PH indicators, fluorescent

FRET sensing based on protic equilibrium in the acceptor that changes its absorption spectrum and thus modulates the overlap integral [35]. There are many fluorescent pH indicators that display pH-dependent absorption spectra in the visible with their different positions depending on ionization state. Thus, the change in pH can be translated into the change of FRET efficiency. 

Cyanine and squaraine dyes with hydrogen substituents on the indolenine-nitrogen in one or both of the heterocyclic end-groups, the so-called norcyanines and norsquaraines, are useful as fluorescent pH-indicators due to the reversible equilibrium between their protonated and deprotonated forms ... 

The sol-gel co-immobilization of a non-fluorescent blue indicator bromothymol blue (BTB) with an europium (Ill)-complex intense antenna mediated lanthanide dye represents a new scheme for the fluorescence analysis38. Luminescence spectra of europium (Ill)-complex shown in Figure 12 were found to be independent of pH changes in the range 1-10. Therefore, BTB, a non-fluorescent pH indicator with alkaline absorption maximum close to main europium emission band was added to the sol-gel mixture to shield reversibly the emission of the europium (Ill)-complex at different pH s without quenching of the antenna function. 

Figure 13 shows pH response of bromothymol blue (BTB) as coimmobilized non fluorescent pH indicator detect by fluorescence. 

An optical sensor for the measurement of carbon dioxide in modified atmosphere packaging (MAP) applications was developed89. It was based on the fluorescent pH indicator l-hydroxypyrene-3,6,8-trisulfonate (HPTS) immobilized in a hydrophobic organically modified (ormosil) matrix. The CO2 sensor was stable over a period of at least 7 months and its output was in excellent agreement with a standard reference method for carbon dioxide analysis. 

Center for Healthcare Technologies at Lawrence Livermore National Laboratory in Livermore, potentially capable to measure pH at or near the stroke site29. The probe is the distal end of a 125 pm fibre tapered up to a diameter of 50 pm. A fluorescent pH-indicator, seminaphthorhodamine-1-carboxylate, is embedded inside a silica sol-gel matrix which is fixed to the fibre tip. Excitation of the dye takes place at 533 nm and the emission in correspondence of the acid (580 nm) and basic (640 nm) bands are separately detected. The use of this ratiometric technique obviates worrying about source fluctuations, which have the same effects on the two detected signals. The pH sensor developed was first characterised in the laboratory, where it showed fast response time (of the order of tens of seconds) and an accuracy of 0.05 pH units, well below the limit of detection necessary for this clinical application (0.1 pH units). The pH sensor was also tested in vivo on rats, by placing the pH sensor in the brain of a Spraque-Dawley rat at a depth of approximately 5 mm30. 

Yao S, Schafer-Hales KJ, Belfield KD (2007) A new water-soluble near-neutral ratiometric fluorescent pH indicator. Org Lett 9 5645-8... 

Class 2 fluorophores that can reversibly bind an analyte. If the analyte is a proton, the term fluorescent pH indicator is often used. If the analyte is an ion, the term fluorescent chelating agent is appropriate. Fluorescence can be either quenched upon binding (CEQ type Chelation Enhancement of Quenching), or enhanced (CEF type Chelation Enhancement of Fluorescence). In the latter case, the compound is said to be fluorogenic [e.g. 8-hydroxyquinoline (oxine)]. 

Fluorescent pH indicators offer much better sensitivity than the classical dyes such as phenolphthalein, thymol blue, etc., based on color change. They are thus widely used in analytical chemistry, bioanalytical chemistry, cellular biology (for measuring intracellular pH), medicine (for monitoring pH and pCC>2 in blood pCC>2 is determined via the bicarbonate couple). Fluorescence microscopy can provide spatial information on pH. Moreover, remote sensing of pH is possible by means of fiber optic chemical sensors. 

In almost all applications, fluorescent pH indicators are employed in a pH range around the ground state pKa (even if the excited state pK is different). Therefore, the absorption (and excitation) spectrum depends on pH in the investigated range. These indicators can be divided into three classes (see formulae in Figure 10.2) on the basis of the elementary processes (photoinduced proton transfer or electron transfer) that are involved. 

