Color control system

• US 5,032,506 A
• Filed: 12/16/1986
• Issued: 07/16/1991
• Est. Priority Date: 12/16/1986
• Status: Expired due to Term

• Current Assignee: Orasure Technologies Incorporated
• Original Assignee: Enzymatics Incorporated
• Inventors: Palmer, John L., Timmerman, Marsha W.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Scheiner, Toni R.
• Application Number:
  US06/942,414
• Time in Patent Office: 1,673 Days
• Field of Search: 435/25, 435/26, 435/175, 435/176, 435/177, 435/178, 435/179, 435/805, 435/810, 435/4
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/008 for determining co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

An assay system useful for the determination of NAD(P)H, NAD(P), or a substrate of an enzyme which reacts with the formation or comsumption of NAD(P)H. Concentrations of organic substrates for example alcohol, cholesterol, uric acid, in a biological fluid such as saliva, blood or urine may be determined. The system includes a diaphorase which catalyzes a NAD(P)H-dependent reduction of a chromogen to cause a visible color change; this color change is indicative of the concentration sought to be determined. The system includes a chromogen which is a first substrate for the diaphorase which causes a color change when reduced by NAD(P)H, and a second substrate which is a competing substrate for the diaphorase; the competing substrate is irreversibly reduced by the diaphorase. The system is capable of measuring colorimetrically without dilution concentrations of organic compounds in biological fluids which previously could not be measured in such concentration. The system provides a convenient, practical sobriety test. The invention also provides a method for such determination and diagnostic kit.

Claims

1. An assay system for colorimetric measurement and determination of NAD(P)H, NAD(P), or an enzyme substrate which reacts to form or consume NAD(P)H wherein the assay system comprises:

a diaphorase which catalyzes NAD(P)H-dependent reduction of a chromogen to cause a visible color change,

a chromogen which is an electron accepting substrate of the diaphorase, and which produces a color change upon reduction by NAD(P)H, and

a non-chromogenic competing substrate of the diaphorase in an amount sufficient to increase the range of concentrations that can be measured by the color change of the chromogen when the competing substrate is irreversibly reduced by the diaphorase,

wherein the change in color caused by reduction of the chromogen occurs in a ratio of less than one molecule of dye for each molecule of NAD(P)H produced and is indicative of the concentration to be determined.

2. The system of claim 1 wherein the color change is an increase in color.

3. The system of claim 1 wherein the diaphorase catalyzes the reduction of the competing substrate and the chromogen with similar efficiency.

4. The system of claim 3 wherein said competing substrate causes the amount of color generated by said chromogen to be linearly related to the concentration of the NAD(P)H.

5. The system of claim 4 wherein a linear relationship occurs over all concentrations measured.

6. The system of claim 1 wherein the chromogen is not reduced until after the non-chromogenic competing substrate is reduced.

7. The system of claim 6 wherein the competing substrate is a benzoquinone.

8. The system of claim 6 wherein sufficient chromogen reduction to generate a visible color change does not occur until virtually all of the competing substrate is reduced.

9. The system of claim 1 which includes means for causing the reduction of the competing substrate to be irreversible.

10. The system of claim 9 wherein the means for causing the reduction to be irreversible is a reactant for the reduced competing substrate.

11. The system of claim 10 wherein the reactant is dihydrolipoic acid present as a supersaturated solution.

12. The system of claim 11 additionally comprising zinc ions.

13. The system of claim 9 wherein the competing substrate is a lipoic acid compound, and the means for causing the reduction to be irreversible is a reactant that reacts preferentially with the reduced lipoic acid compound as compared with the oxidized lipoic acid compound.

14. The system of claim 13 wherein the reactant is selected from the group consisting of iodoacetic acid, oxidized 2-mercaptoethanol, ethanol, chloroacetone, dichloroacetone, methyl iodide, dibenzylsulfide, 2-hydroxy-methyl-6-methoxy-1,4-benzoquinone diiodo-4-pyridone-N-acetic acid, salts of iodoacetic acid, and salts of diiodo-4-pyridone-N-acetic acid.

