Fluorescence and luminescence

Figure 9.13 Fluorescence and luminescence (a) an activator, A, absorbs incident radiation and re-emits it at a lower frequency (b) a sensitisor, S, absorbs incident radiation and passes it to an activator, A, which re-emits it at a lower frequency.

Griseofulvin exhibits both fluorescence and luminescence. A report by Neely et al., (7) gives corrected fluorescence excitation (max. 295 nm) and emission (max. 420 nm) spectra, values for quantum efficiency of fluorescence (0.108) calculated fluorescence lifetime (0.663 nsec) and phosphorescence decay time (0.11 sec.). The fluorescence excitation and emission spectra are given in Figure 7. 

Fluorescence and luminescence spectroscopic studies on imidazo[l,2- ][l,2,4]triazines 54 have been published by a Russian team <1994ZOB327, 1994ZOB497>. The experimental data reveal that protonation of 54 can take place either on N-4 or on N-5 and is strongly dependent on the solvent used. 

Mason WT (ed) (1999) Fluorescent and luminescent probes, 2nd edn. London, Academic... 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Many scientists would argue that the flexibility and reliability of SPA more than offset these potential disadvantages, and SPA is still in wide use for HTS today. Nevertheless, methods that generate fluorescent and luminescent signals without radioisotopes have become favorites in some HTS laboratories. These fluorescence- and luminescence-based methods,... 

W.T. Mason (Ed.), Fluorescence and Luminescence Probes in Biological Activity, Academic Press, 1994. 

Raman scattering cross sections are small ( 10 cm ), thus fluorescence and luminescence can easily interfere with the data. This is particularly acute with surface carbon, which fluoresces strongly when the Raman is excited by visible wavelength lasers. In addition, catalyst... 

Faulkner, S. Matthews, J. L. Fluorescent and Luminescent Complexes for Biomedical Applications, Comprehensive Coordination Chemistry II , Vol. 9 Eds. McCleverty, J. Meyer, T. J. Pergamon, 2003, p. in press, in press. 

J. Hoyland, in Fluorescent and Luminescent Probes for Biological Activity, ed. by W.T. Mason (Academic Press, San Diego, 1999), p. 108... 

Horseradish peroxidase (HRP EC 1.11.1.7) is the most widely used enzyme label for antibodies. It is relatively small (44 kDa), stable, and has a broad specificity that allows is to be measured by absorption, fluorescence, and luminescence. Several of its products are intensely colored, which makes the enzyme convenient to use for immunocytochemistry and immunoblotting applications. 

Fluorescent or luminescent substances are known to be excellent markers for antigen-antibody reactions. The fluorescent and luminescent substances with low molecular weight can be coupled easily to antigen or antibody... 

Because of the complex and often overlapping principles behind kinase and phosphatase assays, I will review the principles of the various fluorescent and luminescent technologies. A textbook by Joseph R. Lakowitz (1999) titled Principles of Fluorescence Spectroscopy is recommended for detailed information on the biophysics of fluorescence. Olive (2004) and Von Ahsen and Boemer (2005) wrote good reviews on the advantages and disadvantages of various luminescent (including fluorescent) technologies for kinase assays. 

Trends in biochemical screening assays seem to favor the use of multi-function PMT-based readers that allow for various MTP well densities (96, 384, and 1536 well plates), can handle a number of readout formats such as prompt fluorescence, luminescence, fluorescence polarization, time-gated fluorescence, and luminescent oxygen channeling or AlphaScreen. Examples of this type including the Perkin Elmer EnVision, TECAN-Ultra, BMG FluoStar, and LJL Analyst GT can be employed for a variety of the assay technologies described above. 

Due to the high specificity and sensitivity of immunoassays, there are bioanalytical methods for the measurement of an analyte of interest, with little or without preconcentration or purification of the samples. The principle behind immunoassays is based on an interaction between an antibody and a corresponding antigen, and the detection of the specific interaction using radiolabels (247), enzyme, fluorescent and luminescent compounds (178, 179,181,183), electroactive markers (177,180, 228, 248), or nanomaterials (249-251). 

Nonradiometric methods iticlude absorbance, fluorescence. and luminescence spectroscopy. Enzyme assays are a common example. The as.suy is usually run at or below the value of thq substrate, with only about S% of the substrate consumed during the assay, and multiple enzyme turnovers occur during the a.ssay. Sometimes enzyme reactions arc coupled, especially if the target reaction does not produce a product that can be detected directly in the assay. An example is carboxypeptidase. which is coupled to the teduction of NADP to NADPH. giving ri.se to absorbance at 340 nm. 

Enzyme immunoassays that require physical separation of free and bound T4 are generally based on principles analogous to those of conventional RIAs except that enzyme activity rather than radioactivity is measured. Most enzyme immunoassays use labeled T4 as antigen. An assortment of photometric, fluorescent, and luminescent substrates have been used to monitor the enzyme activity of the antibody-bound fraction. 

The major different HTS assays can be conveniently classified into biochemical techniques or cellular techniques. Additionally, there are numerous detection techniques, including radiochemical, absorbance, fluorescence, and luminescence. All of these combina-... 

ILA23 A Radioimmunoassays and Enzyme, Fluorescence, and Luminescence Immunoassays... 

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