Biochemical detection methods

Undoubtedly, LC-MS provides the most powerful tool to date, often used in parallel with other chemical or biochemical detection methods for the determination of MCs and their many variants. 

Physical, microchemical and/or microbiological or biochemical detection methods, preparation of pre- or postchromatographic derivatization reagents, derivatization technique, stabilization reagents, heating... 

The detection of spectral sensitizing action often depends on amplification methods such as photographic or electrophotographic development or, alternatively, on chemical or biochemical detection of reaction products. Separation of the photosensitization reaction from the detection step or the chemical reaction allows selection of the most effective spectral sensitizers. Prime considerations for spectral sensitizing dyes include the range of wavelengths needed for sensitization and the absolute efficiency of the spectrally sensitized process. Because both sensitization wavelength and efficiency are important, optimum sensitizers vary considerably in their stmctures and properties. 

Tanabe, S., Hase, M., Yano, T., Sato, M., Pujimura, T., and Akiyama, H. (2007). Real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Biosci. Biotechnol. Biochem. 71, 3131-3135. 

Watanabe, T., Akiyama, H., Yamakawa, H., lijima, K., Yamazaki, F., Matsumoto, T., Futo, S., Arakawa, F., Watai, M., and Maitani, T. (2006). A specific qualitative detection method for peanut (Arachis hypogaea) in foods using polymerase chain reaction. /. Food Biochem. 30, 215-233. 

The discussion above was concerned with the effects of solution conditions on enzyme activity, hence reaction velocity. Equally important for the purpose of assay design is the influence of specific solution conditions on the detection method being used. This latter topic is beyond the scope of the present text. Nevertheless, this is an important issue for screening scientists whose job is often to balance the needs of biochemical rigor and assay practicality in development of an HTS assay. An... 

Gomes, A., Fernandes, E. and Lima, J. L. F. C. (2005). Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 65, 45-80. 

Popov IN and Lewin G. 1996. Photochemiluminescent detection of antiradical activity. IV. Testing of lipid-soluble antioxidants. J Biochem Biophys Methods 31(1-2) 1-8. 

Tao QF, Zeng S. 2002. Analysis of enantiomers of chiral phenethylamine drugs by capillary gas chromatography/ mass spectrometry/fLame-ionization detection and precolumn chiral derivatization. J Biochem Biophys Methods 54 103. 

Dill, K., Song, J. H., Blomdahl, J. A., and Olson, J. D. (1997). Rapid, sensitive and specific detection of whole cells and spores using the light-addressable potentiometric sensor. J. Biochem. Biophys. Methods 34,161-166. 

Dill, K., Stanker, L. H., and Young, C. R. (1999). Detection of salmonella in poultry using a silicon chip-based biosensor. J. Biochem. Biophys. Methods 41, 61-67. 

Hall AG, Hamilton P, Minto L etal. The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase. J Biochem Biophys Methods 2001 47 65-71. Davison JE, McMullin MF, Catherwood MA. Genotyping of thiopurine methyltransferase in patients with acute leukemia using LightCycler PCR. Leuk Lymphoma 2006 47 1624-1628. 

Chevallet, M., Procaccio, V., and Rabilloud, T. (1997) A nonradioac-tive double detection method for the assignment of spots in two-dimensional blots. Anal. Biochem. 251, 69-72. 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

The first two points represent a general motivation for miniaturization in separation science independent of the actual fabrication technology. The benefit of a reduction of the consumption of sample, reagents, and mobile phase in chemical and biochemical analysis is self-evident and does not need to be discussed further (reduced consumption of precious samples and reagents, reduced amounts of waste, environmental aspects). This advantage is, however, sharply contrasted by its severe implications on the detection side, as discussed elsewhere in this volume in detail. The detection of the separated zones of very small sample volumes critically depends on the availability of highly sensitive detection methods. It is not surprising that extremely sensitive laser-induced-fluorescence (LIF) has been the mostly used detection principle for chip-based separation systems so far. 

In the biochemical field, proteins are prepared for NMR analysis by synthesizing them with bN labeled amino acid residues. Since 15N has a natural abundance of 0.368%, labeling increases the sensitivity of the experiment by a factor of about 270, which, when combined with the HSQC type experiment, yields about an 84,000-fold sensitivity increase over natural abundance directly detected methods. Thus, it is possible to obtain quality spectra on proteins up to 50 kDa. 

A state-of-the-art description of broadband ultrafast infrared pulse generation and multichannel CCD and IR focal plane detection methods has been given in this chapter. A few poignant examples of how these techniques can be used to extract molecular vibrational energy transfer rates, photochemical reaction and electron transfer mechanisms, and to control vibrational excitation in complex systems were also described. The author hopes that more advanced measurements of chemical, material, and biochemical systems will be made with higher time and spectral resolution using multichannel infrared detectors as they become available to the scientific research community. 

Nesterenko, M. V., Tilley, M., Upton, S. J. (1994). A simple modification of Blum s silver stain method allows for 30 minute detection of proteins in polyacrylamide gels./. Biochem. Biophys. Methods 28, 239-242. 

Deev, a. a. (2002). Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device. J. Biochem. Biophys. Methods 51, 57-67. 

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