Assay bioluminescence, substrates

CIEEL is of particular interest for the development of modern chemiluminescent bioassays. The most popular clinical bioassays utilize thermally persistent spiro-adamantyl-substituted dioxetanes with a protected phenolate moiety. These designed 1,2-dioxetanes include an energy source, a fluorophore, and a trigger grouping, and are therefore structurally similar to bioluminescent substrates such as firefly luciferin. Three main commercial dioxetanes 75 are available as one-reagent assays for alkaline phosphatase and are sold under the name of AMPPD (R1 = R2 = H), CSPD (R1 = Cl, R2 = H), and CDP-Star (R1 = R2 = Cl) <2006S1781, 2003ANA279>. These substrates are sensitive to 10 21 mol of alkaline phosphatase in solution. 

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). 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

Bioluminescence provides the basis for sensitive enzymic assay methods both for substrate assays and coupled enzyme assays. Firefly luciferase (EC 1.13.12.5) catalyses the production of light (540-600 nm) by the oxidation of luciferin (d-LH2) (Figure 8.18). 

One of the most important quality characteristics of advanced oxidation processes is their ability to reduce the toxicity of an industrial wastewater and to enhance its biodegradability (see Chapter 7.1.5). This criterion can be established by several standardized procedures using different test organisms ranging from microbes to intact animals (cf Tab. 5-1). An easy to perform variant is the bioluminescence assay that uses the inhibition of the bioluminescence intensity of the test organism Vibrio fischeri in the presence of toxic substrates. This is a bioassay used worldwide for the evaluation of toxicity data of individual chemicals or of industrial wastewaters (Froehner et al, 2000, DIN, 1991). Commercial systems are... 

Hsp70 ATPase activity was measured by monitoring the disappearance of ATP substrate over time using a bioluminescence assay as described under Methods. The concentration of the various peptides in the assay mixture was 120 pM. 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Bioluminescence has been used in immunoassays (Terouanne et al., 1986) but its complexity and high background, arising from ATP contamination, thus far make this system less attractive. Biolumines-cent assays have also been proposed with alkaline phosphatase using luciferin phosphate as the substrate (Hauber and Geiger, 1987) and... 

Bacterial luciferase coimmobilized with NAD(P)H FMN oxidoreductase on starch gel has been used for bioluminescent assay of aldehydesCo-immobilization of bacterial luciferase, NAD(P)H FMN oxidoreductase and their substrates is referred to as multifunctional immobilized biosensor and is a new trend for use of bioluminescent analysis, e.g. toxicity biotest and bioassay. The main principle of this luciferase biotest is the correlation between toxicity of the sample being studied and changes in bioluminescence parameters in vitro. Toxicity of the sample is measured by the changes in bioliuninescence intensity compared with that of a control. Multifunctional immobilized biosensors based on luciferase have been used for the following bioassays. 

Chemiluminescence is also found in fireflies. The male firefly uses the reaction of a luciferin substrate and the enzyme luciferase with oxygen, with adenosine triphosphate (ATP) as an energy source, to create the illumination it uses to attract a mate. Because the detection of very minute amounts of light is possible, chemiluminescence and bioluminescence have become the basis of many sensitive analytical and bioanalytical techniques or assays used to quantify particular compounds in samples. Indeed, the use of these techniques is broad enough to justify the existence of a journal devoted to them, the Journal of Bioluminescence and Chemiluminescence. 

Fig. 5 BRET signals of control and positive (27 pM) thrombin cleavage assays, (a) RIuc/Ciz400a bioluminescence signal, (b) GFP2 resonance energy transfer RET) signal. Concentration of biosensor was 1 pM and concentration of substrate Ciz400a was 16.25 pM...

The marine bacterial bioluminescent system when coupled to 7-a-hydroxysteroid dehydrogenase provides a very sensitive (0.5 pmol) and precise method for serum bile acids according to the analytical scheme shown in reaction [XI], This assay is typical of bioluminescent coupled enzyme assays for either a dehydrogenase enzyme or its substrate. 

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