发光分析 Luminescence Analysis－上海科端生物技术有限公司

发光分析 Luminescence Analysis

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Definitions
Certain chemical reactions result in the formation of a fluorescent molecule in its excited state. When this molecule releases its bound energy, a photon, i.e. a light particle, is emitted. The phenomenon is called chemiluminescence or, when appearing in nature, bioluminescence. Bioluminescence is a frequent phenomenon in the marine environment. The fireflies are well-known examples of insect bioluminescence. Bioluminescence is catalyzed by various enzymes called luciferases acting on species specific luciferins.

Analytical Characteristics
In luminescence analysis (or luminometry) one makes use of the fact that light is easy to measure accurately, even at very low levels. The assay is set up in a way that the light intensity or the total amount of light emitted is proportional to the analyte, i.e. the substance one wants to measure. Such assays are have a unique combination of four characteristics:

Low detection limits (often 10^-18 mol or for enzymes even lower).
Low cost equipment (the measuring instrument is called a luminometer and is in principle much more simple than e.g. a spectrophotometer).
Low cost reagents (potential for miniaturized assay systems).
Simple analytical procedure (mix and measure).

Application areas
The firefly luciferin-luciferase system is the most frequently used bioluminescent reaction for analytical purposes. It can be used for assays of ATP and any enzyme or metabolite participating in ATP forming or degrading reactions (cf. ATP monitoring). ATP can be used as a measure of biomass (amount of living material), since the intracellular level of ATP is similar in all living cells, and is rapidly degraded when the cell dies. Rapid microbiology and hygiene monitoring are important applications of the ATP assay. The gene coding for firefly luciferase has become one of the most frequently used reporter genes used in molecular biology to study transfer and expression of genes. Bacterial luciferase combined with oxidoreductase enzymes can be used to monitor NADH and NADPH in a similar way, as is done with firefly luciferase for ATP. Horseradish peroxidase and luminol can be used to measure H2O2 production. Several enzymes and other substances participating in luminescent reactions may be used as labels in immunoassays. Thus luminescence analysis has many applications in a variety of areas. If, e. g., a completely new system for clinical analysis should be built up today, a likely candidate for the analytical system would be luminometry. This would enable all assays (assays of enzymes and metabolites, immunoassays and many bacteriological assays) to be performed using the same instrument).

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