15. The system of claim 13 wherein the reactant forms a chelate compound with the competing substrate.

16. The system of claim 15 wherein the reactant has a greater affinity for the reduced competing substrate than for the oxidized competing substrate.

17. The system of claim 15 wherein the reactant is a metal ion.

18. The system of claim 17 wherein the metal ion is selected from the group consisting of zinc, mercury, chromium and ferric ions.

19. The system of claim 1 wherein the competing substrate is a lipoic acid compound.

20. The system of claim 19 wherein the lipoic acid compound is selected from the group consisting of DL-alpha-lipoic acid, DL-alpha-lipoamide, DL-lipoyl glycine, DL-dihydrolipoyl glycine, DL-lipoyl beta-alanine, DL-lipoyl glycylglycine, DL-carboethoxy lipoanilide, DL-lipoanilide and DL-dihydrolipoanalide.

21. The system of claim 1 wherein the competing substrate is selected from the group consisting of an antiaromatic compound, a disulfide and a dithiobenzene.

22. The system of claim 1 which also includes an organic analyte oxidizable by NAD(P)-dependent dehydrogenase to yield NAD(P)H, wherein the concentration of NAD(P)H is indicative of the concentration of the organic analyte.

23. The system of claim 22 wherein the analyte is in a biological medium.

24. The system of claim 23 wherein the biological medium is saliva, blood or urine.

25. The system of claim 24 wherein the biological medium is urine containing uric acid, and wherein the concentration of uric acid is to be determined.

26. The system of claim 24 wherein the biological medium is saliva containing alcohol, and wherein the concentration of alcohol is to be determined.

27. The system of claim 24 wherein the biological medium is blood containing cholesterol, and wherein the concentration of cholesterol is to be determined.

28. The system of claim 22 wherein the organic analyte is selected from the group consisting of carbohydrates, polyalcohols and ketones.

29. The system of claim 28 wherein the analyte is selected from the group consisting of alcohol, cholesterol, lactic acid and acetone.

30. The method of claim 22 wherein the diaphorase catalyzes the reduction of the competing substrate and the chromogen with similar efficiency.

31. The system of claim 1 wherein the molar proportion of competing substrate to chromogen is at least 1 to 1.

32. The system of claim 1 wherein the competing substrate is present in excess over the chromogen.

33. The system of claim 1 wherein the chromogen is a tetrazolium salt.

34. The system of claim 33 wherein the tetrazolium salt is selected from the group consisting of NTB, INT, 4,5-MTT and DCIP.

35. The system of claim 1 wherein the competing substrate is selected from the group consisting of semicarbazide, hydrazine, hydroxymethyl benzoquinone, hexane-dihydrazine and 1,4-butane dihydrazine.

36. The system of claim 1 wherein the competing substrate is potassium ferricyanide.

37. A method for colorimetric measurement of the amount of an organic analyte in a sample wherein the method comprises:

(a) oxidizing the organic analyte in the presence of NAD(P) and an NAD(P)-dependent dehydrogenase to produce NAD(P)H in an amount proportional to the amount of organic analyte,

(b) irreversibly reducing a chromogen which is an electron accepting substrate of diaphorase in the presence of diaphorase and the NAD(P)H produced in (a) causing a measurable color change,

(c) and irreversibly reducing a non-chromogenic competing substrate of diaphorase in the presence of diaphorase and the NAD(P)H produced in (a), wherein the competing substrate is present in an amount sufficient to expand the range of concentrations that can be measured by the color change of the chromogen,

(d) wherein the color change produced upon reduction of the chromogen is in a ratio of less than one molecule of dye per molecule of NAD(P)H, and

(e) measuring the color change caused by the chromogen, thereby determining the amount of analyte in the solution.

38. The method of claim 37 wherein the irreversible reduction of the chromogen and the competing substrate is concurrent or substantially concurrent.

