Bioluminescence offers huge advantages over conventional chromogenic or fluorescent methods for sensitive detection systems, as it measures light signals against a completely dark background. It is like observing stars in front of a perfectly black night sky. The fantastic sensitivity and the relatively low instrumentation requirements have ensured that bio- and chemiluminescence methods have replaced other measurement methods in many areas in recent years.
Bioluminescence-based methods have also great potential for tests in hygiene monitoring, microbiological food and drinking water control, as well as clinical microbiology. However, the use of luciferase enzyme for in vitro assays has been limited by the availability of a cost-efficient stable Luciferase enzyme. With the new X-Shining™ Luciferase, an inexpensive stable luciferase is now commercially available.
X-Shining™ Luciferase Application Examples
1. ATP Tests
Adenosine triphosphate (ATP) is present in all living cells and plays a central role in the energy balance. The intracellular concentration of ATP is tightly regulated and is maintained at a similar level in all cells. When a cell dies, the ATP is completely degraded; ATP levels therefore reflect the presence of any living cell. The bioluminescence-based assays are extremely sensitive; a standard luminometer can detect as little as 0.1 picomole of ATP. The sensitivity has led to numerous applications as detecting low-level bacterial contamination in samples such as blood, urine and milk. ATP production, measured by the luciferin-luciferase assay has also been used successfully to study the effects of antibiotics on bacterial populations.
X-Shining™ Luciferase in combination with D-Luciferin shows an excellent signal linearity over a wide ATP concentration range, from 8 fmol to 1 nmol in 96-well plate assays with 0.1 mL liquid volume.
2. Detection of Enzyme Activities Using Luciferase with Pro-Luciferins
Luciferase bioluminescence can also be used for ultra-sensitive detection of a broad range of enzymatic activities, using D-luciferin or aminoluciferin coupled with enzyme labile groups as substrates. Luciferin-based substrates have been referred to as “caged luciferins” or “pro-luciferins” in the literature. Pro-lucferins can be used for example for the detection of specific groups of bacteria, for reporter gene assays in animal cells or plant biology.
3. Bacteria Detection with Pro-Luciferins
The introduction of pro-luciferins into microbial analytics has opened the way for new bioluminescent-based bacteria detection methods. In this setup, bacteria cultures are grown to relatively low bacteria concentration and subsequently supplied with the bio-luminescent substrate, i.e. the pro-luciferin. The bacteria-specific enzyme cleaves off the enzyme-labile group from the substrate, leading to the release of D-luciferin. Then, in the presence of ATP, magnesium ions and oxygen, the reaction of D-luciferin with luciferase leads to light emission.
Luciferase can detect luciferin even at very low concentrations, and therefore enables the lowest detection limits for viable bacteria. For example in the identification of bacteria positive for phosphatidyl-choline specific phospholipase C (PC-PLC), such as strains of Pseudomonas aeruginosa and Bacillus cereus.
X-Shining™ Luciferase is supplied as 10 mg/mL aqueous solution with glycerol. 0.1 mL X-Shining™ Luciferase is sufficient for ca. 10’000 assays.
Storage and Stability
Store the stock solution at -15°C.
Stability of luciferase solutions: Filter-sterilized solutions of X -Shining™ Luciferase in assay buffer were stable for at least seven months at room temperature. The same solutions may be used for even longer time if stored in the fridge.
Light emission spectrum:
X-Shining™ Luciferase emission spectrum
. The maximal intensity of light emission with D luciferin and X-Shining™ Luciferase at pH 7.8 is at 560 nm, identical to commercially available wild-type firefly luciferase (from Photinus pyralis).