Fig. 10.2. Various classes of fluorescent pH indicators. (P-1 to P-8 Haugland R. P., Handbook of Fluorescent Probes and Research Chemicals, 6th edn, Molecular Probes, Inc., Eugene, OR. P-9 de Silva A. P. et al. (1989)...

Tab. 10.1. Examples of fluorescent pH indicators allowing ratiometric measurements. It should be recalled that the values of pKa (given here at room temperature for dilute solutions) can be more or less affected by changes in ionic strength and temperature...

The ratiometric measurements are preferable because the ratio of the fluorescence intensities at two wavelengths is in fact independent of the total concentration of the dye, photobleaching, fluctuations of the source intensity, sensitivity of the instrument, etc. The characteristics of some fluorescent pH indicators allowing ratiometric measurements are given in Table 10.1. 

Various pH sensors have been built with a fluorescent pH indicator (fluorescein, eosin Y, pyranine, 4-methylumbelliferone, SNARF, carboxy-SNAFL) immobilized at the tip of an optical fiber. The response of a pH sensor corresponds to the titration curve of the indicator, which has a sigmoidal shape with an inflection point for pH = pK , but it should be emphasized that the effective pKa value can be strongly influenced by the physical and chemical properties of the matrix in which the indicator is entrapped (or of the surface on which it is immobilized) without forgetting the dependence on temperature and ionic strength. In solution, the dynamic range is restricted to approximately two pH units, whereas it can be significantly extended (up to four units) when the indicator is immobilized in a microhetero-geneous microenvironment (e.g. a sol-gel matrix). 

In Chapter 10, fluorescent pH indicators and fluorescent molecular sensors for cations, anions and neutral molecules are described, with an emphasis on design principles in regard to selectivity. 

G. Gabor and D. R. Walt. 1991. Sensitivity enhancement of fluorescent pH indicators by inner filter... 

R. Pal, W. A. Petri, Jr., Y. Barenholz, and R. R. Wagner, Lipid and protein contributions to the membrane surface potential of vesicular stomatitits virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin, Biochim. Biophys. Acta 729, 185-192 (1983). 

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... 

Porphyrin dendrimers are suitable as sensors for small molecular and ionic analytes. Unsubstituted metal-free porphyrins often show poor solubility in water. However, if it proves possible to envelop them in hydrophilic dendrimers they can be used in water as fluorescent pH indicators because they exhibit distinct changes of their absorption and emission bands owing to protonation of... 

Paradiso, A.M., Tsien, R.Y. and Machen, T.E. (1984) Na+—H+ exchange in gastric glands as measured with a cytoplasmic-trapped fluorescent pH indicator. Proc. Natl. Acad. Sci. USA, 81, 7436-7440. 

Luminescence titrimetry has been developed chiefly for acid-base titrations. Therefore, fluorescence pH-indicators are now widely used. Their application is based on changes of fluorescence spectrum upon the addition of a proton or its loss. At present, over 200 fluorescence pH-indicators are available the structural formulae of the most the widely applied indicators are given in Table 8. Some of them (No. 2, 8, 9, 12, 16, 17, 23, 25 and 29) and also, primuline, tripaflavine, and rhodamine 6G are widely used as adsorption fluorescence indicators. The titration end point can be detected in this case because of the differences in of the indicator in the adsorbed state and in solution. Redox fluorescence indicators including rhodamines B and 6 G, 3,6-dihydroxy-phthalic acids, complexes of Ru(II) with 2,2 -dipyridyl or 1,10-phenanthroline and other... 

Oxazine derivatives728 stilbene729,73nitrogen bases of petroleum7311, acridone732,733), indole- and 5-hydroxyindole-2-carboxylic acids734) have been studied as fluorescence pH-indicators. 

More quantitively, Fernandez and Fromheiz investigated the interfacial region of SDS, CTAB, and Triton X-100 micelles (194). They used fluorescent pH indicators, hydroxycoumarin and aminocoumarin dyes substituted by alkane chains, and measured shifts of the pK-values. The authors attributed these shifts partly to the dielectric constant at the micelle-water interface and pardy to the electrical potential at the surface of charged micelles. In this way they calculated... 

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