Fluorescein and its derivatives

The cationic form has a higher fluorescence quantum yield than the neutral form, which allows the excitation ratio (490/435 nm) measurement to be extended down to pH 2. 

With the aim of increasing the apparent p/C, to a value more suitable for intracellular pH measurements, fluorescein derivatives have been designed CF (5(or 6)- 

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Fluorescein and its derivatives represent one of the most popular of all fluorescent labeling agents. Its fluorescent character is created by the presence of a multi-ring aromatic structure due to the planer nature of its upper, fused three-ring system (Figure 9.4). In its most elementary... 

The second family ofxanthene dyes is fluorescein and its derivatives. Fluorescein itself is only slightly fluorescent in alcohol solution. In contrast, the alkali salt obtained by addition of alkali exhibits the well-known yellow-green fluorescence characteristic of the fluorescein dianion (uranin). Fluorescein and its derivatives, e.g. eosin Yand erythrosin Y, are known to be very sensitive to pH and can thus be used as pH fluorescent probes (see Chapter 10). 

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. 

Fluorescein and its derivatives are one set of fluorescent labels that are commonly used to monitor DNA hybridization, and are often used as labels but do not selectively associate with the structure of double-stranded DNA. The most popular fluorescein derivative for nucleic acid labelling is FAM (car-boxyfluorescein). The structure of FAM is shown in Fig. 3. 

An adsorption indicator is typically an organic dye, such as fluorescein and its derivatives. Most adsorption indicators are weak acids. Their use is thus confined to basic, neutral, or slightly acidic solutions in which the indicator exists predominantly as the anion. Some cationic adsorption indicators are suitable for titrations in strongly acidic solutions. In this case, adsorption of the dye and coloration of the precipitate occur if the precipitate particles possess a negative charge. 

PEGs can be used in hpsticks as solubilizers for tetrabromo-fluorescein and its derivatives. According to Nowak (95), the solubility in PEG 400 is about 10%. Higher additions of PEG should be avoided because of their good solubility in water, since dyes then tend to bleed. 

Song A-M, Zhang J-H, Zhang M-H et al (2000) Spectral properties and structure of fluorescein and its alkyl derivatives in micelles. Colloids Surf A 167 253-262... 

Due to their longer wavelength fluorescence and photostability, rhodamine and its derivatives have often been employed for the labeling of probes tested in living cells. Like fluorescein, the chemical structure of rhodamine consists of an upper xanthene ring and a lower benzene ring. In this case, the xanthene ring is substituted... 

The main fluorescent pH indicator probes are based on fluorescein and therefore it is important to understand the pH-dependent ionic equilibria of it and its derivatives, hi aqueous solutions above pH 9 the phenolic and carboxylic acid functional groups in the molecule are almost totally ionised (Figure 3.14). Upon acidification of the dianion, firstly, protonation of the phenolic group occurs (pK 6.4) to yield the monoanion followed by the carboxylic acid (pA < 5), giving the neutral species of fluorescein. On further acidification the fluorescein cation pK 2.1) is generated. In strongly acidic environments fluorescein is non-fluorescent, only the mono-anion and di-anions are fluorescent, with quantum yields of 0.37 and 0.93, respectively. The pH-dependent absorption spectrum of fluorescein exhibits a blue-shift and... 

Evidence supporting this mechanism is presented for the case of acrylamide polymerization sensitized by riboflavin, but not for the case of fluorescein and its halogenated derivatives. Irradiation with a millisecond flash in the presence of air leads to polymer formation after an induction period of one hour. In contrast, when the irradiation is carried out with degassed solutions, polymerization starts only after the sample is exposed to atmospheric oxygen. 

A number of derivatives of fluorescein are useful dyes. Uranin is the disodium salt of fluorescein. ChrysoUn is a salt of benzyl-fluorescein, which is formed from phthalic anhydride and benzyl-resorcinol. It dyes silk and wool a fast yellow, and is used in cotton dyeing. Eosin is the disodium salt of tetrabromo-fluorescein, which is prepared by the action of bromine on fluorescein. Its dilute alkaline solution is rose-colored, and exhibits an intense yellow-green fluorescence. Other dyes related to fluorescein are tetrabromo-dichlorofluorescein and various ethers of fluorescein and its substitution-products. The dyes of this class produce on silk and wool shades from yellow to reddish yellow, cherry-red, and purple. 

Eosin (Tetrabromofluorescein). Place 16 5 g. of powdered fluorescein and 80 ml. of rectified (or methylated) spirit in a 250 ml. flask. Support a small dropping funnel, containing 36 g. (12 ml.) of bromine, above the flask make sure that the stopcock of the funnel is well lubricated before charging the latter with bromine. Add the bromine diopwise during about 20 minutes. When half the bromine has been introduced, and the fluorescein has been converted into dibromofluor-escein, all the solid material disappears temporarily since the dibromo derivative is soluble in alcohol with further addition of bromine the tetrabromofluorescein (sparingly soluble in alcohol) separates out. Allow the reaction mixture to stand for 2 hours, filter ofiF the eosin at the pump, wash it with alcohol, and dry at 100°. The yield of eosin (orange-coloured powder) is 25 g. 

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

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