Fluorescent salt sensitivity

GFP-derived YFPs are not only suffering from photobleaching they are also notorious for their sensitivity to pH and anions, especially chloride [76]. However, the already mentioned improved isoforms Citrine and Venus not only show higher photostability (see above), they also show reduced sensitivity to pH changes and are resistant to chloride. Citrine remains 50% fluorescent at pH 5.7, the lowest pH that has been reported so far for a GFP-derived YFP. As yet, no data about the pH-dependence and salt sensitivity of zoanYFP were published. 

The interface between two immiscible liquids is used as a characteristic boundary for study of charge equilibrium, adsorption, and transport. Interfacial potential differences across the liquid-liquid boundary are explained theoretically and documented in experimental studies with fluorescent, potential-sensitive dyes. The results show that the presence of an inert salt or a physiological electrolyte is essential for the function of the dyes. Impedance measurements are used for studies of bovine serum albumin (BSA) adsorption on the interface. Methods for determination of liquid-liquid capacitance influenced by the presence of BSA are shown. The potential of zero charge of the interface was obtained for 0-200 ppm of BSA. The impedance behavior is also discussed as a function of pH. A recent new approach, using a microinterface for interfacial ion transport, is outlined. 

Kader, M. A. Lindberg, S. Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI. J. Exp. Bot. 2005, 56, 3149-3158. 

The fluorescent compound F, a luciferin, emits blue light (Amax 476 nm Fig. 3.2.4) in the presence of molecular oxygen and the protein P, a luciferase. In the luminescence reaction, F is changed into an oxidized form (structure 8, Fig. 3.2.6). The luminescence reaction is highly sensitive to pH, with a narrow optimal range around pH 7.8 (Fig. 3.2.2) the optimum salt concentration is 0.15 M for NaCl... 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

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). 

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

Tris(bpy) complexes of ruthenium(II) with pendant catechol units are represented by [Ru(bpy)2(114)], isolated as the BF4 salt. Interest in this fluorescent complex stems from its use as a potential skin sensitizer. " In the complex [Ru(bpy)2(115)] + (R = H), the deprotonated catechol unit can act as a binding site for other metal fragments, thereby forming homo- and... 

Manganese in aqueous solution may be analyzed by several instrumental techniques including flame and furnace AA, ICP, ICP-MS, x-ray fluorescence and neutron activation. For atomic absorption and emission spectrometric determination the measurement may be done at the wavelengths 279.5, 257.61 or 294.92 nm respectively. The metal or its insoluble compounds must be digested with nitric acid alone or in combination with another acid. Soluble salts may be dissolved in water and the aqueous solution analyzed. X-ray methods may be applied for non-destructive determination of the metal. The detection limits in these methods are higher than those obtained by the AA or ICP methods. ICP-MS is the most sensitive technique. Several colorimetric methods also are known, but such measurements require that the manganese salts be aqueous. These methods are susceptible to interference. 

Silver metal and its contents in silver alloys and salts can be measured at trace levels by various instrumental techniques such as flame- and furnace-AA, ICP-AES, ICP/MS and x-ray fluorescence methods. It is solubilized by digestion with nitric acid prior to analysis. The AA measurement may be carried out at the wavelength 328.1 nm and ICP analysis at 328.07 nm. ICP/MS is the most sensitive technique while x-ray fluorescence is relatively less sen-... 

Ertekin and coworkers developed an additional optical COj sensor based on the fluorescence signal intensity changes of the pH-sensitive fluorescent dye 8-hydroxypyrene-l,3,6-trisulfonic acid trisodium salt (HPTS) dissolved in ILs [18]. When HCO3 was added to HPTS solution, the fluorescence intensity of the peak centered around 520 nm decreased by 90% in [C4Qlm] [BF4] and by 75% in [C4Cilm]Br. The reported detection limit for CO2 (g) was 1.4% while the detection limit for dissolved COj was 10 M HCO3. The sensor exhibited excellent stability and repeatability over a time period >7 months. 

Fluorescence spectroscopy is probably the most sensitive method for determining the purity of an IL. It has been observed that the fluorescence of ILs can be effectively reduced to zero by a systematic purification of precursor salts. For the lowest fluorescence response, particular care should be taken while carrying out s)mthesis. For convenience it is possible to judge the degree of fluorescence impurity in the IL by exposing a sample to the UV light from a hand scanner at 380 nm. If the IL has significant impurities, it will fluoresce visibly [40]. 

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

The participation of the singlet states of dyes such as rose bengal (RB, tetraiodotetrachlorofluorescein, sodium salt) and MB during the sensitized oxygenation of amines was demonstrated by Davidson (40). Fluorescence quenching Stem-Volmer constants were reported for several amines, halide anions, and for 3-carotene and were consistent with charge transfer stabilization of an exciplex quenching intermediate. 

Only aldehydes with an a-methylene group (i, e., R—CH2CHO) condense with 3,5-diaminobenzoic acid to form quinaldines.92 Consequently, in the carbohydrate series only 2-desoxysugars will react. The usual procedure adopted is to add to the unknown sugar solution an equal volume of a 1.3 percent solution of 3,5-diaminobenzoic acid hydrochloride in 50 percent aqueous perchloric acid. If the carbohydrate solution contains a 2-desoxysugar it will assume a yellow color with a green fluorescence.93 Use of an ester instead of a salt of 3,5-diaminobenzoic acid reduces the sensitivity of the test. 

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