Metabolic biosensor and uses thereof

• US 20060154326A1
• Filed: 12/19/2005
• Published: 07/13/2006
• Est. Priority Date: 06/07/2001
• Status: Abandoned Application

• Current Assignee: McGill University
• Original Assignee: McGill University
• Inventors: Sheppard, John, Vann, Lucas
• Application Number:
  US11/311,898
• Publication Number:
  US 20060154326A1
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 2333/39 from yeasts
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

The present invention relates to a device for analyzing the metabolism of cells involved in a culture or fermentation process. A sample of the culture or fermentation medium is submitted to at least one oxidation-reduction reaction. The device of the invention includes two electrodes that measures the electric conductivity of samples and transmitted a message to an integration electronic system. Thereafter, the difference in the electric conductivity between the untreated and treated samples is indicative of the function of targeted metabolism pathway during the culture of the fermentation process.

Claims

1. A device for measuring a substrate as an analysis of the metabolism of a cell in a culture medium comprising;

a first reaction vessel comprising a first oxidation or reduction reaction mixture containing a first enzyme and a cofactor, said first enzyme being capable of chemically reacting with the substrate to be measured, producing a product and causing reduction or oxidation of the cofactor;

a second reaction vessel containing a second oxidation or reduction reaction mixture containing a second enzyme, said second enzyme being capable of chemically further reacting with the product produced in the first reaction vessel producing a second product and causing reduction or oxidation of the cofactor;

a detector for determination of the cofactor reduced or oxidized in the first and/or second reaction vessel, said first and second reaction vessels and said detector being in fluid connection together in a closed circuit.

2. The device of claim 1, wherein the first enzyme is a diacetyl reductase.

3. The device of claim 2, wherein the diacetyl reductase is OYE1.

4. The device of claim 1, wherein the second enzyme is butanediol dehydrogenase.

5. The device of claim 1, wherein said cell is selected from the group consisting of a microorganism cell, an animal cell, and a plant cell.

6. The device of claim 1, wherein the cofactor is selected from the group consisting of pyridine-linked dehydrogenase, flavin-linked dehydrogenase, iron-sulfur protein, a cytochrome, ubiquinone, NAD(H) and NADP(H).

7. The device of claim 1, wherein the cofactor is NAD(H) or NADP(H).

8. The device of claim 1, wherein the second oxidation or reduction reaction mixture further comprises the cofactor of the first reaction vessel.

9. The device of claim 1, further comprising a third reaction vessel in fluid connection with the first reaction vessel, said third reaction vessel comprising another catalyst assisting in converting a precursor of said substrate into the substrate.

10. The device of claim 1, wherein the detector is a spectrometer.

11. A device for measuring a diacetyl potential in a yeast culture of a brewing process, said device comprising in fluid connection;

a first reaction vessel comprising a catalyst or an enzyme allowing conversion of alpha acetolactate that may be present in the yeast culture into diacetyl;

a second reaction vessel containing a cofactor and an enzyme capable of converting in a redox reaction diacetyl into acetoin, oxidizing the cofactor; and

a detector for determinating the concentration of the cofactor so oxidized in the second reaction vessel, said first and second reaction vessels and said detector being in fluid connection together in a closed circuit.

12. The device of claim 11, wherein the catalyst is aniline.

13. The device of claim 11, wherein the enzyme is a diacetyl reductase.

14. The device of claim 13, wherein the diacetyl reductase is OYE1.

15. The device of claim 11, wherein the cofactor is selected from the group consisting of pyridine-linked dehydrogenase, flavin-linked dehydrogenase, iron-sulfur protein, a cytochrome, ubiquinone, NAD(H) and NADP(H).

16. The device of claim 11, wherein the cofactor is NAD(H) or NADP(H).

17. The device of claim 11, further comprising a third reaction vessel in fluid connection between the second reaction vessel and the detector, said third reaction vessel comprising an enzyme capable of converting in a redox reaction acetoin, oxidizing the cofactor into 2,3 butanediol, the detector determinating the total concentration of the cofactor so oxidized in the second and third reaction vessels.

