Method for detecting pyrophosphate by means of bioluminescence detection
View Patent ↗The invention relates to methods for detecting pyrophosphate by means of bioluminescence detection. In addition, methods for measuring chemical, especially enzyme-catalyzed, reactions in which pyrophosphate is formed or consumed are described. Such reactions are catalyzed for example by guanylyl cyclases, adenylyl cyclases, DNA polymerases or RNA polymerases. The novel methods are distinguished by high sensitivity and low susceptibility to interference, can easily be automated and miniaturized and are additionally suitable for carrying out continuous measurements. The methods can be employed particularly advantageously in the area of medical diagnosis and biomedical research, including pharmaceutical active ingredient research.
1. A method for detecting pyrophosphate, comprising the steps of:
(a) incubating a composition consisting essentially of dehydroluciferin, ATP, and luciferase which is activated by pyrophosphate, to make an incubated composition, the ATP being present in an amount that is in excess of the dehydroluciferin and luciferase,
(b) combining the incubated composition with luciferin to make a detection mix, the luciferin being present in an amount that is in excess of the dehydroluciferin and luciferase,
(c) contacting a test sample with the detection mix, and
(d) measuring luminescence of the test sample, whereby pyrophosphate concentration in the test sample is determined by the intensity of the luminescence.
2. The method as claimed in claim 1 , where the luciferase is selected from the group consisting of:
a non-recombinant or recombinant luciferases,
a non-recombinant or recombinant luciferase from insects of the superfamilies Elateroidea and Cantharoidea,
a non-recombinant or recombinant luciferase from the insects Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis or Luciola mingrelica,
a luciferase derived or mutated from any of the above luciferases, and
a mixture of two or more of the above luciferases.
3. The method as claimed in claim 1 , wherein the test sample originates from a natural source including the animate and inanimate environment.
4. The method as claimed in claim 1 , wherein the test sample originates from a vegetable or animal organism.
5. The method as claimed in claim 1 , wherein the test sample originates from the human body.
6. The method as claimed in claim 5 , wherein the test sample comprises samples or preparations of tissue or body fluids.
7. A method for measuring the time course of a pyrophosphate-forming or consuming a chemical or enzyme reaction, comprising the steps of:
(a) providing a mixture in which pyrophosphate is being formed or consumed in a chemical or enzyme reaction;
(b) adding a detection mix to the mixture, the detection mix comprising an incubated composition consisting essentially of dehydroluciferin, ATP, and a luciferase which can be activated by pyrophosphate, the detection mix further comprising luciferin, the ATP and luciferin being present in an amount that is in excess of the dehydroluciferin and luciferase; and
(c) continuously measuring luminescence of the mixture during the chemical or enzyme reaction, pyrophosphate concentration in the test sample is determined by the intensity of the luminescence.
8. A method for measuring the time course of a chemical or enzyme reaction that forms or consumes pyrophosphate comprising the steps of:
(a) providing a mixture that can form or consume pyrophosphate in an chemical or enzyme reaction, the mixture comprising a detection mix consisting essentially of an incubated composition of dehydroluciferin, ATP, and a luciferase which can be activated by pyrophosphate, the detection mix further comprising luciferin, the ATP and luciferin being present in an amount that is in excess of the dehydroluciferin and luciferase;
(b) starting a chemical or enzyme reaction in the mixture by adding a substrate to the mixture; and
(c) continuously measuring luminescence of the mixture during the chemical or enzyme reaction, pyrophosphate concentration in the test sample is determined by the intensity of the luminescence.
9. The method as claimed in claim 7 or 8 , where the luciferase is selected from the group consisting of:
a non-recombinant or recombinant luciferase,
a non-recombinant or recombinant luciferase from insects of the superfamilies Elateroidea and Cantharoidea,
a non-recombinant or recombinant luciferase from the insects Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis or Luciola mingrelica,
a luciferase derived or mutated from any of the above luciferases, and
a mixture of two or more of the above luciferases.
10. The method as claimed in claim 7 or 8 , where the chemical or enzyme reaction is selected from the group consisting of:
pyrophosphate-forming or pyrophosphate-consuming enzyme reactions, guanylyl cyclase-catalyzed reactions,
adenylyl cyclase-catalyzed reaction,
squalene synthase-catalyzed reaction,
pyrophosphatase-catalyzed reaction,
DNA polymerase-catalyzed reaction, and
RNA polymerase-catalyzed reaction.
11. The method as claimed in claim 7 or 8 , characterized in that the enzyme reaction is catalyzed by soluble vascular guanylyl cyclase.