Indirect Fluorescence

The high sensitivity and selectivity of fluorescence detection make this the obvious choice for improving detection of proteins. Three approaches have been used direct detection of intrinsic protein fluorescence, indirect fluorescence detection, and protein derivatization for fluorescence detection. 

When the ascending solvent-front has reached a convenient height, the strip is removed, the position of the solvent-front marked, and the paper strip dried. The positions of the various solutes, if they are coloured compounds, now appear as clear separate spots. Frequently however, the solutes are colourless, and the position of their spots must be determined by indirect methods, such as their fluorescence in ultraviolet light, or their absorption in such light (when the spots appear almost black), or by spraying the paper with a dilute solution of a reagent which will give a coloured insoluble derivative with the solutes. 

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for the direct or indirect quantitative analysis of analytes in a variety of matrices. A direct quantitative analysis is feasible when the analyte s quantum yield for fluorescence or phosphorescence is favorable. When the analyte is not fluorescent or phosphorescent or when the quantum yield for fluorescence or phosphorescence is unfavorable, an indirect analysis may be feasible. One approach to an indirect analysis is to react the analyte with a reagent, forming a product with fluorescent properties. Another approach is to measure a decrease in fluorescence when the analyte is added to a solution containing a fluorescent molecule. A decrease in fluorescence is observed when the reaction between the analyte and the fluorescent species enhances radiationless deactivation, or produces a nonfluorescent product. The application of fluorescence and phosphorescence to inorganic and organic analytes is considered in this section. 

Spectroscopic methods such as uv and fluorescence have rehed on the polyene chromophore of vitamin A as a basis for analysis. Indirectly, the classical Carr-Price colorimetric test also exploits this feature and measures the amount of a transient blue complex at 620 nm which is formed when vitamin A is dehydrated in the presence of Lewis acids. For uv measurements of retinol, retinyl acetate, and retinyl palmitate, analysis is done at 325 nm. More sensitive measurements can be obtained by fluorescence. Excitation is done at 325 nm and emission at 470 nm. Although useful, all of these methods suffer from the fact that the method is not specific and any compound which has spectral characteristics similar to vitamin A will assay like the vitamin... 

Zirconium is often deterniined gravimetrically. The most common procedure utilizes mandelic acid (81) which is fairly specific for zirconium plus hafnium. Other precipitants, including nine inorganic and 42 organic reagents, are Hsted in Reference 82. Volumetric procedures for zirconium, which also include hafnium as zirconium, are limited to either EDTA titrations (83) or indirect procedures (84). X-ray fluorescence spectroscopy gives quantitative results for zirconium, without including hafnium, for concentrations from 0.1 to 50% (85). Atomic absorption determines zirconium in aluminum in the presence of hafnium at concentrations of 0.1—3% (86). 

However, the direct determination of absorption at the wavelength of maximum absorption is more sensitive (or in the worst case at least as sensitive) as the indirect measurement of absorption by fluorescence or phosphorescence quenching. 

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). 

E. Jurkevitch, Y. Hadar, Y. Chen, M. Chino, and S. Mori, Indirect utilization of the phytosiderophore mugineic acid as an iron source to rhizosphere fluorescent Pseudomonas. BioMetals 6 119 (1993). 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Richmond, M. D. and Yeung, E. S., Development of a laser-excited indirect fluorescence detection for high-molecular weight polysaccharides in capillary electrophoresis, Anal. Biochem., 210, 245, 1993. 

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. 

Lovdahl, M. J. and Pietrzyk, D. J., Anion-exchange separation and determination of bisphosphonates and related analytes by post-column indirect fluorescence detection,. Chromatogr A, 850, 143, 1999. 

Marti, V., Aguilar, M., and Yeung, E. S., Indirect fluorescence detection of free cyanide and related compounds by capillary electrophoresis, ]. Chromatogr. A, 709, 367, 1995. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

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