Fluorescence alternatives

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Fig. 1 The fluorophore-spacer-receptor paradigm. It is expected that the interaction of the desired analyte with the receptor portion drastically modifies the emission of the nearby fluorophore. In the case illustrated, the recognition process restores the quenched fluorescence. Alternatively, the fluorescence before recognition may be on and after recognition be off.

Hatsuo, M., Matsuno, H., Ushida, M., Katayama, K., Saeki, K. and Itoh, N. 2, 4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman s reagent in thiol-quantification enzyme assays. Angeiv. Chem. Int. Ed. 44 2922—2925, 2005. 

In Raman spectroscopy, polymer samples often exhibit fluorescence due to contaminants on their surfaces. Wiping the sample with a solvent, e.g. acetone or alcohol, can reduce this fluorescence. Alternatively, taking a thin slice off the surface of the sample can also be helpful. 

If neither of the above approaches works, indirect detection may be employed. The inorganic ion may quench the fluorescence of a second compound, in which case the analyte is detected as a decrease in the fluorescence of the second compound. For example, most assays for fluoride (F ) are based on the decrease in fluorescence of an A1 or Zr chelate due to subsequent fluoride complex formation (AlFg or ZrFg ). Quenching is generally less sensitive and less selective than fluorescence. Alternatively, the analyte may cause the release of a ligand which then reacts to form a fluorescent product. 

Quantitative analysis may be performed directly on the thin-layer plate in a variety of ways by visual comparison by measurement of spot areas by the transmittance of spots that are colored, are charred, or that absorb ultraviolet light by reflectance or by fluorescence. Alternatively, the zone of absorbent containing the constituent can be scraped from the plate and extracted. The extract is then analyzed by... 

An alternative type of spectrometer is the energy dispersive spectrometer which dispenses with a crystal dispersion element. Instead, a type of detector is used which receives the undispersed X-ray fluorescence and outputs a series of pulses of different voltages that correspond to the different wavelengths (energies) that it has received. These energies are then separated with a multichannel analyser. 

An alternative mechanism of excess energy release when electron relaxation occurs is through x-ray fluorescence. In fact, x-ray fluorescence favorably competes with Auger electron emission for atoms with large atomic numbers. Figure 16 shows a plot of the relative yields of these two processes as a function of atomic number for atoms with initial K level holes. The cross-over point between the two processes generally occurs at an atomic number of 30. Thus, aes has much greater sensitivity to low Z elements than x-ray fluorescence. 

Because aH bonds within the polymethine chain of symmetrical PMDs are significantly equalized and change slightly on excitation, relatively smaH Stokes shifts (500 600 cm ) are observed in their spectra. In unsymmetrical PMDs, the essential bond alternation exists in the ground state. However, bond orders in the excited state are found to be insensitive to the symmetry perturbation. As a result, the deviations of fluorescence maxima, are much lower than those of absorption maxima, (3,10,56—58). The vinylene shifts of fluorescence maxima of unsymmetrical PMDs are practicaHy constant and equal to 100 nm (57). 

Quantitative aluminum deterrninations in aluminum and aluminum base alloys is rarely done. The aluminum content is generally inferred as the balance after determining alloying additions and tramp elements. When aluminum is present as an alloying component in alternative alloy systems it is commonly deterrnined by some form of spectroscopy (qv) spark source emission, x-ray fluorescence, plasma emission (both inductively coupled and d-c plasmas), or atomic absorption using a nitrous oxide acetylene flame. 

As an alternative to radiation, a stain such as ethidium bromide is used to visualize DNA. The ethidium may be incorporated into the stmcture of DNA either before or after electrophoresis. The gel is then visualized under a fluorescent lamp. 

Confirmation of the formation of the radicals during combustion reactions has been made by inuoducing a sample of dre flames into a mass spectrometer. The sample is withdrawn from a turbulent flame which is formed into a thin column, by admitting a sample of the flame to the spectrometer drrough a piidrole orifice, usually of diameter a few tenths of a millimetre. An alternative procedure which has been successful in identifying the presence of radicals, such as CHO, has been the use of laser-induced fluorescence. 

Note A 5% solution of polyethylene glycol 4000 in ethanol can be sprayed onto the chromatogram [2, 4] for the purpose of increasing and stabilizing the fluorescence instead of dipping it in liquid paraffin - -hexane (1 - - 2). If this alternative is chosen the plate should not be analyzed for a further 30 min since it is only then that the full intensity of the fluorescence develops [6]. 

Note The alternative fast blue salt BB produced the most intensely colored chromatogram zones for visual analysis in daylight, while fluorescence quenching in UV light (A = 254 nm) was greater with fast blue salt B and fast blue salt RR (Figs. 1 and 2). 

Equations (4-5) and (4-7) are alternative expressions for the estimation of the diffusion-limited rate constant, but these equations are not equivalent, because Eq. (4-7) includes the assumption that the Stokes-Einstein equation is applicable. Olea and Thomas" measured the kinetics of quenching of pyrene fluorescence in several solvents and also measured diffusion coefficients. The diffusion coefficients did not vary as t) [as predicted by Eq. (4-6)], but roughly as Tf. Thus Eq. (4-7) is not valid, in this system, whereas Eq. (4-5), used with the experimentally measured diffusion coefficients, gave reasonable agreement with measured rate constants. 

The similarity of the ultraviolet spectrum of 4,5-diphenyloxazol-2-one (91) with those of both alternative methyl derivatives preclude application of the spectral comparison method to the elucidation of their structures, but the fluorescence spectra of these compounds indicate that 91 exists in the oxo form. ° Infrared data for a number of substituted oxazol-2-ones support this conclusion. ... 

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