Immobilized luciferases

The LDH+ALT reactor provided a linear response from 0.1 to 50 pmol/L lactate, thereby increasing lactate conversion by 117-183% relative to LDH alone. The intra- and inter-assay CV were both less than 5%, and recoveries ranged from 93 to 106%. Even though roughly 100% of the LDH and ALT added bound to the support under the immobilization conditions used, the activities of the immobilized enzymes were ca. 3% of those of the free enzymes, which is consistent with previous results obtained by the same [67] and other authors [69,70]. Jointly immobilized LDH and ALT preserved ca. 50% of their original activity after 60-90 days of intermittent use. On the other hand, immobilized luciferase was less markedly inhibited than that in the free solution by substances present in the biological samples assayed [71]. 

Optoelectronic biosensors based on immobilized dyes have been developed for the determination of glucose, urea, penicillin, and human serum albumin (Lowe et al., 1983). Other promising approaches use immobilized luciferase or horseradish peroxidase to assay ATP or NADH or, when coupled with oxidases, to measure uric acid or cholesterol. These principles have not yet been generally accepted for use in routine analysis. Most probably, the first commercial optical biosensors will be those for immunological assays. 

Immobilized luciferase and other enzymes must retain substrate specificity, with their kinetic constants unaltered. 

Entrapment of luciferase from Photobacterium leiognathi in starch gels increases it s K , for dodecanal and tetradecanal to 1/3, but the change is insignificant for decanal. Also, K of aldehydes with different chain length is smaller for immobilized luciferase than for the soluble enzyme. ... 

The effect of blood serum, lymph and other biological liquids on bioluminescent reactions has been studied. The immobilized luciferase shows lower sensitivity than the soluble enzyme in human clinical tests, and in analysis of com and bread infection by fungi. But this problem is overcome by using larger samples, and hence this new biosensor can be successfully used for toxicity bioassays. 

Brovko L, Ugarova N, Kinetics and mechanism of inactivation and reactivation of immobilized luciferase from fireflies Luciola mingrelica and the role of sulfhydryl in these processes. Biokhimiia (in Russian) 1980 45 794-801. 

Fluorescence from luminescent bacteria Bioluminescence from immobilized luciferase evanescent wave changes due to antibody-antigen binding... 

Eigure 2 shows a perforated microwell chip to obtain an array of immobilized luciferase. The microfluidic chaimels on top and bottom surface provide access for reagent and sample injection... 

Bioluminescence and Bioluminescence Resonance Energy Transfer Assays in Microfluidics, Fig. 2 3D microfluidic chip for immobilized luciferase reaction [2] (With permission from MicroTas2012)... 

M Tricine-NaOH, pH 8.4. ATP was determined by bioluminescence using the immobilized luciferase from Luciola mingrelica (8). The yield ATP amounted to about 2% of the initial ADP. 

A direct determination of the ATP content in P. shermanii at different growth phases (Gaitan and Vorobjeva, 1981) was carried out using the luciferin-luciferase method (Strehler and McElroy, 1957) with immobilized luciferase (Brovko et al., 1978). In cells growing on glucose the ATP level increased continuously up to 5 nmol per mg dry weight (Fig. 3.3). The ATP level dropped toward the end of the log-phase, but remained above 4 nmol/mg. The decrease was most prominent in the mid-log phase in the stationary phase the ATP level stabilized. The decline in ATP levels was accompanied by the accumulation of polyphosphates (Gaitan and Vorobjeva, 1981). 

A new trend in BL analysis is die appheation of immobilized luciferases. These are very stable, can be used more dian once, are not sensitive to the inhibitors of the sample (blood plasma, serum, etc.) and cheap. These make BL-based analysis preferable to other clinical methods [14]. 

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. 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

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. 

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... 

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.)...

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

The first insoluble derivatives of bioluminescent enzymes were prepared by Erlanger et al. by reacting luciferases they investigated the properties of these immobilized enzyme preparations and their potential for studying the mechanism of bioluminescence [54]. 

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