Chemiluminescence research methods

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. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

McKee, H. C., R. E. Childers, and V. B. Parr. Cdlabroative Study of Reference Method for Measurement of Photochemical Oxidants in the Atmosphere (Ozone-Ethylene Chemiluminescent Method) EPA 650/4-75-016. San Antonio, Texas Southwest Research Institute, 1975. 49 pp. 

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. 

Because of concerns about the safety of radioisotope use, researchers are developing fluorescent and chemiluminescent methods for detection of small amounts of biomolecules on gels. One attractive approach is to label biomolecules before analysis with the coenzyme biotin. Biotin forms a strong complex with enzyme-linked streptavidin. Some dynamic property of the enzyme is then measured to locate the biotin-labeled biomolecule on the gel. These new methods approach the sensitivity of methods involving radiolabeled molecules, and rapid advances are being made. 

Methods based on chemiluminescent labels are other non-isotopic IAs and ILAs that continue to be the focus of active research. Most common approaches under this category are direct measurements of the chemiluminescence accomplished by the action of the imprinted polymer (e.g., catalytic effect) or by indirect methods, such as competitive CILAs. 

Spectrophotometric techniques combined with flow injection analysis (FIA) and on-line preconcentration can meet the required detection limits for natural Fe concentrations in aquatic systems (Table 7.2) by also using very specific and sensitive ligands, such as ferrozine [3-(2-bipyridyl)-5,6-bis(4-phenylsulfonic acid)-l,2,4-triazine], that selectively bind Fe(II). Determining Fe(II) as well as the total Fe after on-line reduction of Fe(III) to Fe(II) with ascorbic acid allows a kind of speciation.37 A drawback is that the selective complexing agents can shift the iron redox speciation in the sample. For example, several researchers have reported a tendency for ferrozine to reduce Fe(III) to Fe(II) under certain conditions.76 Most ferrozine methods involve sample acidification, which may also promote reduction of Fe(III) in the sample. Fe(II) is a transient species in most seawater environments and is rapidly oxidized to Fe(III) therefore, unacidified samples are required in order to maintain redox integrity.8 An alternative is to couple FIA with a chemiluminescence reaction.77-78... 

Cyalume sticks are chemiluminescent when you bend the flexible tube enough to break the barrier that separates two substances, the tube glows for several hours until the chemical reactions are completed. A method called enhanced chemiluminescent detection, developed by researchers in Paris, offers a non-radioac-tive way of keeping track of genes and is being used in the international Human Genome Project. 

Although AFP was traditionally measured by RIA, newer methods use lEMA or chemiluminescent immunoassay (CIA) because of their lower detection lunits, better precision, speed, avoidance of radioactivity, and ease of automation. Most laboratories in the United States measure AFP by use of automated systems available from Abbott Laboratories (Abbott Park, 111,), Bayer (Medfield, Mass.), Beckman Coulter (Fullerton, Calif.), Diagnostic Products (Los Angeles), and Perkin Elmer (Norwalk, Conn.). Judging from the results of proficiency testing sponsored by the Foundation for Blood Research and College of American Pathologists (Survey FP), all of these systems perform satisfactorily. ... 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

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