Biochemical detection

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. 

T. Schenk, G. J. Breel, P. Koevoets, S. van den Berg, A. C. Hogenboom, H. Irth, U. R. Tjaden, J. van der Greef Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization, f Biomol Screen 2003, 8, 421-429. 

J. van der Greef, R. Verpoorte High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products. J Chromatogr A 2000, 872, 61-73. 

Long, L. M. Olson, S. C. Bobzin, H. Irth High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system, f Biomol Screen 2001, 6, 291-303. 

J. van der Greef Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography-biochemical detection, coupled to electrospray mass spectrometry. 

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. 

Choi, J.W., Oh, K.W., Thomas, J.H., Heineman, W.R., HalsaU, H.B., Nevin, J.H., Helmicki, A.J., Henderson, H.T., Ahn, C.H., An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities. Labchip 2002, 2, 27-30. 

X. Ling, R. Nagai, N. Sakashita, M. Takeya, K. Takahashi, and S. Horiuchi, Immunohistochemical distribution and quantitative biochemical detection of advanced glycation end products in rats from fetal to adult life, in G, 2002, 137-142. 

Enantiomeric excess can be detected in a number of ways. Direct observation of optical activity, that is, determination of the specific rotation [a]d of a compound, is cumbersome. Biochemical detection is also possible, although methodologies are generally specific to individual compounds or compound types. Indirect measurement through a chromatographic procedure, for example, gas chromatography or gas chromatography-mass spectrometry on a chiral stationary phase, has wide applicability and is very sensitive. 

Zeck, A., Weller, M.G., and Niessner, R. 2001b. Multidimensional biochemical detection of microcystins in liquid chromatography., 4 na/yf/ca/ Chemistry ly.5509-5511. 

However, whether the interaction is direct or indirect remains an open question. Although there is biochemical evidence suggesting a direct interaction of one pilot protein (gpl6) with the coat protein (Thomas and Prevelige, 1991), attempts to biochemically detect interactions between the scaffolding and the minor and portal proteins have met with failure (P. E. Prevelige, unpublished data). 

Table 2 Reagents for biochemical detection of Ras and Ras effector pathway activation ...

R.G. Rudnitsky, E.M. Chow and T.W. Kenny, Rapid biochemical detection and differentiation with magnetic force microscope cantilever arrays. Sensors and Actuators A. Physical, 83 (2000) 256-262. 

D.A. van Elswijk, U.P. Schobel, E.P. Lansky, H. Irth, J. van der Greef, Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using online biochemical detection coupled toAdS, Phytochem., 65 (2004) 233. 

Additional markers of catecholamine overproduction have been employed to improve the biochemical detection of neuroblastomas. Free dopamine may be abnormal in urine from neuroblastoma patients with VMA and HVA excretion. Combined testing for VMA, HVA, and dopamine may therefore improve tumor detection, and in 1993 an international consensus report on neuroblastoma diagnosis added dopamine to the Hst of acceptable measurements to document the adrenergic nature of the tumor. Plasma measurements of dopamine and L-dopa, the amino acid precursor of dopamine, may also have clinical value and allow the alternate use of plasma. Measurement of methylated metabolites, especially normetanephrine, has also been explored. When urinary normetanephrine, metanephrine, methoxytyra-mine, dopamine, norepinephrine, VMA, and HVA were measured, clinical sensitivity for detection of neuroblastomas was 97% to 100% when results of normetanephrine testing were coupled either with VMA in the infants or with HVA in children greater than age 1. Even with an extended panel of catecholamines and metabolite measurements, a low incidence of nonsecreting tumors continues to be identified and should be considered in the interpretation of a negative test result. 

To determine the Cl-nt of compound X, we are able to use the in vitro half-life method, which is simpler than finding all the component Cl nt values. When the substrate concentration is much smaller than Km, the Michaelis-Menten equation simplifies from velocity (V) = Vmax([S])/(Km + [S]), because [S] (substrate concentration) becomes negligible. Furthermore, under these conditions, the in vitro half-life (7) 2 = 0.693/Xel) can be measured, and this, in turn, is related to the Michaelis-Menten equation through the relationship velocity (V) = volume x Kel (where volume is standardized for the volume containing 1 mg of microsomal protein). When both V and Vmax are known, then Km is also found. Although simpler than finding a complicated Cint, one caveat of the in vitro half-life method is that one assumes that the substrate concentration is much smaller than Km. It may be necessary to repeat the half-life determinations at several substrate concentrations, and even model the asymptote of this relationship, because very low substrate concentrations that are beneath biochemical detection may be needed to fulfill the assumptions needed to simplify the Michaelis-Menten equation. 

Enzymes modified with carbohydrates (neoglycoenzymes) can be used in cytochemistry as described above or in biochemical detection of lectins in solid-phase assays to gain greater sensitivity in analysis. For example, bacterial 3-galactosidase modified with p-aminophenyl a-D-mannopyranoside via amide linkage was useful in determination of Con A immobilized on plastic microtiter plates, and lactose-modified P-galactosidase was effective in histochemical detection of galactoside-specific lectins [63]. Other enzymes frequently used for these applications are alkaline phosphatase and horse radish peroxidase. There are a number of colorimetric, fluorometric, and chemiluminescent substrates available for these enzymes. 

Peristaltic pump (Gilson) for maintaining a constant flow during all the biochemical detection process. 

Johnson. D, E., Seidlcr, F. J. and Slotkin, T, A. (1998). Early biochemical detection of delayed neurotoxicity resulting from developmental exposure to chloropyrifos. Brain Res. Bull. 45, 143-147,... 

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. 

Allen, J.N., Abdel-Aty-Zohdy, H.S., Ewing, R.L., Chang, T.S. Spiking networks for biochemical detection. In The 2002 45th Midwest Symposium on Circuits and Systems. MWSCAS 2002, vol. 3, pp. 129-132 (2002)... 

Josse F., Bender F., and Cernosek R. W., Guided shear horizontal surface acoustic wave sensors for chemical and biochemical detection in liquids, Anal. Chem., 73,5937-5944, 2001. 

Haake H.-M., de best L., Irth H., Abuknesha R., and Brecht A., Label-free biochemical detection coupled on-line to liquid chromatography. Anal. Chem., 72, 3635-3641, 2000. 

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