (a colorimetric assay, in this case)...

A fluorimetric assay method for the determination of acetylcholine with picomole sensitivity was reported by MacDonald [44]. The method is based on the hydrolysis of acetylcholine to choline and acetate, catalyzed in the presence of acetylcholineesterase, oxidation of choline to betaine, and H202 in the presence of choline oxidase, and oxidation of 4-hydroxyphenylacetic acid by H202 to a fluorescent product, catalyzed by peroxidase. The interference in the analysis of brain homogenates was discussed. 

Stead, D. 1984. Evaluation of a fluorimetric assay on the lipases from strains of milk psychro-trophic bacteria.. / Dairy Res. 51, 123-130. 

Grilli S, Ancora G, Rani P, et al. 1991. In vivo unwinding fluorimetric assay as evidence of the damage induced byfenarimol and DNOC in rat liver DNA. J Toxicol Environ Health 34 485-494. 

Klein, G. and Reymond, J.L. (1998) An enantioselective fluorimetric assay for alcohol dehydrogenases using albumin-catalyzed beta-elimination of... 

Total thioridazine plus metabolites determined by a non-specific fluorimetric assay. 

The reaction can be followed by O2 uptake (manometrically or oxygen electrode), by determination of ammonia, or by determination of hydrogen peroxide. A fluorimetric assay procedure for determining the hydrogen peroxide produced permits determinations of n-amino acids in the 1-100 /xg/ml range (53). 

CG Knight. Fluorimetric assays of proteolytic enzymes. Meth Enzymol 248 18— 34, 1995. 

Among different coumarin derivatives used, 7-Amino-4-trifluoromethylcoumarin (ATFMC) revealed the most promising characteristics as an efficient fluorescent emitter. AFTMC is used in the synthesis of a substrate for fluorimetric assay of proteolytic enzymes and for use as a laser dye. We have recently investigated the chemiluminescence reactions of some peroxyoxalate esters, hydrogen peroxide and AFTMC. In this paper we report the solvent effects on the kinetics of the chemiluminescence process of the peroxyoxalate chemiluminescence in the presence of AFTMC. 

Of the various fluorimetric assays available for sialic acid analysis, the method which allows the discrimination between sialic acids with or without 0-acyl groups at C8 and/or C9 may be of interest [6,265], After mild periodate oxidation, formaldehyde, derived from C9 in the case of non-substituted H09 and H08 is derivatized with acetylacetone in the presence of ammonium acetate, leading to the fluorigen 3,5-diacetyl-l,4-dihydro-2,6-dimethylpyridine (410nm excitation, 510nm emission). It is evident that all contaminants producing formaldehyde under the influence of periodate will interfere with this sialic acid analysis. 

The fluorescence of the ortho-aminobenzoate motif was utilized to elaborate a continuous fluorimetric assay based on intramolecular fluorescence quenching through isomer-differential collision of the fluorophore and a quencher, i.e. nitro-phenylalanine or a para-nitrophenyl moiety at the C-terminus [57]. Relative fluorescence recorded at 410 nm (2ex = 317 nm) is cleared from the participation of the preexisting trans isomer, but this assay is limited to peptides bearing fluoro-phores and properly positioned fluorescence quencher moieties. 

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