Technology Overview
Luminescence Analysis
Certain chemical reactions result in the formation of a fluorescent molecule in its excited state. When this molecule releases its bound energy, a light particle or photon is emitted. The phenomenon is called chemiluminescence or, when appearing in nature, bioluminescence. Bioluminescence is a frequent phenomenon in nature and is catalyzed by enzymes called luciferases acting on species specific luciferins.
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, the substance one wants to measure. Such assays have a unique combination of four characteristics:
- Low detection limits (often 10-18 mol, or even lower for enzymes).
- Low cost equipment (the measuring instrument is called a luminometer and is in principle much more simple than a spectrophotometer).
- Low cost reagents (potential for miniaturised assay systems).
- Simple analytical procedure (mix and measure principle).
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.
- ATP can be used as a measure of the amount of living material or biomass, since the intracellular level of ATP is similar in all living cells, and ATP 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.
- 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 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 assays of enzymes and metabolites, immunoassays and many bacteriological assays to be performed using the same technology and instrumentation.
Sensitivity of ATP measurement and stable light emmission
BioThema has developed kits which measure ATP levels at different sensitivities. The most sensitive can measure at the level of a single bacterial cell (using the ATP Reagent SS). On the other hand the ATP Reagent SL, while still able to measure a single eukaryotic cell, gives a stable light emmission which makes manual measurements easy and convenient.
