Luminogenic and nonluminogenic multiplex assay
View Patent ↗A method to detect the presence or amount of at least one molecule for an enzyme-mediated reaction in a multiplex luminogenic/nonluminogenic assay is provided.
1. A method to detect the presence or amount of a first molecule for a first enzyme-mediated reaction and a second molecule for a second enzyme-mediated reaction, comprising:
a) contacting a sample with a reaction mixture for a first enzyme-mediated reaction to detect a first molecule, and with a reaction mixture for a second enzyme-mediated reaction to detect a second molecule, wherein a reaction mediated by a first enzyme in the first reaction yields a bioluminogenic product, and wherein a reaction mediated by a second enzyme in the second reaction yields a fluorogenic product; and
b) detecting the presence or amount of the first and the second molecules in the sample.
2. The method of claim 1 wherein the first molecule is a substrate for the first enzyme-mediated reaction.
3. The method of claim 1 wherein the second molecule is a substrate for the second enzyme-mediated reaction.
4. The method of claim 1 wherein the first molecule is an enzyme for the first enzyme-mediated reaction.
5. The method of claim 1 wherein the second molecule is an enzyme for the second enzyme-mediated reaction.
6. The method of claim 1 wherein the first molecule is a co-factor for the first enzyme-mediated reaction.
7. The method of claim 1 wherein the second molecule is a co-factor for the second enzyme-mediated reaction.
8. The method of claim 1 wherein bioluminescence is employed to detect the presence of the first molecule.
9. The method of claim 8 wherein the bioluminescence increases in the presence of the first molecule.
10. The method of claim 1 wherein fluorescence is employed to detect the presence of the second molecule.
11. The method of claim 1 wherein the presence or amount of the first and second molecules is detected sequentially.
12. The method of claim 1 wherein the sample is a cell lysate, suspected of comprising either the first or second molecule or both.
13. The method of claim 1 wherein the sample is contacted with the reaction mixture for the first reaction before the reaction mixture for the second reaction.
14. The method of claim 1 wherein the sample is contacted with the reaction mixture for the second reaction before the reaction mixture for the first reaction.
15. The method of claim 1 wherein the sample is contacted with the reaction mixture for the first reaction and the second reaction at the same time.
16. The method of claim 1 wherein the bioluminogenic product is a substrate for a beetle luciferase.
17. The method of claim 1 wherein the fluorogenic product comprises fluorescein, Cy3, BODIPY™ (4,4 -difluoro-1,3-dipropyl-4-bora-3a,4a-diaza-s-indacene), a rhodol, Rox, 5-carboxyfluorescein, 6-carboxyfluorescein, an anthracene, 2-amino-4-methoxynapthalene, a phenalenone, an acridone, fluorinated xanthene derivatives, α-naphtol, β-napthol, 1-hydroxypyrene, coumarin, 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin (AFC), TEXAS RED™ (sulforhodamine 101), tetramethyirhodamine, carboxyrhodamine, rhodamine, cresyl, rhodamine-110 or resorufin.
18. The method of claim 1 wherein the first molecule is a protease.
19. The method of claim 1 wherein the second molecule is a protease.
20. The method of claim 1 wherein one of the molecules is a glycosidase, phosphatase, kinase, dehydrogenase, peroxidase, sulfatase, peptidase, or hydrolase.
21. The method of claim 1 wherein the presence or amount of the first and second molecules is detected simultaneously.
22. The method of claim 11 wherein bioluminescence is employed to detect the presence or amount of the first molecule prior to detection of the second molecule.
23. The method of claim 11 wherein bioluminescence is employed to detect the presence or amount of the first molecule after detection of the second molecule.
24. The method of claim 1 wherein bioluminescence is employed to detect the presence of the first molecule and fluorescence is employed to detect the presence of the second molecule.
25. The method of claim 24 wherein the bioluminogenic product is a substrate for a beetle luciferase.
26. The method of claim 24 wherein one of the molecules is a glycosidase, phosphatase, kinase, dehydrogenase, peroxidase, sulfatase, protease, or hydrolase.
27. The method of claim 1 wherein the sample comprises mammalian cells, suspected of comprising either the first or second molecules or both.