Our technology

BioThema is entirely focused on analytical applications of the firefly luciferase reaction whether it is detecting cells, measuring enzyme activity or something else. In this reaction luciferin is activated with ATP (adenosine triphosphate) and thereafter oxidized to oxyluciferin and CO2. The oxyluciferin is formed in an excited state and emits light when going to its ground state. The light is normally measured in what is called a luminometer.

ATP+Luciferin+O2—Luciferase—>AMP+Pyrophosphate+Oxyluciferin+CO2+light

In assays of ATP the light emission may be essentially constant or decay rapidly. Thus there are stable light and flash type ATP reagents based on the firefly luciferase reaction. At low levels of luciferase a negligible proportion of the ATP is consumed per minute and the light can then be almost constant but also low. At high levels of luciferase it is inevitable that ATP is consumed resulting in a rapidly decreasing light but initially the peak light is high. Actually the peak height and the decay rate are both proportional to the luciferase activity. BioThema has developed three types of ATP reagents66:

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Markets

Research institutes

In biochemistry, molecular biology and biology there is a need to measure cells, enzymes, metabolites and DNA/RNA. The luc gene coding for firefly luciferase is a reliable in vivo or in vitro reporter gene. ATP (adenosine triphosphate) participates in a large number of these reactions. Using the firefly luciferase reaction emitting a light proportional to the ATP concentration makes it possible to follow the reactions in a simple and highly sensitive way. Calibration of the assays with our certified liquid-stable ATP standard provides robust and reliable results.

 

 

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Healthcare

 

Clinical diagnostics as e.g. measurement of bacteria in urine with a luciferase based assay of bacterial ATP can be used in the diagnosis of urinary tract infection (UTI). Screening for Duchenne’s Muscular Dystrophy (DMD) has been performed in 528,410 newborn boys in a German study.

Luciferase based assays of ATP and ATP+AMP is more and more used to control hospital hygiene.

 

 

 

 

 

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Pharma and biotech industries

In the development of new drugs high throughput screening (HTS) is often used to screen large libraries of compounds for inhibitors or stimulators of various reactions. The firefly luciferase reaction can be performed reliably in a small reaction volume and is therefore highly suitable for HTS. Our Protein Kinase Kit RR can be used to measure not only protein kinase, but also all ATPases and aminoacyl tRNA synthetases. Growth inhibition from e.g. antibiotics can be measured. Gene expression can be monitored.

 

 

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Cleaning industry

 

In cleaning industry luciferase based assays of ATP and ATP+AMP is used for hygiene control in food and beverage industry, restaurants, hospitals and pharma industry. It is also used to develop improved cleaning tools and procedures and for education of cleaning personnel. The advantages area that an objective result is obtained, that seller and purchaser can agree on a measurable degree of cleanliness and that the cleaning personnel can perform the test in less than one minute using a portable instrument immediately seeing whether the work done is satisfactory or not.

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Applications

Enzyme assays and metabolite assays

All ATP producing and ATP degrading enzymatic reactions can be continuously monitored by measuring the light from firefly luciferase/luciferin being present in the reaction mixture. Examples include protein kinases67, adenylate kinase14, pyruvate kinase6, pyruvate phosphate dikinase, glycerol kinase43, 44, creatine kinase isoenzymes8, 10, 18, 22, ATPases14, ATP synthetases49, aminoacyl tRNA synthetase. Substrates to such enzymes can be measured kinetically or with endpoint assays. Examples include ADP, AMP, pyrophosphate.

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Rapid cell counting

 

All living cells contain ATP and when the cell dies ATP is degraded by intracellular enzymes to ADP and finally AMP. The intracellular ATP concentration is similar in different cell types. This means that bacterial cells contain 1-2 attomoles per cell while animal and plant cells contain 100 to 10 000 times as much ATP. Extracellular ATP may be degraded by adding ATP consuming enzymes or by physical separation. Determination of intracellular ATP can then be used to estimate biomass. If the amount of ATP per cell is known the cell number may also be estimated.

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Reporter gene assays and in vivo imaging

The luc gene coding for firefly luciferase may be attached to another gene of interest. The expressing of this gene may then be estimated from the level of luciferase expression measured as light. This may be done in vitro, i.e. after lysing the cells adding ATP and D-luciferin, or in vivo. In the latter case D-luciferin has to enter the cells. Even whole live animals or plants may be studied, “in vivo imaging”, by injecting D-luciferin into the organism (ATP is already in the cells).

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Hygiene control

All biological residues contain ATP. When living cells die ATP is degraded to ADP and finally AMP. Measurement of ATP+AMP is a proof of biological residues being present. This can be done using the unique Kikkoman LuciPac Pen (surfaces) or LuciPac Pen Aqua (liquids) with a portable instrument PD-30. Hygiene control measuring ATP only may underestimate the contamination if ATP has been degraded in the biological residues. On the other hand ATP is an indicator that the residues are still alive. Neither ATP nor ATP+AMP are a reliable measure of bacteria as these contain much less ATP than cells from animals or plants.

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