PH-sensitive fluorescent probes

S. Jin, Z. Xu, J. Chen, X. Liang, Y. Wu, and X. Qian, Determination of organophosphate and carbamate pesticides based on enzyme inhibition using a pH-sensitive fluorescence probe. Anal. Chim. Acta 523, 117-123 (2004). 

Two commercially available pH-sensitive fluorescent probes were used to monitor the photooxidation of the UV-cured polyurethane-acrylate adhesive L350 Oregon Green (OG) and />-dimethylaminosalicylic acid (p-DASA). During the period of UV-exposure, the photostability of these fluorescent compounds was assessed by UV-absorption monitoring and the fluorescence emission was measured at several... 

At equilibrium, the pH in the internal solution depends on the concentration of carbonic acid produced upon hydration of the permeated CO2, which in turn is proportional to the parti pressure of the analyte in the sample. Various pH-sensitive fluorescent probes have been used for measurements of carbon dioxide based on the pH change in an inner buffer solution caused by permeation of carbon dioxide through different gas-permeable membranes (68-74). 

With the same mechanism, a series of unique, acidic pH-sensitive fluorescence probes have been eonstructed for bioimaging viable cancer eells (Scheme 7.9). All these bear various anilines at the meso-position of the BODIPY fluorophore. Similarly, they are almost non-fluorescent in the non-protonated form due to the efficient PET from the aniline moiety to the BODIPY fluorophore. In contrast, they are protonated in acidic medium, and become highly fluorescent, along with a 300-fold increase in emission. With these promising features in hand, fluorescence turn-on probe-antibody eonjugates have been successfully exploited in specific imaging of viable eaneer eells. 

Rather than to draw up an exhaustive list of pH sensing fluorescent probes which can be found in several reviews 112,119) we will try to define different classes of compounds whose fluorescence is sensitive to protonation and give some illustrative examples. 

A large number of sensitive fluorescent probes are now available for the measurement of pH and of the concentration of ions. 

Novel fluorescence probes for nitric oxide (DAMBO-P ) and for Zn " (ZnAB) have been developed based on BODIPY structure (Fig. 3). DAMBO-P is a pH-independent and more highly sensitive fluorescence probe for nitric oxide than DAF-2. ZnAB has the advantages of less sensitivity to solvent polarity and pH than ZnAF-2, fluorescein-based Zn " probe, and is also not influenced by other cations such as Na", K, Ca and Mg, which exist at high concentration under physiological conditions. 

USE In sunscreen lotions and creams as intracellular and pH sensitive fluorescent indicator and blood-brain barrier probe. 

Fluorescence enhancement can be based on the change in environment, such as change of polarity, dielectric strength, pH, viscosity, formation of hydrogen bonds. One principle makes use of polarity sensitive fluorescent probes such as dansyl derivatives or anilinonaph-thalene sulfonic acid, which show fluorescence enhancement properties upon binding to proteins, e.g., antibodies. The second variation is based on the fact that the fluorescence of a fluorescein-labeled analyte is quenched in the presence of heavy atoms, such as iodine. When the labeled probe binds to the antibody, this quenching is partially reversed. 

Abstract We discuss applications of selected fluorescence spectroscopy techniques for the studies of block copolymer micelles in aqueous solution, focusing on solvent relaxation measurements using polarity-sensitive fluorescent probes, oti fluorescence quenching studies, and on using fluorescent pH indicators for studies... 

An alternative approach is the use of pH-sensitive fluorophores (Lichtenberg and Barenholz, lOSS). These probes are located at the lipid-water interface and their fluorescence behavior reflects the local surface pH, which is a function of the surface potential at the interface. This indirect approach allows the use of vesicles independent of their particle size. Recently, techniques to measure the C potential of Liposome dispersions on the basis of dynamic light scattering became commercially available (Muller et al., 1986). 

Another example of fluorescence intensity modulation in cou-marins is the 3-azido substitution that quenches the fluorescence completely. These compounds are used as starting material for the synthesis of fluorescent triazolocoumarins by click chemistry [31], Interestingly, the fluorescence of some coumarins depends strongly on the solvent. This is the case for 7-alkoxycoumarins that have been used as probes for microenvironments [32], 7-hydroxycoumarin that is pH sensitive, and 7-NR2 substituted coumarins such as coumarin 120 whose quantum yield is reduced in nonpolar solvents due to a change in the 3D structure [33],... 

Ratiometric luminescent probes make a smart use of the excitation wavelength effect on the emission intensity for extended optosensor performance. For example, the fluorescence from 8-hydroxy-l,3,5-pyrenetrisulfonate (HPTS) and other pH-sensitive dyes in water comes only from its (photo)excited basic form, but the absorption spectra of HPTS and PTS (pAa 7.3) differ considerably (Figure 3). 

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). 

The first pH indicators studied possessed the acid-base site (phenol, aniline, or carboxylic acid) as an integral part of the fluorophore. Structurally, in the most general sense, pH sensitivity is due to a reconfiguration of the fluorophorets re-electron system that occurs on protonation. Consequently, the acid and the base forms often show absorption shifts and also, when the two forms fluoresce, emission shifts or at least, when only one form emits, a pH-dependent fluorescence intensity. This class of compounds has been reviewed 112 and the best structures have to be designed according to the medium probed and the technique used. After a short consideration of physiological pH indicators we will describe the main photophysical processes sensible to protonation. 

We demonstrate the Mil method, which couples the sensitivity of multiphoton excitation on the spectral phase of the laser pulses to probe microscopic chemical environment-induced changes in the multiphoton excitation spectrum of sensitive reporter molecules. We carry out the optimization of the required phase functions in solution and provide theoretical simulations. We show experimental images whereby pH-selective two-photon microscopy is achieved and demonstrate how selective excitation can be used to enhance contrast and, consequently, to achieve functional imaging, using fluorescent probes sensitive to changes in their local environment. 

Fluorescent compounds are sensitive to changes in their chemical environment. Alterations in media pH, buffer components, solvent polarity, or dissolved oxygen can affect and quench the quantum yield of a fluorescent probe (Bright, 1988). The presence of absorbing components in solution that absorb light at or near the excitation wavelength of the fluorophore will have the effect of decreasing luminescence. In addition, noncovalent interactions of the probe with other components in solution can inhibit rotational freedom and quench fluorescence. 

A number of derivatives of fluorescein have been synthesized. Dichlo-rotriazinylamino fluorescein (DTAF) (1) is spectrally identical to FITC, but provides a more consistent and stable antibody conjugate. Succinimidyl esters of fluorescein are also available for conjugation to antibodies. BODIPY FL2 (Molecular Probes, Inc., Eugene, OR) has been offered as a substitute for fluorescein. It has a short Stokes shift, but offers higher fluorescence intensity, and is claimed to be more photostabile and less pH-sensitive than fluorescein. 

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