Sensitivity shift procedure

It is of interest to note the use of the sensitivity shift procedures, illustrated in Figure 9.55. Arrows on the figure indicate where detector sensitivity was increased to allow for the detection of low levels of the product. 

The second procedure is to measure the luminescence intensities at various Ca2+ concentrations and plot log (light intensity) against —log [Ca2+] for each aequorin. Examples of this method are shown in Fig. 4.1.14. This method provides more detailed information on the sensitivity of each aequorin. Generally, an increase in Ca2+ sensitivity shifts the curve to the left. 

The analysis of a sample from an incubation mixture that contains significant differences in substrate and product concentrations presents some problems, since the detector must be set so that the substrate peak is on scale, but also it must be set to detect small amounts of product. Usually different sensitivity settings are required to put both components on scale. For these cases we have adopted a procedure that might be called the sensitivity shift. In this procedure, the injection is made with the sensitivity set at a value that allows for the detection of one of the compounds. If this is the product, the detector is set at its maximum sensitivity. As soon as the product has emerged, the sensitivity of the detector is changed (either manually or electronically by computer) to a value that allows the substrate peak to appear completely on scale. 

Once production of the desired secondary compound is demonstrated in cell cultures, the emphasis can be shifted toward inducing and selecting genetic variants that synthesize increased levels of the compound. However, before one can embark on a program of genetic modification for enhanced production capacity, rapid, but sensitive assay procedures must be developed for the detection of the desired compounds (10). These methods should be geared to handle a large number of samples quickly, but should minimize the amount of tissue required for analysis. 

Beasley et al. developed a panel of immunoassays to monitor DDT, its metabolites, and structurally related compounds, but they found that milk has a severe effect on the assay performance. They found that when directly utilizing whole milk, color development was completely inhibited. Even when using 1 100 dilutions of whole milk, the assay sensitivity was reduced by 90% (based on the IC50 shift, not simply the dilution factor). A number of procedures were evaluated to eliminate the interferences from the fat-soluble analytes. However, many of the procedures that removed interferences also removed the analytes. Extraction with a mixture of solvents and the use of similarly processed blank milk to prepare the standards ultimately yielded more accurate results. This article demonstrates the difficulties encountered in analyzing lipid-soluble analytes. 

Hassan et al. [39] used a sensitive color reaction method for the determination of primaquine in pharmaceutical preparation. Primaquine was treated with diazo-p-nitroaniline in acidic medium to give an orange-yellow product with an absorbance maximum at 478 nm. When the medium was made alkaline, bathochromic, and hypochromic shifts occurred the new maximum was located at 525 nm. The mean percentage recoveries for authentic samples amounted to 100 and 100.21 by the acid and alkaline procedures, respectively (P = 0.05). Both reactions could be used to determine primaquine salts in pharmaceutical preparations. The results obtained were in good agreement with those of the official methods. Recoveries were quantitative by both methods. 

The most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye s absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also, it exhibits little quantitative variation between different proteins. 

Sodium contamination and drift effects have traditionally been measured using static bias-temperature stress on metal-oxide-silicon (MOS) capacitors (7). This technique depends upon the perfection of the oxidized silicon interface to permit its use as a sensitive detector of charges induced in the silicon surface as a result of the density and distribution of mobile ions in the oxide above it. To measure the sodium ion barrier properties of another insulator by an analogous procedure, oxidized silicon samples would be coated with the film in question, a measured amount of sodium contamination would be placed on the surface, and a top electrode would be affixed to attempt to drift the sodium through the film with an applied dc bias voltage. Resulting inward motion of the sodium would be sensed by shifts in the MOS capacitance-voltage characteristic. 

Colorimetric assays are commonly used in molecular biology and biotechnology laboratories for determining protein concentrations because the procedures and their instrumentation requirements are simple. Two forms of assays are used. The first involves reactions between the protein and a suitable chemical to yield a colored, fluorescent, or chemiluminescence product. Second, a colored dye is bound to the protein and the absorbance shift is observed. Disadvantages of both these methods include limited sensitivity at below 1 pg/mL, interferences from buffers, and unstable chromophores (Jain et al. 1992). 

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