18. The device of claim 11, wherein the detector is a spectrometer.

19. A method for monitoring metabolic rate of cells in a cell culture preparation comprising the steps of:

a) contacting a sample of cell culture preparation containing a product to be measured as an indicator of said metabolic rate of said cells with a first oxidation or reduction reaction mixture containing an first enzyme and a cofactor, said first enzyme transforming the product to be measured causing reduction or oxidation of the cofactor to obtain a once-reacted sample containing a first transformed product and a reduced or oxidized cofactor;

b) contacting said once-reacted sample of step a) with a second oxidation or reduction reaction mixture containing a second enzyme, said second enzyme transforming the first transformed product of step b) causing reduction or oxidation of the cofactor to obtain a second transformed product and the reduced or oxidized cofactor;

c) detecting a concentration of cofactor reduced or oxidized in the first and/or second reaction vessel.

20. The method of claim 19, wherein the first enzyme is diacetyl reductase.

21. The method of claim 20, wherein the diacetyl reductase is OYE1.

22. The method of claim 19, wherein the second enzyme is butanediol dehydrogenase.

23. The method of claim 19, wherein the cofactor is selected from the group consisting of pyridine-linked dehydrogenase, flavin-linked dehydrogenase, iron-sulfur protein, a cytochrome, ubiquinone, NAD(H) and NADP(H).

24. The method of claim 23, wherein the cofactor is NAD(H) or NADP(H).

25. The method of claim 19, wherein the second oxidation or reduction reaction mixture further comprises the cofactor of step a).

26. The method of claim 19, wherein the concentration of the reduced or oxidized cofactor in step d) is determined by measuring light absorbance or electric conductivity, and correlating said measuring with a measurement of light absorbance or electric conductivity of a known concentration of the cofactor.

27. The method of claim 19, further comprising before step a) a step of pre-contacting the sample with a further oxidation or reduction reaction mixture comprising another catalyst assisting conversion of a precursor of said product into the product.

28. The method of claim 19, wherein said metabolic rate is selected from the group consisting of physiological state, cell age, growth rate, and vitality.

29. The method of claim 28, wherein said physiological state is selected from the group consisting of reduction reaction rate, oxidative reaction rate, glycosylation, acetylation, methylation, and carboxylation.

30. The method of claim 19, wherein said cells are selected from the group consisting of microorganism cells, animal cells, and plant cells.

31. The method of claim 30, wherein said microorganism is yeast or bacteria.

32. The method of claim 19, wherein said culture preparation is a culture medium, a culture broth, a fermentation medium, or a fermentation broth.

33. The method of claim 32, wherein said fermentation medium is an alcoholic or a lactic fermentation medium.

34. A method for the determination of diacetyl concentration as an indicator of cell metabolic rate in a brewing fermentation process, said diacetyl being measured in a sample of a medium obtained from said fermentation process, said method comprising the steps of:

a) contacting said sample in a first reaction mixture containing a catalyst for transforming alpha-acetolactate into diacetyl;

b) contacting said diacetyl produced in step a) with a second oxidation reaction mixture containing a cofactor and an enzyme for transforming diacetyl into acetoin causing oxidation of the cofactor; and

c) detecting the concentration of the oxidized cofactor of step b) with correlation to known standards.

35. The method of claim 34, wherein said catalyst is aniline.

36. The method of claim 34, wherein said enzyme is a diacetyl reductase.

37. The method of claim 34, wherein said diacetyl reductase is OYE1.

38. The method of claim 34, wherein the cofactor is selected from the group consisting of pyridine-linked dehydrogenase, flavin-linked dehydrogenase, iron-sulfur protein, a cytochrome, ubiquinone, NAD(H) and NADP(H).

39. The method of claim 38, wherein the cofactor is NAD(H) or NADP(H).

40. The method of claim 34, wherein the concentration of the oxidized or reduced cofactor in step c) is determined by measuring light absorbance or electric conductivity, and correlating said measuring with a measurement of light absorbance or electric conductivity of a known concentration of the electron acceptor.

41. The method of claim 34, further comprising between step b) and c) a step of contacting the second transformed product with a third oxidation or reduction reaction mixture comprising a further cofactor and an enzyme for transforming said second transformed product into 2,3 butanediol.

42. The method of claim 41, wherein said further cofactor is selected from the group consisting of pyridine-linked dehydrogenase, flavin-linked dehydrogenase, iron-sulfur protein, a cytochrome, ubiquinone, NAD(H) and NADP(H).