Detection methods chemiluminescence

Concerns over safe handling of radioactive materials and issues around the cost and disposal of low level radioactive waste has stimulated the development of nonradiometric products and technologies with the aim of replacing radioactive tracers in research and medical diagnosis (25). However, for many of the appHcations described, radioactive tracer technology is expected to continue to be widely used because of its sensitivity and specificity when compared with colorimetric, fluorescent, or chemiluminescent detection methods. 

Separation and detection methods The common methods used to separate the Cr(III)/(VI) species are solvent extraction, chromatography and coprecipitation. In case of Cr(VI) from welding fumes trapped on a filter, a suitable leaching of the Cr(VI) from the sample matrix is needed, without reducing the Cr(VI) species. The most used detection methods for chromium are graphite furnace AAS, chemiluminescence, electrochemical methods, ICP-MS, thermal ionization isotope dilution mass spectrometry and spectrophotometry (Vercoutere and Cornelis 1995)- The separation of the two species is the most delicate part of the procedure. 

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

Optical sensors have the advantage of an easily measured signal that can be seen by the naked eye in some cases. Optical detection methods include fluorescence, surface plasmon resonance spectroscopy, Raman, IR, and chemiluminescence (Fabbrizzi and Poggi 1995 deSilva et al. 1997). However, the fabrication and development of optical MIP sensors requires that a colored, emissive, or fluorescent monomer... 

Other detection methods have been used in optical MIP sensing systems. An MIP-based chemiluminescent flow-through sensor was developed for the detection of 1,10-phenanthroline (Lin and Yamada 2001). A metal complex was used to catalyze the decomposition of hydrogen peroxide and form the superoxide radical ion that can... 

Electrogenerated chemiluminescence has rapidly gained importance as a sensitive and selective detection method in analytical science. During last decade, several review articles describing primarily different analytical applications of ECL have... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

E. S. Yeung and W. G. Kuhr, Indirect Detection Methods for Capillary Separations, Anal. Chem. 1991, 63, 275A J. Ren and X. Huang, Indirect Chemiluminescence Detection for Capillary Electrophoresis of Cations Using Co(III) as a Probe Ion, Anal. Chem. 2001, 731, 2663 M. Macka, C. Johns,... 

A modification of these coloring systems has recently been developed that leads to more sensitive detection. Chemiluminescent substrates have been designed that are converted by the enzymes to products that generate a light signal that can be captured on photographic film. This increases the level of sensitivity about 1000-fold over standard color detection methods. 

Kricka, L. J. and Thorpe, G H G. (1986) Photographic detection of chemiluminescent and bioluminescent reactions. Methods Enzymol 133,404-420... 

The technique described above is not useful for removing colored peroxidase reaction products (e.g., diaminobenzidine oxidation products) from blots Thus, we avoid detection methods based on these reactions. On the other hand, peroxidase-based luminescent assays (20) do not deposit a chemical reaction product on the blot and are compatible with this erasure method (21). An example of the use of this erasure method (Section 3 3, steps 1-5) after chemiluminescent detection is shown in Fig 2. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

PCR-ELISA [37]) were described. The immobilized hapten-labeled amplification product was subsequently detected by an antibody-enzyme conjugate similar to conventional ELISA. By choice of a chemiluminescence- or fluorescence-inducing substrate, the sensitivity of the IPCR essay is further enhanced [37]. A comparison of different detection methods is given schematically in Fig. 6 typical results are compared in Fig. 5. 

Method of detection Fluorescence Chemiluminescence Fluorescence Change in pH... 

There is an increasing trend to replace radioactive detection methods by approaches which exploit spectrophotometry, particularly UV/VIS absorption, fluorescence and chemiluminescence (Levy and Herrington 1995 Howard 1993). So, for example, radioimmunoassays (RIA) have been effectively replaced by enzyme-linked immunosorbent assays (ELISA) (Sect. 6.2.4) in which the enzyme-catalysed liberation of a... 

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