39. The method of claim 38 wherein the sample is not diluted prior to adding the sample of the system.

40. The method of claim 38 wherein the concentration of the organic analyte in the sample is at least 0.5 mM.

41. The method of claim 37 wherein the irreversible reductions of the competing substrate and chromogen are sequential, the reduction of the chromogen following that of the competing substrate.

42. The method of claim 41 wherein a visible color change does not take place until virtually all of the competing substrate is reduced.

43. The method of claim 37 wherein the biological medium is urine containing uric acid, and wherein the concentration of uric acid is to be determined.

44. The method of claim 37 wherein the biological medium is saliva containing alcohol, and wherein the concentration of alcohol is to be determined.

45. The method of claim 37 wherein the biological medium is blood containing cholesterol, and wherein the concentration of cholesterol is to be determined.

46. The method of claim 37 wherein the biological medium is human serum containing beta-hydroxy butyrate, and wherein the concentration of beta-hydroxy is to be determined.

47. A diagnostic device for the quantitative or qualitative determination of an organic analyte in a biological medium, which comprises:

in combination, support means for a dehydrogenase capable of oxidizing the organic analyte, NAD(P), a chromogen which produces a color change upon reduction by NAD(P)H in the presence of diaphorase, a non-chromogenic competing substrate which is irreversibly reducible by NAD(P)H in the presence of diaphorase, and diaphorase,

wherein the color change produced upon reduction of the chromogen occurs in a ratio of less than one molecule of dye per molecule of NAD(P)H.

48. The device of claim 47 wherein the organic analyte is selected from the group consisting of carbohydrates, polyalcohols and ketones and the biological medium is selected from the group consisting of blood, serum, saliva and urine.

49. The device of claim 47 wherein the diaphorase catalyzes the reduction of the competing substrate and the chromogen with similar efficiency.

50. The device of claim 47 wherein the analyte is selected from the group consisting of alcohol, glucose, ketone and lactic acid.

51. A test kit for colorimetric measurement of the amount of an organic analyte is solution wherein the analyte is capable of being oxidized in a NAD(P)-dependent dehydrogenase reaction to produce NAD(P)H, wherein the test kit comprises at least one reaction area means, containing NAD(P), a dehydrogenase capable of oxidizing the organic analyte, diaphorase, a chromogen which produces a color change upon irreversible reduction by NAD(P)H in the presence of diaphorase and a non-chromogenic competing substrate which is irreversibly reduced by NAD(P)H in the presence of diaphorase, wherein the amount of color produced upon reduction of the chromogen is less than that produced in the absence of the competing substrate and the color change occurs in a ratio of less than one molecule of dye per molecule of NAD(P)H produced, said color change being indicative of the concentration of the analyte to be determined.

52. The test kit of claim 51 which comprises two reaction area means, a first reaction area means containing the NAD(P) and the dehydrogenase and a second reaction area means containing the diaphorase, the chromogen and the non-chromogenic competing substrate.

53. The test kit of claim 52 which further comprises a known amount of reduced chromogen, reduced non-chromogenic competing substrate and oxidized organic analyte.

54. The test kit of claim 51 which comprises a collection means for oxidized analyte.

55. The test kit of claim 51 wherein the reactions are carried out in a liquid.

56. The test kit of claim 51 wherein the reactions are carried out on a solid medium.

57. The test kit of claim 51 wherein the affinity of the diaphorase for the competing substrate is greater than that for the chromogen, whereby the non-chromogenic competing substrate is reduced without production of color prior to reduction of the chromogen, and wherein color is produced upon subsequent reduction of the chromogen.

58. The kit of claim 51 wherein the diaphorase catalyzes the reduction of the non-chromogenic competing substrate and the chromogen with similar efficiency.

59. The test kit of claim 51, wherein the reaction area means comprises a test strip of absorbent material.

60. The test kit of claim 59 wherein the reaction area means is a multilayer test strip.

61. The test kit of claim 60 wherein the dehydrogenase and the NAD(P) are positioned in a first layer of the test strip and the diaphorase, the chromogen and the non-chromogenic competing substrate are positioned in at least one other layer, and wherein NAD(P)H produced in the first layer diffuses into at least one other layer for detection.