Bioluminescence, 240 also

Cell responses to physical or chemical cues are measured in microfluidic devices primarily via optical or electrochemical means. Huorescence is the most widely used optical detection technique, because absorbance detection (commonly used for macroscale assays) is of limited value in microchannels because of the short path lengths. Fluorescence detection, characterized by its unparalleled sensitivity, is easy to implement in microfluidic systems. Chemiluminescence and bioluminescence also offer low detection limits and have less background noise than fluorescence [8]. Electrochemical detectors are even more easily integrated with microfluidic devices and often are much less expensive than optical systems. However, fabrication of electrodes in microchannel devices is a technical challenge, and the electrical fields used in detection can interfere with on-chip processes such as electrophoresis. Electrochemical techniques include potentiometry, amperometry, and... 

The technique of detection via light absorption [9] exploits results from UV-visible spectrophotometry, especially in the use of monochromatic radiation and a reference system. This requires the presence of at least two fibers, to separate the outward and inward light beams, and so this method is rarely used. In contrast, fluorescence [10] can be measured directly using a single fiber and very low concentrations can be detected. Furthermore, coupling the immobilized enzyme on an optical fiber with a fluorescent cc actor, such as NADH, opens up greater possilnlities. The system is even simpler in the case of bio/chemiluminescence because the reference and excitation beams are obsolete, and the light can be emitted directly to the sensitive component [11]. The excellent quantum yield of bioluminescence also facilitates the detection of low concentrations. 

Dubois [7] in 1885 had shown that bioluminescence also involved the oxidation of a relatively small organic molecule, although this initiative also lay fallow for over fifty years. 

The study of both bioluminescence and chemiluminescence which led to our present understanding was inaugurated by Harvey [8] in 1953 with the publication of Bioluminescence . Also about this time White [9] made the first thorough and convincing study of the mechanism of luminol chemiluminescence. A review of the chemistry of the hy dr azides, with special reference to their chemiluminescence [10] also added to the foundations. 

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). 

The carbonyl compound (43) has also been synthesi2ed, and its fluorescence spectmm has been shown to match the bioluminescence spectmm under equivalent conditions (214). The details of the excitation step are unclear and a dioxetanone mechanism (59,215) may apply to the reaction. 

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 autoinducer is a low molecular weight compound that is easily leached from the cells into the culture medium. By the propagation of bacterial cells, the concentration of the autoinducer in the medium increases. When the concentration reaches a certain threshold, the biosynthesis of bioluminescence system begins, and the bacteria become luminescent. The process is also called quorum sensing (Fuqua et al., 1994). 

The product coelenteramide is not noticeably fluorescent in aqueous solutions, but is highly fluorescent in organic solvents and also when the compound is in the hydrophobic environment of a protein. When coelenterazine is luminesced in the presence of Oplophorus luciferase, the solution after luminescence (the spent solution) is not fluorescent, presumably due to the dissociation of coelenteramide from the luciferase that provided a hydrophobic environment at the time of light emission. An analogous situation exists in the bioluminescence system of Renilla (Hori et al., 1973). 

Coelenterazine emits chemiluminescence when dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) containing a trace amount of base. It also emits bioluminescence in aqueous media in the presence of a coelenterazine luciferase, such as Renilla luciferase or Oplophorus luciferase. In both cases, the luminescence reactions require molecular oxygen. The capability of coelenterazine to produce luminescence is attributed to the presence of the imida-zopyrazinone structure in the molecule. 

The decomposition of dioxetanone may involve the chemically initiated electron-exchange luminescence (CIEEL) mechanism (McCapra, 1977 Koo et al., 1978). In the CIEEL mechanism, the singlet excited state amide anion is formed upon charge annihilation of the two radical species that are produced by the decomposition of dioxetanone. According to McCapra (1997), however, the mechanism has various shortfalls if it is applied to bioluminescence reactions. It should also be pointed out that the amide anion of coelenteramide can take various resonance structures involving the N-C-N-C-O linkage, even if it is not specifically mentioned. 

Latia luciferase is colorless and normally nonfluorescent. However, the luciferase fluoresces visibly in alkaline solutions. The fluorescence is most prominent in a KCN solution, showing an emission spectrum that is very close to the bioluminescence spectrum and also to the fluorescence emission of a flavin (FAD) except for the 370 nm... 

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

The substances involved in bioluminescence reactions are usually unstable. Thus, the extraction and purification of bioluminescent substances should be carried out in the shortest possible period of time, usually at a low temperature. It is known through experience that luminescent substances are almost always more stable in the original animal tissues than in extracts when preserved at a low temperature. Therefore, before starting extraction and purification, the stability of the extracts and purified substances should be investigated by carrying out a small-scale pilot experiment. A pilot experiment is also essential in the course of purification to avoid an unexpected loss of the target substance. If a component of the luminescence system is insoluble in common buffer solutions, it must be solubilized to purify it (see C1.3). 

Laetmogone, 301, 337 Latnpadena, 163, 339 Lampito, 216, 234, 335 Lampteroflavin, 270 Lampteromyces, 267, 270 Lampyridae, 1, 2 See also Fireflies distribution, 2 morphology, 2 Lampyris, 2, 337 Latia bioluminescence, 189 activators and inhibitors, 189 light emitter, 191 luminescence spectrum, 192 reaction scheme, 190 Latia luciferase, 183-189, 343 assay, 184... 

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