Spectroscopy, fluorescein

In the literature, fluorescence spectroscopy in OFD has been limited to the use of ultraviolet (UV) or visible dyes as molecular probes.(1) The most common fluorescent dye used in OFD is fluorescein and its derivatives/21 23) Fluorescein possesses a good fluorescence quantum yield and is commercially available with an isothiocyanate functionality for linking to the polymeric support/24-26 Additionally, selective laser excitation can be performed because the absorbance maximum of fluorescein coincides with the 499-nm laser line emitted from an argon laser. Unfortunately, argon lasers are costly and bulky, thus limiting the practicality of their use. Similar difficulties exist with other popular commercial dyes. 

Techniques which allow one to monitor the boundary layer as a function of time, such as total internal reflection fluorescence (TIRF) spectroscopy 4 43), permit a quantitative evaluation of interfacial mass transport processes using, for example, fluorescently-tagged macromolecules which do not adsorb, such as fluorescein-labeled dextran 40 ... 

Optical absorption to a higher triplet has afforded further evidence that the emitting state in phosphorescence is a triplet. Intense irradiation of a boric acid glass containing fluorescein leads to the appearance of a new absorption band due to triplet-triplet absorption. Flash photolysis, in which a sample is exposed to a brief, intense flash of light, can be used to produce high transient concentrations of triplet species kinetic absorption spectroscopy of the system enables the build-up and decay of several singlet and triplet levels to be followed as a function of time. 

The effect of an electrical field on the absorption/emission of spectra of a probe such as fluorescein or a coumarin derivative. It is derived from the interaction of the induced dipole(s) in the probe interacting with the charged group. See Sitkoff, D., Lockhart, D.J., Sharp, K.A., and Honig, B., Calculation of electrostatic effects at the amino terminal of an helix, Biophys. J. 67,2251-2260,1994 Pierce, D.W. and Boxer, S.A., Stark effect spectroscopy of tryptophan, B/opfiyx. J. 68,1583-1591,1995 Klymchenko, A.S., Avilov, S.V., and Demchenko, A.P., Resolution of Cys and Lys labeling of a-crystalUn with site-sensitive fluorescent 3-hydroxyflavone dye. Anal. Biochem. 329, 43-57, 2004. 

When photo-electron transfer is used with a dye, such as eosin or dibromo-fluorescein in combination with NADH (and the dithionite concentration is low, ca 4 X 10" m), no dissociation of proteins is detected. Kinetic laser spectroscopy helped Syrtsova et al. [9] to follow electron transfer from the Fe protein to the MoFe protein and it was shown that, unlike the situation with dithionite, Fe protein in the complex with MoFe protein could undergo reduction by the photodonor as efficiently as in the free state in solution and electron transfer proceeds in the complex of two proteins without dissociation. 

Fluorescence polarization has proved to give high sensitivity in determination of avidin and biotin by means of the fluorescein conjugate. Synchronous derivative spectroscopy can also be useful as exemplified by measurement of pesticide compositions . 

Total internal reflection fluorescence spectroscopy has been used to assay the fluorescence of tryptophan in proteins or of fluorescence markers. Morphine has been determined in this manner with a detection limit of 0.2 pmol/l on a quartz support bearing immobilized fluorescein-labeled antihapten (Kronick and Little, 1973). 

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