Fluorescence intensity, fluorescein isothiocyanate

Further functionalization of mesoporous films with the pH-sensitive dye fluorescein was accomplished by Wirnsberger et al.204 The organosilane used for the actual co-condensation reaction was first prepared by reaction of fluorescein isothiocyanate (FITC) with APTES. The possible use of the dye-modified films as pH sensors was investigated by measurement of the fluorescence after excitation with an Argon laser (488 nm) a dramatic change in fluorescence intensity was observed around pH 8 with a response time of a few seconds. 

Signal intensity can be increased by double antibody labelling, a procedure in which both the primary and secondary antibody are tagged with fluorescein isothiocyanate (FITC). As these two fluorescent signals are additive, the signal intensity will be markedly increased (Aurell et al. 2004). 

Fuh et al. (1988) devised an enzyme optrode for penicillin. (5-Lactamase was immobilized on a fluorescein isothiocyanate-labeled porous glass particle which was glued to the tip of a fiber optic bundle. Excitation was carried out by an argon laser. pH changes resulting from the enzyme reaction led to changes of the fluorescence intensity. The response time of the sensor was 20-45 s, the detection limit being 0.1 mmol/1 penicillin. 

Fluorescent dye-polypeptide conjugates were prepared by labeling with either fluorescein isothiocyanate adsorbed onto Celite (51) or with 1-dimethylaminonaphthalene sulfonyl chloride (DNS) dissolved in ethanol (65). The degree of labeling was determined from the amount of dye on the polypeptide estimated by fluorescent intensity or ultraviolet absorption measurements and from the concentration of the polypeptide determined by Kjeldahl nitrogen. There were generally two to four dye residues per polypeptide molecule. 

FIGURE 32.13 Microfluidic multi-injector, (a) 3D schematic representation of the device shows two small-diameter channels under the control of microfluidic valves that pneumatically eject fluid into a cell culture reservoir to form soluble molecule gradients, (b) Top view of the device in operation forming gradients of fluorescein isothiocyanate (FITC)-conjugated dextran. (c) 3D plot of the fluorescence intensity within the cell culture reservoir. (Adapted from Chung, B. G., et al., Lab Chip, 6, 6, 764, 2006. Reproduced with permission from The Royal Society of Chemistry.)... 

Sulphhydryl groups react with fluorescein isothiocyanate, and phenolic groups (of tyrosine) react to a small extent under the above reaction conditions. Fluorescein thiohydantions in amounts exceeding 1 nmol are visible as yellow spots on TLC plates and show up as intense greenish-yellow fluorescent spots under UV light. 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

FITC is probably the most popular fluorescent probe ever created. An isothiocyanate derivative of fluorescein is synthesized by modification of its lower ring at the 5- or 6-carbon positions. The two resulting isomers are nearly identical in their reactivity and spectral properties, including excitation and emission wavelengths and intensities. Their chemical differences, however, may affect the separation of modified proteins from excess reagent or the analysis of tagged molecules by electrophoresis. For this reason, most manufacturers purify the carbon-5 derivative as the FITC reagent of choice. 

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