Fluorescence nature

Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373 663-664... 

The detection of the individual C vitamers is complicated by their distinctly different properties. Although AA and DHAA are both ultraviolet (UV) absorbers, the absorbance maximum of DHAA is between 210 and 230 nm (15,18,42,43). For practical detection purposes, this makes DHAA particularly susceptible to interferences from a number of naturally occurring food constituents and limits the choice of reagents and solvents. In contrast, AA exhibits a pH-dependent absorbance maximum of 245-265 nm, which makes UV absorbance an ideal choice for detection. On the strength of its reducing capacity, AA can be detected electrochemically, but DHAA is electrochemically inactive. Neither AA nor DHAA fluoresce naturally. However, DHAA readily forms a fluorescent quinoxaline derivative upon reaction with o-phenylenediamine. As a result, chemical derivatization is often used to achieve the sensitivity needed to detect the naturally occurring vitamin C in food. 

The riboflavin vitamers all have similar UV absorbance spectra. They all fluoresce naturally, although the quantum yield of FAD s fluorescence is 10 times less than that of riboflavin and FMN... 

Detection of the B6 vitamers is complicated by the low levels at which they occur in foods (102,103). The sensitivity and specificity of the detection methods is therefore critical. All of the principal B6 vitamers are UV absorbers (70). Although their spectra are similar in 0.1 M hydrochloric acid, this is not the case at higher pH. These vitamers fluoresce naturally in slightly acidic to neutral solution and under strongly alkaline conditions (42,70). However, the individual vitamers exhibit some qualitative dissimilarities in their fluorescence spectra and significant differences in the intensities of their quantum fluorescence response PLP is significantly less fluorescent than the other five vitamers. In general, fluorescence is the preferred method of detection, due to its increased sensitivity and specificity relative to UV absorbance. Derivatization has been used to enhance and standardize the fluorescence properties of the B6 vitamers. Detailed reviews of the spectral properties of the B6 vitamers have been published (102,103). 

A high degree of sensitivity and selectivity can be obtained with certain biomolecules by the chemical attachment of fluorophores. The most common fluorescent derivatization reagents include fluorescamine, dansyl chloride, pyridoxal, pyridoxal 5-phosphate, dansyl hydrazine, and pyr-idoxamine. Such derivatization procedures can be used to enhance the fluorescence of compounds with low quantum yields as well as impart fluorescent properties to compounds that do not fluoresce naturally. 

Whitcombe, D. Theaker, J. Guy, S.P. Brown, T. Little, S. Detection of PCR products using self-probing ampli-cons and fluorescence. Nature Biotechnology. 1999, 17, 804-807. 

Not many compounds fluoresce naturally. However, some compounds, when added to another compound, cause that compound to fluoresce. These compounds are called fluorophores. Dansyl chloride and fluorescamine are two that are used to react with primary amines. o-Phthalaldehyde is used as a postcolumn reactant to produce fluorescent compounds. Other compounds are naphthalenedialdehyde (NDA), fluorescein isothiocyanate (FITC), and phenylthiohydantoin (PTH). See Chapter 19, p. 203, for more detail. 

One of the developments in the area of confocal microscopy is the use of fluorescence to highlight or visualise certain features that either fluoresce naturally or are derivatised with probes prior to microscopic examination. Another new development is two-photon microscopes, which allow operation in the UV spectrum and, hence, deeper penetration of structures such as living cells, skin and other biological samples. 

The most important fluorescing natural compounds are porphyrins, flavins, and reduced nicotinamide. The most important quenchers are triplet oxygen, redox active dyes, radicals, and transition metal ions. 

Borson, N.D. Strausbauch, M.A. Wettstein, P.J. Oda, R.P. Johnston, S.L. Landers, J.P. Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis. Biotechniques 1998,25,130-137. Lu, W. Han, D.S. Yuan, J. Andrieu, J.M. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence. Nature 1994, 368, 269-271. 

Sessler and coworkers expanded the calix[4]pyrrole binding cavity by employing bipyrrole in place of pyrrole, yielding new calixpyrrole analogs such as calix[n]bipyrroles" (n = 3, 4), a calix[2]bipyrrole[2]furan, and a calix[2]bipyr-role[2]thiophene. The carbazole moiety was also an attractive linker to include as the fluorescent nature of... 

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