Bioluminescence Assay
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Bioluminescence assays use the property of bioluminescence, or light production by living organisms, to measure various biological processes. They involve reactions between luciferin and luciferase enzymes that produce light, which can then be measured to assess factors like cell proliferation, apoptosis, drug effects, and kinase activity. Specific techniques include using luciferase-labeled cells or tissues to noninvasively image processes inside live animals via bioluminescent imaging, employing luciferase genes as reporters to study the activity of other genes of interest, and monitoring tuberculosis bacteria in mice treated with anti-tuberculosis drugs.
Bioluminescence Assay
- 1. BIOLUMINESCENCE ASSAY L. Sanathoiba Singha M. Pharm 1st Semester Department of Pharmaceutical Analysis Karnataka College of Pharmacy Bangalore- 64 Karnataka, India.
- 2. 1 Bioluminescence is the production and emission of light by a living organism. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some bioluminescent bacteria and terrestrial invertebrates such as fireflies. Bioluminescence assays involve the use of the property of bioluminescence for measuring cell proliferation, apoptosis, drug metabolism, kinase activity, etc. PRINCIPLE: Bioluminescence is a form of chemiluminescence where light energy is released by a chemical reaction. This reaction involves a light-emitting pigment, the luciferin, and a luciferase, the enzyme component. Because of the diversity of luciferin/luciferase combinations, there are very few commonalities in the chemical mechanism. For example, the firefly luciferin/luciferase reaction requires magnesium and ATP and produces carbon dioxide (CO2), adenosine monophosphate (AMP) and pyrophosphate (PP) as waste products. Other cofactors may be required for the reaction, such as calcium (Ca2+ ) for the photoprotein aequorin, or magnesium (Mg2+ ) ions and ATP for the firefly luciferase. Generically, this reaction could be described as: Luciferase Other cofactors Oxyluciferin Light energyO2Luciferin HARNESSING BIOLUMINESCENCE: 1. Food testing using ATP (Bioluminescence) Technology- The test kit for this test contain firefly luciferase and luciferin which are used to detect the presence of ATP, a compound that is found in all living cells; this includes any live microbes like Salmonella or E. coli that might be present in food and food products. The intenisty of the light produced from the reaction is detected by a luminometer. The more the ATP, the brighter the light, so the intensity of luminescence reveals how many bacteria are present. This test is able to detect even tiny amounts of microbial contamination, using very senstitive instruments to measure light production. It requires mere minutes instead of the days needed to detect contaminated food by growing bacterial cultures. 2. Bioluminescent imaging- Fireflies have helped scientists develop real-time, noninvasive imaging to see what's happening inside living organisms. When LUC (luciferase) genes are used to label particular cell or tissue types, very sensitive cameras can be used to detect their light inside the live animal.
- 3. 2 Mice bearing tumors are tagged with LUC gene which express luciferase and are further injected with luciferin. Researchers then use a sensitive camera system to view, without killing the mice, the tumor and any effects of the different cancer agents. Tumor cells can be grown in culture medium and then treated with different drugs. Using luminescence-based tests to measure cell viability, those drugs most effective at killing tumor cells can be quickly identified. This way, potential new chemotherapies for treating cancers can be tested. 3. Luciferase (LUC) gene as reporter for the activity of other genes- Here, the researchers splice the LUC gene together with a specific gene they want to study, and then insert this spliced DNA into living cells. Whenevr the spliced DNA gets transcribed, the cells will manufacture luciferase. When luciferin is added, these cells will respond by lighting up. This technque has been used, for instance, to find out exactlty when and where specific plant genes get turned on. To learn about particular genes regulating plant growth, biologists have spliced the LUC gene into different bits of plant DNA. When plants are sprayed or fed with luciferin-containing water, the leaves will glow whenever LUC gene gets turned on. This allows researchers to identify specific genes regulating plant growth at different times and locations. Such reporter genes have also provided tools for studying diseases, for developing new antibiotic drugs and for gaining new insights into many human metabolic disorders. 4. Development of new treatment for antibiotic-resistant tuberculosis- To help discover new treatments for antibiotic-resistant tuberculosis, scientists have infected mice with luciferase-labeled tuberculosis bacteria. They then treat the mice with various anti- tuberculosis drugs and use bioluminescence imaging to monitor the bacteria inside. 5. Measurement of calcium changes inside cells- The photoprotein aequorin requires Ca2+, it is often used to measure calcium changed indside cells. 6. Discovery of green protein (GFP)- One of the most well-known developments to come out of bioluminescence research is the discovery of the green fluorescent protein (GFP). While GFP is not a bioluminescent protein, it serves as an accessory emitter by receiving energy from a luciferin-luciferase reaction and re-emitting it as green light.