Enzymes bioluminescent detection

Simple or automated offline or online biodegradability tests can be performed by measuring CO2 or CH4 gas production or O2 consumption [13]. Biosensors may utilize either whole bacterial cells or enzymes to detect specific molecules of hazardous substances. Toxicity can be monitored specifically by whole-cell sensors whose bioluminescence may be inhibited by the... 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Chemiluminescence. Chemiluminescence (262—265) is the emission of light duting an exothermic chemical reaction, generaUy as fluorescence. It often occurs ia oxidation processes, and enzyme-mediated bioluminescence has important analytical appHcations (241,262). Chemiluminescence analysis is highly specific and can reach ppb detection limits with relatively simple iastmmentation. Nitric oxide has been so analyzed from reaction with ozone (266—268), and ozone can be detected by the emission at 585 nm from reaction with ethylene. 

The bioluminescence of the American firefly (Photinus pyralis) is certainly the best-known bioluminescent reaction, thanks to the work of Me Elroy and coworkers and E. H. White and his group (for references see P, p. 138, 6,168,169)) The substrate of this enzyme-catalyzed chemiluminescent oxidation is the benzothiazole derivative 107 (Photinus luciferin) which yields the ketone 109 in a decarboxylation reaction. The concept of a concerted cleavage of a dioxetane derivative has been applied to this reaction 170> (see Section II. C.). Recent experiments with 18C>2 have challenged this concept, as no 180-containing carbon dioxide was detected from the oxidation of 107 171>. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

The second one contained the bacterial bioluminescent enzymes. This system made it possible to reach a detection limit of 0.5 [imol/L. 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

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. 

Directly labeled chemiluminescent systems produce <1 photon/label and require complex chemical synthesis to produce each new labeled molecule (1,2). In contrast, chemiluminescent detection of enzyme labels combines the advantages of a high specific activity label with the convenience of relatively simple coupling chemistries, which use commercial reagents. A number of enzyme labels can be detected via chemiluminescent or bioluminescent reac-... 

Identification of the varying biological functions, classification of the bioluminescent relationships between different organisms, elucidation of the detailed reaction pathway, and the possibility of convenient study of the effect of enzyme or substrate modification have all been prime motivations for the study of bioluminescence (McCapra, 1976 Henry and Michelson, 1978 Hastings and Wilson, 1976 Cormier et al., 1975). Interest in chemiluminescence has been stimulated by its remarkable sensitivity and often selectivity as an analytical tool. As a result, chemiluminescence has found extensive application in the detection of trace metals in solution (Montano and... 

Geiger, R. Miska, W. Bioluminescence enhanced enzyme-immunoassay new ultrasensitive detection systems for enzyme immunoassays. J. Clin. Chem. Biochem. 1987, 25, 31-38. 

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