Luciferase immobilized, bacterial

Irregular and flat surfaces Firefly luciferin/luciferase HRP/H202/luminol AP/dioxetanes Firefly luciferin/luciferase Bacterial luciferin/luciferase Detection of ATP as an indicator of microbial contamination Evaluation of the spatial distribution of immobilized biomolecules... 

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

Holzman, T. F., and Baldwin, T. O. (1982). Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose phosphate-mediated binding to immobilized substrate analogue. Biochemistry 21 6194-6201. 

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. 

Sensitive flow-injection analyses of aspartate, glutamate, 2-oxoglutarate, and oxaloacetate were developed using immobilized bacterial luciferase enzymes. 

Figure 2 Effect of enzyme immobilization on luminescent image spatial resolution evaluated using coupled enzymatic reactions on nylon net as a model system, (a) Immobilized 3a-hydroxysteroid dehydrogenase (b) immobilized 3a-hydroxysteroid dehydrogenase and FMN-NADH oxidoreductase (c) immobilized 3a-hydroxysteroid dehydrogenase, FMN-NADH oxidoreductase, and bacterial luciferase. (From Ref. 47. Copyright John Wiley Sons Ltd. Reproduced with permission.)...

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. 

A. Nabi and P. J. Worsold, Bioluminescence Assays with Immobilized Bacterial Luciferase Using Flow Injection Analysis. Analyst, 111 (1986) 1321. 

Of comparable general importance is the bacterial luciferase system [226-228], which opens up the opportunity to combine any NAD(P) " -dependent enzyme-catalyzed reaction with a luminometric measurement. Even the chemiluminescent luminol reaction can be used for biosensing, because it can be coupled to any oxidase reaction that produces HjOj [225, 229]. The logical further development of these systems towards real optical biosensors has recently been reported by Blum et al. [230], who immobilized the light-producing systems onto the tip of optical fibers and thus obtained fiber-optic luminescence probes. 

Marine bacterial luciferase can be immobilized on to a variety of solid supports (e.g., Sepharose, nylon) and it has also been coimmobilized with NAD(P)H FMN oxidoreductase. The close proximity of the two enzymes leads to efficient channeling of FMNH2 produced by the oxidoreductase to the luciferase. More extensive coupled coimmobilized systems have been prepared in which all of the enzymes in the enzymatic conversion of glucose to alcohol were coimmobilized with bacterial luciferase and the oxidoreductase, and the bioluminescent enzymes were used to monitor different stages in the biotransformation of glucose. 

Chemiluminescent reactions through the immobilization of chemiluminescent reagents on different supports in OF or FIA format, as with the use of NADH oxidoreductase and bacterial luciferase for NADH determination at subpicomolar concentration. 

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