Sensitivity-enhancement techniques isotope labelling

In more recent years, it has become less essential to use radioactivity, which, though very useful, is also hazardous. As we have mentioned, there are also stable, rare isotopes. In the case of hydrogen we have deuterium in the case of carbon we have carbon 13 (seven neutrons). It is also possible to label a biochemical compound with a stable isotope. Some of these isotopes ( C being one) can be monitored by nuclear magnetic resonance (NMR) all of them can be detected by mass spectrometry, which, as the name implies, relies on measuring the differences in mass of different molecules or fragments of molecules. The improved sensitivity of these two techniques in recent years has enhanced the usefulness of stable isotopes. 

Microarray detection methods In the early days of protein microarray analysis, mostly radioisotope-based labeling was applied, especially for phosphorylated proteins [99]. Later, stable isotope-coded amino acids (SILAC) proved useful in in vivo incorporation to cell cultures [100]. Both methods were very sensitive, requiring only minute sample amounts to reveal protein expression differences [101]. The use of an enhanced chemiluminescence (ECL) technique using X-ray film exposure or a phosphor imager instrument was. 

Of the two isotopes of nitrogen, N with a spin quantum number of 1/2 is the most widely used. It is very insensitive (1 x 10" ), and has a low natural abundance of 0.37%. While some studies have used the direct detection of N-labelled compounds, the indirect detection of N through H by techniques such as INEPT (insensitive nuclei enhanced by polarization transfer) and HMQC (heteronuclear multiple quantum coherence), which considerably enhance sensitivity, have increased the applicability of N in biological systems. 

Differentiation between labeled and unlabeled flavor compounds is based on the difference in nuclear masses of the isotopes and on employment of the mass spectrometry method. One of the main advantages of isotope dilution analysis is the high selectivity of the gas chromatography/mass spectrometry combination. The selectivity and the sensitivity of the determination can be further enhanced by the use of different ionization techniques. In most cases, differentiation between the internal standard and the analyte is achieved with the aid of chemical ionization with reactant gases, with the intensities of the molecular ions typically being measured by selective ion monitoring (SIM mode). 

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