Of fluorescent indicators

The scintillators are a special type of fluorescence indicators they are employed for the fluorimetric detection of radioactively labelled substances. They are stimulated by ) -radiation to the emission of electromagnetic radiation and will be discussed in Volume 2. 

The reasons for the above phenomena are to be found in differing configurations of hydrogen bonds, the effect of pH, differences in the structures of fluorescence indicators and binders and differences in surface area. For example, silica gel 60 possesses a surface area of 500 m /g [211] while that of Si 50 000 lies below 5 m /g [212],... 

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. and Miyawaki, A. (2004). Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554-9. 

Romoser, V. A., Hinkle, P. M. and Persechini, A. (1997). Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270-4. 

Indicator displacement assays (IDAs) - or, in the specific case of fluorescent indicators, F-IDAs - are based on the next alternative concept described here. A receptor with an affinity for a given analyte is loaded with an indicator, usually a fluorescent or colored dye, whose spectral properties undergo a change upon complexation with the receptor. Treatment of this indicator-receptor complex with the analyte results in the displacement of the indicator from the receptor and a restoration of the indicator s original spectral properties, indirectly reporting analyte coordination (Fig. 27). For effective detection, two main requirements have to be fulfilled (1) the receptor/indicator interaction must be reversible and weaker than the interaction of the receptor with the designated analyte and (2) the indicator must show significantly different optical properties when bound to the receptor and when freely dissolved in solution. 

Dual lifetime referencing (DLR) is another powerful technique that enables referenced measurements in case of fluorescent indicators [23]. In this method, the analyte-dependent signal from an indicator is referenced against the signal from an inert luminophore. This can be realized in both the time domain [24] and in the frequency domain [25]. Often, a luminescent reference dye is embedded into gas blocking nanobeads to avoid oxygen quenching. Polymers with very low gas permeability such as poly(acrylonitrile) [24] or poly(vinylidene chloride-co-acry-lonitrile) [26] are the best choice here. 

Figure B3.6.12 Depolarization of fluorescence indicates rotation of the chromophore. Monochromatic radiation from the source (S) has all but the vertically polarized electric vector removed by the polarizer (P). This is absorbed only by those molecules (see Fig. B3.6.5) in which the transition dipole of the chromophore is aligned vertically. In the case where these molecules do not rotate appreciably before they fluoresce ( no rotation"), the same molecules will fluoresce (indicated by shading) and their emitted radiation will be polarized parallel to the incident radiation. The intensity of radiation falling on the detector (D) will be zero when the analyzer (A) is oriented perpendicular to the polarizer. In the case where the molecules rotate significantly before fluorescence takes place, some of the excited chromophores will emit radiation with a horizontal polarization ( rotation ) and some with a vertical polarization. Finite intensities will be measured with both parallel and perpendicular orientations of the analyzer. The fluorescence from the remainder of the excited molecules will not be detected. The heavy arrows on the left of the diagram illustrate the case where there is rotation.

Determination of acid strengths by means of fluorescent indicators (21) and still another class of indicators, the arylcarbinols (24), has also been reported. Comparison of these indicators for the titration of surface acidity will be discussed in the following section. 

At present the dye techniques are very useful and economical but are somewhat approximate. Advances in use of indicator dyes for measuring surface acidity and basicity may be expected to include a two-parameter measure of acid or base strength similar to the E and C equation of Drago, and the use of fluorescent indicators for colored solids. 

Figure 3 Fluorescent sensors of Surface and Dipole potential in membranes. Fig. 3a (LHS) indicates the position in the a single bilayer leaflet of fluorescent indicators of the membrane dipole potential (upper chemical structure) and the surface potential (lower chemical structure). The RHS profile indicates how the profile of surface potential varies with distance from the membrane surface. Fig. 3b indicates the use of FPE as a surface potential indicator that responds to the addition of a charged peptide (P25) as it interacts with simple membranes (see 25 for more details).

In ethanol, the 31,850 cm maximum (A) is reduced to a shoulder near 33,000 cm. Whereas in heptane a normal "Weller shift" of fluorescence indicating Intramolecular proton transfer is observed, in alcohols we note a fluorescence band at higher frequency with a maximum at 23,800 cm. The latter is due to the anion (D) and corresponds to the absorption maximum of D at 29,400 cm The intensity of this emission increases with increasing pH and remains constant above pH = 9 (in ethanol). The anilide of 2-methoxy benzoic acid shows a ultraviolet fluorescence that was too weak to be recorded (21). Low quantum efficiency may, therefore, be the reason that the fluorescence of C has not been detected. The only evidence of the presence of C is the close similarity of the absorption spectra of SAN and the methoxy derivative in ethanol (21). 

The lifetime of the state responsible for this emission could be determined by single photon counting and varied from 45 ns for n=2 to less than 10 ns for n=5. Kinetic analysis by quenching of the reaction and quenching of fluorescence indicated that the exclmer formation lies on the reaction coordinate leading to product. An analogous statement was made by Aladekomo in the dimerization of 9-methylanthracene (106). An interesting point is the observation of excimer emission in a photochemical reactive system in which the chain contains more than four... 

This rule has a very decided bearing on the application of fluorescence indicators to pH measurements. It is, obviously, imperative that the indicator exhibit a decided fluorescence in extremely small concentrations (as do bivalent quinine ions), so that one may work in the region of direct proportionality. Addition of larger amounts of indicator is to be avoided because, just as color indicators possess acid or basic properties, fluorescence indicators may change the pH of a solution (cf. especially Chapter Ten, section 1, unbuffered liquids). 

This brief and necessarily incomplete review of fluorescence indicators must sufl ce. A more quantitative discussion of the subject would be premature at present because too many factors, such as salt error, effect of indicator concentration, etc., remain yet to be studied exhaustively. Chlorides, for example, diminish considerably the fluorescence of the quinine cations and of the naphtholsulfonic acid anions. Fluorescence indicators are as yet... 

Since the publishing of the 1st edition of this book, some new precoated layers have been introduced. To simplify the visual evaluation for the user, Macherey-Nagel and Merck have developed precoated silica gel 60 layers with an increased amount of fluorescence indicator that go by the trade names of Adamant - and Lux -plates. Furthermore these plates contain a higher amount of binder which improves the abrasion resistance. Figme 7 shows the comparison at UV-hght 254 nm between a normal TLC-and a Lux -plate. 

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