Fluorescence detection methods

Fig. 1.14. Multiphoton excitation scheme in Hj showing the laser-induced fluorescence detection method...

M.A. Lage Yusti and J.L. Cortizo Davina, Supercritical fluid extraction and high-performance hquid chromatography-fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil. Food Cont. 16 (2005) 59-64. 

Cholerton, S., Idle, M E., Vas, A., Gonzalez, F.J. Idle, J.R. (1992) Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorimetric method for the determination of 7-hydroxycoumarin in human urine. J. Chromatogr., 575, 325-330 Cohen, A.J. (1979) Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food chem. Toxicol, 17, 277-289... 

Since the effect they produce is actually an irritation to the nerve endings responsible for heat and pain sensation in the mouth, standardization of the level of heat is essential. Methods for their analysis include organoleptic and spectrophotometric methods. Historically the organoleptic one has been preferred by the food industry since it is a direct measure of levels of heat. Nowadays a variety of GC (after derivatization) and HPLC with UV or fluorescence detection methods have been proposed. 

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. 

Cholerton S, Idle ME, Vas A, et al. Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorometric method for the determination of 7-hydroxycoumarin in human urine. J Chromatogr 1992 575 325-330. 

The work discussed above demonstrates that it is possible to use molecular monolayers on silicon for label-free electrical detection of DNA hybridization. More work remains to fully understand the details of the observed response, optimize the sensitivity of this approach and benchmark it with respect to fluorescence detection methods. However, this work points... 

Li, B., Gutierrez, P. L., and Blough, N. V.. (1999) Trace determination ofhydroxyl radical using fluorescence detection, Methods in Enzymology 300 (Oxidants and Antioxidants, PartB), 202-216. 

Crespi, C.L., Chang, T.K., and Waxman, D.J. CYP2D6-dependent bufuralol 1 -hydroxylation assayed by reverse-phase ion-pair high-performance liquid chromatography with fluorescence detection. Methods Mol. Biol. 2006, 320, 121-125. 

Sora, D.I. et al. Vahdated ion-pair liquid chromatography and fluorescence detection method for assessing the variability of loratadine metabolism occurring in bioequivalence studies. Biomed Chmmatogr. 2007, 21, 1023-1029. 

GuL. KelmM. Hammerstone J.F. Beecher G. Cunningham D. Vannozzi S. Prior R. L. 2002. Fractionation of polymeric procyanidins from lowhush blueberry and quantification of procyanidins in selected foods with an optimized normal-phase HPLC-MS fluorescent detection method. J. Agric. Food Chem. 50 4852-4860. 

Robbins, R.J. Leonczak, J. Johnson, J.C. Li, J. Kwik-Uribe, C. Prior, R.L. Gu, L. 2009. Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography-fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. J. Chromatogra. A 1216 4831 840. 

Recently, a small molecule fluorophore phosphosensor technology referred as Pro-Q Diamond dye has been developed to detect and quantitate phosphorylated amino acids within peptides and proteins in microarrays. ° In addition to binding assays, fluorescence detection methods have also been developed for functional assays. For example, microarrays of quenched fluorescent substrates can be used to detect protease or esterase activities in the analytes. In this method, quenched fluorescent substrates are prepared by coupling the peptide substrate to coumarin, a fluorescent dye. These peptide substrates are then spotted onto the solid support... 

The following sections give an overview and some examples of the fluorescence detection methods currently used in HTS fluorescence intensity (FI) fluorescence polarization (FP) or fluorescence anisotropy (FA) fluorescence resonance energy transfer (FRET) Hfetime-based measurements (TRF and FLT) fluorescence corre-... 

The most common type of capture molecules used on a protein chip are antibodies, though other proteins, such as peptides, nucleic acids, enzymes and receptors, have been spotted on to chips. Protein chips can be difficult to prepare, as the proteins need to be kept in an active state, in a high concentration and in a moist environment. Typical samples investigated are cell lysates for general research or blood and urine for diagnostic applications. Fluorescence detection methods are preferred since they are simple and sensitive. Other detection methods, where labelling is not required, can take the form of SELDl or AFM. 

Lee and Field reported a selective fluorescence detection method for the determination of some A -nitrosamines after a postcolumn reaction. The nitrosamines eluted from the column are first hydrolyzed to produce the nitrite anion, which is then oxidized with Ce" to give the fluorescent Ce. The detection limit for this method is at the ppb level. A more sensitive fluorimetric method has been developed based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the highly fluorescent product l-(H)-naphthotriazole. About 10 nmol/1 of nitrite can be detected by this procedure. 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

Munro et al. showed separation and detection of amino acids on microchips using an indirect fluorescence detection method. Figure 36.10 shows application of this method to urine samples with no pretreatment other than dilution in the appropriate separation buffer. Abnormal amounts of amino acids can easily be detected in the two patient samples compared to the healthy control sample. An absorbance detection based approach was utilized for the clinical analysis of calcium ion in serum, which is important in the regulation of a number of physiological processes. Beads with an immobilized calcium reactive dye were placed into the detection region, and the samples mobilized past the beads using electrophoretic flow. While a true separation was not intended, the interference... 

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